BLG/UMIP/IT. 1 Rev. l

ENCLI SH ONLY

MANUAL FOR THE PRODUCTION AND CONTROL OF VACCINES

DIPHTHERIA TOXOIlJ - ---

Ih* lssur o f this dncdmcnt does no1 CO11GtltUte

b m a ~ PUbl lCLl j~n I\ should not bt rsu~cwed cbttrlctsd or wilh.~ul the arireement 0 1 *a World Health Organrra!~nn Authnrl &:Ono

'Olpnnslbie IQr views exptA5sed h r . ssgned ~ 7 l f l r ~ I ~ .

CC docurncnl ne constitlle poa uns publ1Cab0.l

11 nv 0 0 1 7 Ialrc I'obiel d'x.r.un Comolr! rendu Ou

r l s u m e "I d rucune c#tallun sans I'aulorr~ntmon ds l'Oryanis3l1on Mnnctabt 0 8 ln S3nle \ c? ~)[nr . i~I: ,

eaprl.ro6os dans le5 arltclba %.me-. "cng:igerll

q c ~ s loura auleurs

B L G / O N D P / ~ ~ . 1 Rev. 1 I

Appendix U.28 I ! 17

K6Ul.i lRI'MIINI'S !;OK BIt)I.UGff:AI. SlmS1'.4N(.I:S ( [%S)

...

The mixt irnpur~icnt iniurriltion concerl~i l l~ tllr *Pty o f s biologic; 1 w h t m c c is given by evnsiste~lq of praiualion, r h i c l l is col~lp[c~ncn~;i;,i

the tL.ns Lipplied to the tin31 lillcd material. The .qppro\:;ii (1s mim;!. fcld~rillg n~ctllodr. t l l ~ 111:iintcli;lnce of iicolratc rfcordr. and tllr inyxctiuli

of p r ~ l u f l i o s hy the ni~tioll;~l contml ;~~ilborily pl:~? ;I n3:ij.r in cieisill; -.

collfrdeucc in i hc s3i'r.t-r; <>I. ttlc pro~j11i.t.

- In m i d c ~ l l situ;ition t11c rjnw contml n1p:isurcl a ( ~ ~ ~ l d he cxcf i . i rd hi

1111' g ~ ~ ~ ~ n I l I i ' l t 1 ~ or 311 L , ~ U I I ~ I i i s 1 s SLJ;II c i r n u ~ r , e i ; ~ ~ , t larc u.l,u],j problcn~ i l l rlw I)ec F A C ~ I ~ I I I ~ C 01 iilbil)gi~al \~b!;t:~gl~(. tydl~:Pefi countrie~,

ILL. cl'tiiri?l ~ ~ L h o r i l ) In ally onr would be T.lt:rJ c~lkly with ihc pmbleiji Of ~ ~ " t ' D " i n g S Y ~ S ~ ~ ~ I I C L ' S 111rllllf~~cf11rn~ vilhitl i t s own jurisdic~inn, 11 is. ~ s Y I ' I ~ ~ P I 10 r~it l izc tha l i t Ull! 1~ m;ll,)

hrforc sucjl ;lll idrill siiuatiun c m possibly b brovgllt :~L)ut ; ill inlcrim it will continilc '0 JlUXs~iUy for the nrfi(m:ii canfml uuthorfty to deal not only with th,.

subslrncrs n ~ m u f i t c t s r c i i W I ~ I I ~ I I its or11 jul ixl ic~i l js , bllt wt,. stances inrporrcd frorn i?t her count lies.

.- ~

BLG/WP/?~ . 1 Rev. l

Appendix D.28

REH)RT OF A. WilO EXPERT GROUP 13

1. Pcrsonne1 ~lnuf*c~uril lg shall be in charcc of if peslon who h" brcn trained in

the l ec~niqLirs u sd in mdnufaduriog biolugica~ substasces 2nd the aicn- lific knoviedgc upon which the mnnuhcture of ~ s C product\ is ~ ~ S R I . hi^ havs aufijuient iiu~.hority to enforce d i ~ i p l i n ' among c,ployees, wllo rllull include specialists with training npp~op'i'v the

made i l l [he cstablirhment, The names ZHId q~l l i f ica l lon~ of such spccialidr, espccial\y thoa rcsponsiblc h r signing 2 protocol, illall jqirtered with the nationill control ;luthority.

~ h ~ , , in d t a l i n g ~ j t h the p r v h ~ n n vf mlmufucture. r jruinirir i * nccdc,j is sol,,r. i lr all of thc hitowing f ic lr l i : b;icicriviQgY, banwlr,,, &.llli$ry, i i c phi~~'mdi). p h l r m a ~ . I ~ g ~ + vererlniiry rnctlicinc and vltolo!+y.

,rhc aaE making sr,nirOi lusts shauid hc sCp;ll.~lc t'rdin mnnuf:iclul.ir>6 U ~ L : in~ i not r t s p l ) n s r b l ~ 10 lhe pcn(m in char!?

2. Buildings and wuipmert

B ~ G / w ~ p / 7 7 . 1 Rev. 1

Appendix D.28

1 6 I ~ ~ ( L ~ ' I H 1 M1:NTS 1.OK BIOIDGI(.AI. SL'BSTANC~S {lqt,r)

~'lUp1~~ycc.r should Stag in $heir or$-11 work arras, .rnd ncur prOtccli+c rftkhing, including shoes, caps etc., which shoul,j rr?m~ill ill thc ;!red. Employres sufkring f r ~ m rn inrccri\u illfIcss ~h0uhl not he ptrmltlnl to wad until c<>n~plddy recor- untl.

Vi%i(urs bhothtl bc as 1 . e ~ as poss~b~u and t h q shnulct not ntlrmnllg hc pcrrnitled t o enter sterile rooms,

AnllnaJs intended for 11se in productiol~ or in tcsrs slioultj bc "hscr\rJ daily dnring a quarantine period or not less thAln onr "cch. in SOmC inr1;tnccs it is desirable to maintain the ;tnimal rll('nl* L'()llsldlltly al lhc oplimum lemp'ature for the parrluular ~Pccic% m d test. m d a b( wsslrl m a i n l a i n strains 01' lest animals.

*'l~tll*lr 11\81 t f l t I'rom i r l lcnl~.~~ arc d , - , l , ~ , ~ ~ r j , Frrfclhj~ 111 a11 I ~ C I I I ~ K I I , ) ~ .

4. Filling and contait~rrs

4 . l J-i//il?\c r~lo,,,.,,

1 1 l c i n I O S r i s l r p s , . ~h~~ shall be sterile ro l ln l r quippcd sprcific;~llj lar tnnsRmng mcijr~lncd qll;ll,.

titics of liljishcd hiulogirrl ruhst:~aces fro~n bulk containcrs t o ,hc fin;ll Stri~f

C(3ntml InCilSl!rcr ;lnd aseptic techniqllrr ~ b ; ~ l ] br ~ " l ~ r ~ ~ l l efisllrc lhrt tilt? pridt ict is nut a ,n tnminard dllr inc the liltin!: Pri'ccss.

L.

BI.G/UNDP/ 7 7 . I Rev . 1

The filling process should be chcckod I 1 iwrc ~ u c h Year

at ,he md of 1 m k i l ~ g dry by d l l i n ~ not than .~P~)LJICS

nu,nent mcijium containing no antibintics or baclcrio and incubatin$ the complde Dntch

fillel'

mpou\us. NO^ mnre th~. 1% of the ampoules 611rd in this 5h,lw s i p of ~onraminat~un and all conVamin*llls

4.3 Coirtniner~

=he fitul Eonlaiorr be sealed ss soon 25 p o ~ ~ i b l e after fifling-

~ ~ s ~ ~ c s be of material that does not have a d ~ ~ e t e r i o ~ s cliect *pDn the bic>logicrl subsranrer, and shall be designed to munlain a hermaic throuphold tbe d:~ting period.

. S. Tests

test, of a v i f i e biological substance. rcquHng the of living

lni~~o-org~nisms. shall bc carried out in ranns % p r a t e from those uwd f"r production.

prcfernbly, tests should bc carried out in such sfparhtc

rooms. me dacrip,,, 'h. tcws neccssury to establish the 5afsi)l.

and of ~ c h Ic)t of a bi~>loCal subSp.nCc ~111 & girt" in the, requirements to be fomulated (0' the ~ a ' l i ~ ~ ~ ~ ~ hiologicrl subrtittlcf

6. Records

6.1 Pr~ducrio,r p r ~ t ~ c o h grid disrrihutiort recofA shall pcrmancnt and dearly indicats all Steps in p r ~ ~ s ~ i n ~ ~

t.*i.g. fillins and distfibution Wri t ten W O ~ ~ S shall kept of tests itrcrpcctirr of their results. The records shlll be of a appr~ved by the llalioml control U h o r i t y . Thcy shall be retained thro%hovt Ihc

dating prriod a l o t or bateh of a hiologicicd product a n d awiiablc

all times for inspsctioo by thc national ~0nlfOl authority. ~~~~d~ must it ponsibk m trPCc ,111 steps the

nmurn.ttlrc waing of s hash, and rhodd inclultc records

of S;lcrilinrlion of apparatus and materi;~ls U-d In its manu- hciun, L ) i ~ ~ ~ i h v l i ~ n WONIS must he ~ c p in I rnln1.r h n t pcrm,w rc,c;il~ l d ;my puticuli~r hl1.h. if nwsyrr) .

6 .7 Rccorrls of c~vilvrer words rh;l\l bp rnaiotain4 uf the c f l m p l ~ t o PSSaP history Of ql'

c ~ l t m n kepi in the ertubli&mt. C u l t t i r ~ ~ shall be LaMled and stored In

a safe, ordvrly m n e r .

B L ~ U N D P / ~ 7 . l R e v . 1

Appendix D.28

"'F' B ~ G / U N D P / ~ ~ .L Rev . 1

I 8 Y1WJIRW.NIS FOR BI~~L<)<;Ic*L S U H ~ ~ , , N C ~ ~ ( I ~ L ~ ~

7. Samples

S C ~ ~ P ~ C S front each lot ~ l ~ s l l bi: takcn in sufficient amount to for samplc~ 0f t h ~ natlunrl ~ u n l r o l h h r a i o r y , Additional

mlm~lc!+ shall be retained tltrouphoui the d;lfing pried ,Is reference in a minner which ensures the ldcnfity ofthe lot.

Mafiumcltlrrrs should relri~l sufin.ll[ .&jllioIlal Ul,,,,~cl l. prr1llit reprulion ol. the cl,ntrol tcrtr,

8. Labelling

A l pmducts shall be clurdy idontilied by i;lh.l,. The inhrmalioh given on the label on the container or the label on 1 1 % ~ p2kckabL 'I shall .

bc delermi~lctI by the national control ;iuftl,.lrity. The labcl on the contuincr shall ~ h u u ;,I iwa( :

Ule of the p r o d ~ ~ t (Le. . ttie intrnl;lla~n.~/ li,\mc and,/ur the p r o p r name) : mme of the IXii l~huturtr (;sid addrcn if required) ;

the number ot. the fin:tl . .-- I-. 3 the re~~n7mcnded human dose a ~ t d route or n&lliilirlmtiun : conrlilion of storkg~ aild expiry dillC.

The label on the package shall. in addition to the iaformdtioll shown on tfre labcl on thc containtr, sI1ow ; ~ t fist +

tllc llaturc 3 4 amount of ally pesovrlni. 0, r&Jd soblt;lnce prcsinl in 1 he product :

l f t h f i ~ d l wntaincr i s not s ~ ~ i t n h l r f i ~ r Irhfiill~ (hr cxalllplr, "uupillary l uhc) , i t should trc in :t labcflctl packitpc

9- Distribution m d shipping

9 c 1 Rt+a.rc, fur ciirrrifirrrio,,

A lot of l biolop.c:~l substancc shall not he re1carrd ufltll all ttle rcquird tests have been performed, summarized and reviewed

undl m\ elilci

Appendix D.28

REPORT ~ i : A WHO EXPERT GROUP

.fiCir\ rtquirmellt is saiaficd. These teas shlll a lr l ) , i"llidc ;l1'

identity lest performed 0" the contents d r finished piick;*$c f ~ o m crcl1 f i l l io~, to confirm the rcuraiy of the i ~ b c l l i n ~

biologic;il sub"anrc &a\\ be rc1c:ixd until cansislmuy of

pro~uctian has b s o utabl\shcd.l In routine production, faiiurc of si11!$' a3,ch to r ~ q u i r ~ m ~ ~ safety shall he ~ o n s d c r e d as a ~ ~ ~ ~ k ~ 1 3 ~ n in pto~uct~o.tioo, and shall be rc-established to thc wtishlclioll 01.

the nakioml ccn~rol ruthori{y hefo" any fufthtl b l k h ~ s released.

9.3- Shipping ~ i ~ l ~ ~ i ~ ~ l rubstaocer shall be

with precovlions (0 CnSUrc th:ll

repdins its p(ytacy upon arrival L its destinatl~n. annot be hid down to corer i l l [ ~ l t u d i i l l l * : 11115

,qnirss the continuous exercise of l u d g t m e l ~ ~ .

10. s t o w a d expiry date me atatcmenrr EOIKeming rtorag~ ~ L M ~ C T " Y ~ C S

e x ~ i r ) .

apFing on the hbal and the packing klRer. ns rcqdred in part 4. a- ,,, g, (x based on crpcrimefital evidence a11d drill hc 5.hmilled for approvd to the cont rd authority.

10.1 Storage conditions ' produds be rtowd at all tims at eontn)llcd anl)rrdk~nr

within a range which ensures opimd st~bil i t~. okng d-ibution, & ~ f l periods rt an~"imt w m p r ~ l l l ~ r ~ ~

may have w be @ttdtd.

10.2 Expiry dale ' 7hs npjFi d a Of a b i o l o P d produck shdl he dehncd fire* with

the approval of the nntiawal mnlrol .uthoriiy*

B. maed p~quiremcRts for Control L a M a i ( * b

1. AclminisCra~on ULd -1

~1~~ eonlrol luhc,rilrory h. ndminiaared by 0' ('1'1

t"t1dlf tllc

,,jnnal wrd atithori{y, (he cveilt 0f W U ~ ~ C ~ U ~ C

""til)nr'

being dc,,nu in the same cstabli~hment, thc rolltrd i ~ l h l ~ r i ~ t ~ r ? *haLi h an idrpndcnt

dimf ly rcspp~sibl. to the ndion.1 confrolauthori t~.

A ~ ~ M ~ ~ for taking mwst)rn de,ignol 10 CIIW~ that hu,t,b,cyi rubs,..o.. YIJ in a roudry 3'e S ~ P . Potent

. -. - .. .ut.\tr\i\i~ed for tht l i l ~ t

I ln roun&s, c~n"sttn~y 01. ~ ~ < L I Y C I I O I ~ 111s (0 *c L,

five production butcha.

BLG/'uNDP/'~~.~ Rev. 1

Appendix D. 28

20 vc'ulRf?bfEh. 1s FOR BIOI (XiICAL S I R f l ~ ~ c f i (~th~]

bivlo~lca[l~ l'u". ~ 1 ~ ~ & f h rest3 Yprn I h hralth depdmml ,* the EoV(mnml (g lh.2 muntry. This aulbority mull; hclhch,er, be dclearlnl 10 the n p e r t in char@ of t h ~ control ~rboralorr, whl. shcltlld j1.W r ~ l l a lho r i ty and full mponribi l ig . -

The ilcillf of the ~ C W ~ I O ~ laboraton shall a pr,,, qualified alld c * ~ ' i ~ n r . t d ill the control of hiulogiral rubstanws,

stiif the cCWKd bboratory shall include expens in all discipline\

required to cover the biolvgical substmuc. the labratoy ,,,, colltml, h,tfl ~ I ~ U W tli;lt manufanurcd in fhr counfqand lbon imponcd for use.

2. bildiqs and equipment

requir~rncnts in respect of buildings and quipmat d s s c r i ~ ill A . balon 2 , shall apply in it general way to a control laborntnrv.

" C s)\rtctn of contml shall includc l ims ing or m a n u h t u r e n and '01liin~ inspwtion of their esshlishnmts. T,,

arldition, the national control JU'"O"V sb;ilf demnine the extent o~coonlro~ testing ofindit,aual produelr, . ".

Mmuhcfurcrs shall be liccnsed in respacl of cacti indiudual biologicill subsfaner which they ItXdllufact~re and mrthodr shall exist for rithdmrinp 'he licence for thnt substance in tIw tvcnt of failurc to meet the rpprop~;rintc gcn~t:ll i1116 bpe~.i:11 r ~ q l i i ~ ~ l t l C n t S , I.$ rnanufiicturing area anlf p r ( ~ c e s 5

dull lrlilde rccrssihlr t o the nallunrj mnlrol ra[horjl). inhpCc, tint1 :it aH re:isttn;thlc times.

finishccl Druductr, its ~ ~ 1 1 :is on samples taken at an in~fmediate

~h~ lrhoirtoy devir c k 3 1 ~ c int~rnal c0"twL inFayFs to ,,it objecUve interPTnation of tests and e ~ d ~ a t i o n of its

rcltabl"t~

K

in pcrforrni& all tests.

.rhc iwluuon of replicate coded umples in pwducn LIV .imu)taneous i n d e ~ n k n l lcsling of

btch

of lub.(anc., and mutine chcrkr on ~ ~ n ~ i t i v i l y and fnlibr.tlot1 of insmmnts are m e ~ ~ u r e s that my M applied as self.im~a=d .' coIlkrols " for the control laboratory.

~ ~ ~ l t h ~ of wrious species md unqucstioncd strains ,hall

available in adequal= numbers far an effective performance Of fhc tests

be undertaken. T~.~

c ~ n f n r m to strictcr requirtmentr "a"

used in mafiufitcturing ronlrol W a u X the n u m a r yf pmphs jr ~ i ~ l d marimsm reliability of lm1s 'S

d,,mdoc,e~, bnlmd1s s h ~ u W be nuintaiord o~limvrn

nutntiunr, e ~ v i m n n ~ e n t a l cond~tions and during lerlr 1, nMual b r animals tu br kept frar ln*m ink. Y<)u5 is he51 acc~>rnplYhcd by hrrcding '"(1 n13intcnuxc anlmsl~ n r t n ~ r d y free fmm s p i f i c V~'~QF" and prylc,.led again. inf~<t~tums h c c > ~ a m ~ a l ~ J food. air and

or by c O L l t a ~ t wit)) ercturs, a~umais and man. he ,,*ionat cc,otrYl ilvfh~trny should i nvn i iy l c thr 3 s of

s l , , ,c l~ pthogen.frc* (s.P.F.) animals for c ~ n l r u i 1cll.-

~ ~ ~ r ~ ~ l authoriris Should be familiar with intcmati0na1 smn dards for the of potency and, where national standads e*s t -

they should be

calibrated in terms of the in(cma1onal ~ ~ ~ n d a r d s .

DL.,~! ULYULJ/ //.L KeV. 1

Appendix D.28

3 . 3 Release unil c~r!$cation

A lot of a biological siibstance shall be relcased only i f i t fulfils the requirements iidoptcd by the national control laboratory.

In certain circumstattccs, the oficial in charge of the national control laboratory shall provide a statcmcnt. at the request of the manufacturing Iaboral.ory, crrlifying whether or n o t a given lot of a biolugiral substance rnccts all appropriate requiretnents.

It IS in ~cncral impracticahlc and may in the futurc bccornc untlcccssary for o colllrol hboratory adeql~ately t o iut l i l thc rcquiremcilts fc~r licensing and inspeclion of manubctnring establislitnents nutside its own jurisdiction. t'wr the control of. iniportcd proclucts it is thtrcfore primarily dependent upc>~r tcritr on ihc find products thenisei\-e\. suppleii~cntetl by pro- tocois of [eslr cilrricd out hy the m:rnr~lhcturer and, in certiiir., ~ i r c u n ~ s t a n ~ ~ s , h\. a cert~ric,~lo to the ewwt that the control authorhy of the outtntry 01' origin has found the proriuu~ in fulfil sprcifictf requircnicnts.

I t is desirable so to urwnirc the control laburattrry th:~t opportuniiy is prav~dcd I'm ?esesrch in addition to routine testing. Encouragcnrcnt of restarch activities will nnt only Itad ra the dcvelopmeni of better methods of control, bu t will atsn help thc luborntnry l0 relain an inleresled, efficient, a13d highly quslificd staff. 7%~: nunihcr o f spcriniisis in the control r l l ' biological subsranccs needed hy any country is ~ o o slnall i r i

justify specific ulliversily courses in this field. It is thcrefi>~c nweaa ry fur thc curttral lahoratory itself to adopt a vigcrrous training progrmime, covering hnth the techniciil anti ~ h c administrative aspects of colllrol prwcdures. In general, this will bc hest accon~ptished hy direct supervision of junic,r sfntl. during thc actual prrf~>rmince of tiuties, buf auch supervision may he supplcnlented wherc cnndilio~\s permit by mare furniid instruction.

A number of countries are wishing to produce veccines a t the nat ional level f o r which they require the n cessary technical infomation. Y I t h t h i n aim the present manualf has been p r e p a r e d .

I t should be s t r e s s e d that t h i s mnual is intended only to provide general iof~rmetion on methods - preferably the least complicated ones - for the production of a diphtheria vaccine of acceptable meeting the W O llequirements2 and t o d e ~ c r l b e the necessary t o s c s . There e x i s t other production methods, which may l e a d t o e simller r e s u l t . The manual , therefore , does not necessarily express any preference for the methods choaen and should not be regarded as a description of those production methods preferred by WHO. T h i s is true also for all (biolchemicai preparetinns, specified in the manual as well BS for the mantlfacturers and epecial equipment mntiorled.

I f c l d r i f l c a t i o n In r e q u i r e d an any p o i n t , reference shodld be made to t h e WHO Secretariat { C h i e f , Biologicals ) .

L The manual was prepared by the foll~wing coasul tants and members of the WHO S e c r e t a r i a t :

Or J . Cameron, Connaught Laboratories, Willowdale, Ontario, Canada ( ~ o n s u l t a n ~ l

Dr S . ~ u ~ t a r a k , GavernrnenL Phaweceutical organization, Rangkok, Thailand {ConbuItant)

l)r 1.j. H i g y - M n d i f , Hiologicals, WliO, Geneva, s w i r z e r l a n d

Dr P . Knight, Tire Wellcome Research Laboratories, Betkenham, Kent, UK (Consultant)

Ur Y . S. Nirnbkar, H a f Ekine Aiophermacebt i cal Curpora t i o n , Ro~nbay , I n d i a (Consultant)

Dr F. T. P c r k i n s ,chief, Biologlcals, W l i O , Ceneva, Switzerland

Dr . I . D. v a n Rurnshorsr., r,loloy.icals, WilO,Gcneva, SUI Lzcr land

I. - continued D, N gksricb, i-nologiral In6titute, zaErcb.

~ ~ ~ ~ * ~ a v i a (consultant)

2wo Technical ~ p ~ f f Ssrles 1979 ( t o b. p u b l i s h e d ) i AppenJixD.26 of th i s manual.

B I C / U N D P / ~ ~ . ~ R e v . 1

page 4

CONTENTS

DL, INTRODUCTION . . . . , . . . , . . . . . . . . . 7

D L . 1 D e s c r i p t i o n a£ t h e v a c c i n e . . , . . . , . 7 01.2 E f f i c a c y of t h e vaccine . . . . , , , . , .

7 01.3 Suggested method o f p r o d u c t i o n . . . . . . 8 D1.4 WHO Requirements . . . - . . - . - . . 8 D1.S CLclssary of t e r m s . . . . . . . , , . , . .

g

D2.1 Des ign of p r e m i s e s . - -

D 2 , 2 Q u a l i t y of mate r ia l s . . " " . * - * * 12

D2.3 Changing facilities . , . . . . . . , . . . 1 7 . . L L

D2.4 P r e m i s e s f o r breeding and k e e p i n g a n i m a l s . 13

D 3 . EQUIPMENT FOR T O X l N PRODUCTION . . . . , D3.1 P r u d u c t i o n rnerhods . . , . . , . . . . . 14

D3.2 S e p a r a t i o n o f t h e toxic f i l t r a t e . . , . . 1 5

D3.3 P u r i f i c a t i o n of t h e a n t i g e n . , . , , , , . 15

D 9 . L B l e n d i n g of bulk . , . . . . . . . . . . . 16

STAFF

D4.1

D4.2

D 4 . 3

D4.4

. . . . . . . . . * . . . . , . . . , . . . Q u a l i f i c a t i o n s and numbers . . . . . . . . E x p e r i e n c e . . . . . . . . . . , . . . . . H e a l t h . + . . . . . . . . . . . . . . , ,

O r g a n i z a t i o n o f a c t i v i t y . . . . . . . . .

l I D6. STRAINS OF C . DlPHTHERIAE m - . . - - . 19

D 6 . 1 O b t a i n i n g t h e s t r a i n . . . . - . . . . . . 19

D 6 . 2 Maintenance of t h e s t r a i n . . , . . . . . . . 19 ; I

D6.3 Preparat ion f o r p r o d u c t i o n . . . . . . . . . 19

I D7. PRODITCTION OF VACCINE . . . . . . . . . . - S .

2 1 I t I

D7.1 Growth of C. d i p h t h e r i a c . . , , , . . . . a

2 1

D7.2 H a r v e b t i n g of t h e t o x i n . . , . . . - . . 22

D 7 . 3 SLorage of t o x i n . . . . . , . . . . , . . . 2 3 I

D7.4 P r o d u c t i o n of b u l k t o x o i d . . . . . . . . A . 2 3

Wethod A. P u ~ i f i c a t i o n of t o x i n . . . . . . 24

Method E . D e t o x i E i c a t i o n o f t o x i n . . . - . 25

D 7 , 5 "ln process" c o n t r o l . . . . . . . . . . . . . 2 7

t D8. PDDLlNG AND STClWIGE OF BULK TOXOID , , . . . . 30

i D9. DILUTING AND FILLING . , . - . . . . . , . . , . . 3 0

I D9.L P r e p a r a t i o n of adsorbed toxoid . . . . . . . 30

D9.2 P r e p a r a t i o n and t e s t s on f i n a l b u l k . . . + . 31

1 * D 9 . 3 Filling i n t o C i n a l c o n t a i n e r s . . . . . . . . 3 3

D9.4 I n b p e c t i n n . . . . . . . . , . . . . . . . 35

b, 010. TESTS I)W FI~;AI. PRODUCT . , . . . , . . . . . . . 36

. . ..:,:X, , P10.1 Tests

. . , - , , . . + . . . . . . . . 3h

. ,

. - ,; .S>

!.)5;i;: D10.2 R e p o r t i n e of d a t a . . , . L . . , . . . . , . 36

* b.,'

3;; *,y.: , g$$

. .,;<::.&; ...~%5'~=-,< . . >

..'F

BLG/UNDP/ 7 7.1 Rev. 1

page 6

E!%!? D l l . IARELLINC AND PACKAGING . . . . . . . . + . , . , . 37

D13. RETATNEL) SAMPLES . . , . , . . . . . " " * . * 38

I D14. SLIRVElLULNCE OF VACCINES . . . . , . . . . . . . 3 9

IIL5. WHO UEQ11IREENTS . . . . . . . , . . . . . . . . 40

B L G ~ U N D P / ~ 7 . l Rev. 1

page 7

(Requirements f o r B i o l o g i c a l Subs t e n c e s No. 10 [ r e v i s e d 1978)) See Appendix D - 2 6

DL,L D e s c r i p t i o n o f the v a c c i n e ( P a r t A . S e c t i o n 2 , page 31) Sec Appendix D.26

D i p h t h e r i a t o x a i d i s p r epa red f r m the t o x i n produced by t h e growth of a s u i t a b l e s t r a i n nf C o r y n c b a c ~ e r i u m diph~l ier i8 .e i n a s u i t a b l e medium. The s u p e r n a t a n t f l u i d , which contains t h e t o x i n , Is s e p a r a t e d from t h e o rgan i sms ; t h e t o x i n so s e p a r s t ed £rum the

organisms i$ d e r a x i f i e d and p u r i f i e d .

D1.2 E f f i c a c y of the v a c c i n e

D i p h t h e r i a t oxo id i s the i m n i z i n g a n t i g e n p r o t c c c i n ~ against the a d v e r s e e f f e c t s of i n f e c t i o n by d iph the r i a a r g 8 n i s m . Although t h e t o x o i d i n d u c e s the p r o d u c t i o n of d i p h t h e r i a anti- t o x i n and w i l l , t t t e rehy , p r o t e c t a g a i n s t the l e tha l e f f e c t s of d i p h t h e r i a toxin, i t does not n p c e a s a r i l y p r o t e c t a g a i n s t the growth of the urganisms . I n t h i s diseaue, theratore, t h e r e may

be a c a r r i e r scate and t h e organisms may c i r c u l a t e in a cormnunicy . . u n t i l such t ime as a h e r d immunity i s b b i l t up.

Although t h e t o x o i d i s qui te a good ant igen i t cannor be r e l i e d upon t o give p r o t e c t i o n if used f o r p r imary i m u n i z a t i o n Without an a d j u v a n t . For this pu rpose , t h e r e f o r e , t o x o i d

adsorbed on to a n ad j u v a n t ( u s u a l l y aluminium hydroxide o r 6 3

i s phosphate) i s used . Fu r the rmore , d i p h t h e r i a t o x o i d e i t h e r . p l a i n or adso rbed i s rarely used a l o n e . I t is much more common

. V .

, + to include i t i n a combined v a c c i n e w l th t e t a n u s t o x o i d and

>X-. P e r t u s s i s vaccine . *l,. c':< ? " A d i p h t h e r i a t o x o i d complying w i th t h e WHO Requi rements

Z.;:a' and given i n t h r e e dose s s u i t a b l y spaced f o r prinvlry ~mnuniza - . ',- .*, . t i o n w i l l p r o t e c t a g a i n s t d i p h t h e r i a , and a reinforcing dose

a 7 '?=?g ~ . ~ i e h ~ ~ l d be g iven a t s c h o o l e n t r y ( a b o u t f i v e years o f a g e ) .

D1.3 S u g g e s t e d method of p r o d u c t i a n

T h i s manual describing t h e p r a d u c ~ i o n and c o n t r o l n f d i p h t h e r i a t o x o i d suggests methods of p r o d u c t i o n t h a t have been s h o r n t o g ive a snLisfactory p r o d u c t . The method v i l l not n e c e s s a r i l y u t i l i z e t he m o s t r e f i n e d , e x p e n s i v e a n d modern e q u i p - ment t h a t may be i n u s e i n sane l a b o r a t o r i e s having many year.5' e x p e r i e n c e i n t h e p r o d u c c l o n of t h e tnroid, but the suggested method is known t o y i e l d t o x b i d oi c o n s i s t e n t l y good q u a l l t y . IL i s o n l y a f t e r a new p r o d u c t i o n area has llad a mmber

of y e a r s 01 e s p c r i e n c e i n Lhr p r o d u c t i o n nl the t u x o i b b y a r e l a t i v e l y simple method that mtdificatiuns should be considered.

The controls a p p l i e d both d u r i n g p r o d u c t i o n and on ~ h c final p r o d u c i follow cIose ly t h o s e i ~ ~ c l u d e d i n t h e WHD RequiremenLs (see Appendix D.26). A most i m p a r t a n t f a c t o r i n vaccine manu- Lactur t l i s consistency a t p r o d u c t i o n oL quaiity v a c c i ~ l e and a d d i t i o n a l c o n t r o l s t o e n s u r e his a r e suggesled.

I n a c L e m p t i ~ ~ g ta make the manual a5 w i d e l y a p p l i ~ d b l c a s p o s s i b l e i c has been n e c e s s a r y i n some ~ n s t a n c e : , t o s u g g e s t a1 t e r n a t ~ v e c o n t r o l L e s t s . Theselurc, if a l a l u r a t ( ~ r y has no experience ot m y oi the s u g g c s ~ c d t e s t s tllc u z ~ c of chuicc s h o u l d he che one most e a s i l y c a r r l c d o u t , I:av~np r e g a r d t o t he a v a i t a b i l l Ly nf marerials and r e s o u r c e s .

Tn g e n e r a l , t h e methods r e c o m r n d e d will cr)nply w i t h the r c q u i r e m c n t s o f most n a t i o n a l a u t h u r i t i e s f a r the p r o d u c t i o n

a n d contrvl of biological s u b s t a n c e s , 1 2 c h e r e i s a d i v c r - gence i n any p a r t i c u l a r c o u n t r y , p e r m i s s i o n t 3 u s e t h e nrethuds o t h e r t h a n those recommor.ded must be obtained from t h e national control a u t h o r i t y .

D1.4 WHO Requirements ( T e c h n i c a l Repor t S e r i e s (to b e p u b l i s h e d 19791 S e e Appendix D . 2 6

B L G / U N D P / ~ ~ . ~ R e v . 1

page I)

The WHO Requi rements f o r D i p h t h e r i a Toxoid were f i r s t fo rmula ted i n 1964; s i n c e then c h e r e have been s e v e r a l d e v e l o p n e n t s i n vacc ine p r o d u c t i o n and t h e Requi rements uere t h e r e f o r e r e v i s e d i n 1978.

The c o n t r o l p r o c e d u r e s suggested i n the manual f o l l ~ u them c l o s e l y . Al though t h e method of p o t e n c y c o n t r o l s u g g e s t e d i n t h e manual may n o t be i d e n t i c a l w i t h t h a t used i n meny c o u n t r i e s , t h e q u a l i t y of the f i n a l t ~ x o i d w i l l satisfy the - - -

r e q u i r e m e n t s of a l l c o u n t r i e s .

i

i DL. 5 G l o s s a r y of terns_

i K f = F l o c c r l l a t i o n t i m e ( i n a i n u t e a ) as

observed i n thc f l o c c u l a t i o n r e a c t i o n ,

i L f Limes f l o c c u l e t i o n i s ; the amount of toxin or toxoLd which when mLxed w i t h 1 Int r r -

I n a t i o n a l U n i t oL a n t i t o x i n g ives a Ramon f l o c c p l a t i n n i n the s h o r t e s t time.

L £ / U I ~ N = F l o e c u l a t i o n u n i is per m i l l i g r a m of total n i t r o g e n as detemlned by the Kjeldahl a n a l y s i s .

~ f / m ~ PN = F l o ~ c u l a t i ~ n unit^ per milligrau of protein n i t r o g e n (PN) which i s usua l ly d e t e r m i n e d bv t h e p r e c i p i t a t i o n o f the p r o t e l n s w i t h t r i c h l o r o a c e t i c acid .

I.U. = International U n i t is the specific a c t i v i t y oE a s t a t e d amount of the I n t e r n a t i o n a l S t a n d a r d as d e f i n e d by the WHO E x p e r t Committee on B i u l o g i c ~ l S t a n d a r d i z a t i o n .

= The minimum amount of t o x i n which when combined w i t h 1 1 . U . of a n t i t a x i n k i l l s an a n i m a l of a d e f i n e d weigh t i n Tour days ( t h e Li i s d e p e n d e n t cpon t h e an imal species u s e d ) .

" . . - C :; : iT . .. G '., , -

L+- / l0 = The minimum amount of toxin which when ctm- , . . . ,<. L .,. . , . ; ? . 3 . , r . * .

b i n e d w i t h 0.1 I.U. of a n t i t o x i n kills an a n i m a l of a d e f i n e d r ~ e i g h t i n [our days .

page l0 page 11

L r = The minimum amount o t t o x i n which when

combined with a f i x e d a m o t ~ r ~ t ot a n t i toxi11

( u s u a l l y 0.002 I . U . 01: a n t i t o x i n ) i t 1 a volume of 0 . 2 m1 causcs a l o c a l s k i n r e a c t i o n t h a t i s j u s t v i s ib l e .

= The amount of toxin t h a t k l l s 5o;b of a group of animals w i t h i n l o u r days (the LD d i f f e r s f a r d i f f e r e n t a n i m a l species?!

M . L . D . = Minim1 l e t h a l d o s e , the a m o u n t o r Lvxin w h i c h k i l l s a n i m a l s w i t h i n f o u r days ( M . L . D . i s different Tor d i f I e r c n t a n i m a l s p e c i e s ) . I n g e n s r a l t h e M . l . . U , has bee0 r e p l a c e d by the LD

50' M . T . V . = Maximum t o x i n v a l u e , t h e q u a n t i t y of a n t i -

t o x i n t h a c can b i n d toxin; t h ~ s i s d e t c r - mined Ly t h e n e u t r a l i z a t i o n of t h e tox ic e f f e c t i l l a n i m a l s of toxin-a11Li toxin m i x t u r e s .

A . B . V . = Anti t o x i n b i n d i n g v a l u e , a v a l ~ i e w h i ~ h

d c t i r l c s t h e t o x i t ~ p l . u s coxuid it1 a m i x t u r c ( d t t c r r n i r ~ e t l in a n i m a l s ) .

D. U. = O p a c i r y OnfL i s t h e rneasurr OF o p a c i t y a s d c t e r n ~ i n e d i n cornparisnn w i t h t h e Intcr- n a t i o n a l R e t e r e n c e P r e p a r a t i o n [ IF Oyavi Ly.

T.C.1'. = 'rota1 r n m b j n i ~ ~ g powcr, the r e c i p r n c a l o f

t h e vnlurne of t u x o i d which when d l l u t c l d i.;

founrl ~o he c~qrliva1r:nt L(> 1 i n k e r n a t i o n a l llni r ,

E l1 $0 Tile d c l h c nC a bacclnc w h i c h p r o L a c t e 50'%.

" f t l~c! in~ir l r~~liec~d o ~ i r n a Is a g a i r ~ s t a c h a l l engc do:+e 1 3 1 v i r u lcn t bactcria nr t o x i n .

S i n ~ l a h a r v e s t . The c o x i c t i l t ra te o b t a i n e d from one batch of cu1tur-S -

i n o c u l a t e d , h a r v e s t e d and processed t o g e t h e r ,

Bulk p u r i f Led t o x o i d , The processed

p u r i f i e d m t e r i a l p r e p a r e d from e i t h e r a s i n e l c h a r v e s t o r a p o o l of a number U

oC s i n g l e h a r v e s t s . It i s the p a r e n t

material from which t h e f i n a l bulk i s p r e p a r e d .

F i n a l b u l k , The f i n a l t o x o i d p r e s e n t i n a e i n g l e c o n t a i n e r from which t h e f i a a l c o n t a i n e r s a r e f i l l e d e i t h e r d i r e c t l y or through one o r more i n t c t - m e d i a t e c o n t a i n e r s .

F i n a l l o t or f i l l i n g l o t . A c o l l e c -

t i o n of s e a l e d final c o n t a i n e r s chat i s h m o g e n ~ w s u i c h r e s p e c t t o t h e risk o f c o n t a m i n a t i o n d u r i n g filling. A f ina l l o t must, t h e r e f o r e , be f i l l e d i n one work ing s e s s i o n .

( p a r t A , S e c t i o n 2 , page 13) Sce Appcndix Q . 2 8

Fnr more d e t a i l s s e c a l s o : b k n u s l f o r the Dcsign , Equippink and S t a f f i n g of F a c i l i t i e s f o r P r o d u c t i o n

and q u a l i t y C o n t r o l b c r e r i a l VaccLnes (BLG/UNUP/IB.I) t L - ,

02 .1 Design of p r e m i . L:;$ ' >\L<;,,, . ; 4 g:- ,> 0

, ,c1 , ' , lke most i m p o r t r i a L requirements f o r t h e premises i n v h i c h

~ $ 5 C A, L

t o r i n i s t o he produced are t h a t t h e y should be clcan, c o m f o r r a b l e . & C . . '

., .v,::. ..L c 5 ~ I : . t o work in, have control of t c m p r r a t u r e and h u m i d i t y , i n c l u d e all >$,F: .;. the essent ia l f a c i l i t i e s , and have h i l t - i n safety m e a s u r e s . ./6 .,A.:' .iiji~;;vI .&.in? t hazards s u c h as s o n t a p i n a t i u n . The liphrlag shou Id be

.$%;?:

BL@CMIY!~I.~ Rev. L page L2

such t h a t a l l equipment can be seen and Opera ted without d i f l i - cu lLy . I t i s a d v i s a b l e tliat Lhe l a b o r a t o r i e s s h o u l d be s u p p l i e d w i t h Elltered a i r or t h a t work s h o u l d he done i n larnit~ar-f IOW c a b i n e i s t h a t are m o n i t o r e d r e g u l a r l y t o e n s u r e t h a t t h e y are o p e r a t i n g correctly. I t s h o u l d be s t ressed , however, Lhat the use of l a m i n o r - f l o 4 cabinets d o e s n o t obvia te the need for a h i g h rlandard af m i c r o b i o l o g i c a l t e c h n i q u e ,

D 2 . 2 Q u a l i t y of m a t e r i a , l s

The main o b j e c t i v e i n t h e cho i ce of building materials Ear a l a b o r a t o r y i s t h a t the sheddlng or c o l l e c t i o n o f dust s h a l l bc avo ided . T h i s a p p l i e d t n all surlacrs i n c l u d i n g t h e f l u o r wh ich s h o u l d hc c o v e r e d with an inpervious epoxy

r e s i n s u r f a c e . Where t h e r e is heavy t r a f f i c qua r ry t i l c s mr Cerraeoo f l o o r s a r e ~u be preferred .

The walls and ceiling s h o u l d be u F a high quality hard p l a s t e r sealed by p.air~Ling wish a washable m a t e r i a l that does no t f l a k e . Lcdges, t h a t i n e v i t d b l y c o l l c c ~ d ~ r s t , rhoold be avuidcd , and benches s h o u l d be i m p e r u i [ ~ u s - w i t h o u t cracks o r crevices. S u i t a b l e c o n s L r ~ c c i o n m a t e r i a l s f u r b c ~ i c h r s include l a m i n a t e d p l a s t i c s , metal and wood covered uict: an eoyxy resin.

They s h o u l d be able t u w i t h s t a n d i requent swabbing and d i s i n f e c - t i ~ n w i r h o v t d e t e r i v r a t i u n . L t must be p o s s i b l ~ t o s p r a y t h e whole arca viLh a disinlectant r i t h o u L damage t o any of the m a t e r i a I s .

Modern praslrices i n t h c p roduc t iu l t of v i r u s vacc inc s mode from a t t e n ~ l a t c d s t r a i ~ ~ s of l i v i n g o r g a n i s n ~ s demtld that, Cos e n t r y i n t o a p r o d u c t i o n a r c a , a l l ou tdoor c l o t h i n g be removed, and t h a t t h r ope ra to r s bc cocooned i n sterile c l o t h i n g . This is bccausti t h e r e arc no p u r i l i c a t i u n o r i n a c t i v a t i o n s t e p s i n v o l v e d i n t h e p r a d u c ~ i o n P ~ O C C F S .

Althvup,h s u c h srringenL precautions a r e I f s s imporlant i n the p r o d u c t i o n of 3 t o x u i d o r b a c t e r i a l v ~ c c i n c i;~ v h l c l : ?he organisms are k i l l e d h y a chcmica l rrr i n w h i c h p u r i f l c a - t i o n i s i r l v c r l ~ c d , i t is r s k e n t l a l t o h a v e some 5arricr

B L G ! ' U N D P ~ ~ ~ . I Rev. 1

page 13

between t h e outside d u s t y con tamina ted atmosphere a n d the produc- tion area. The areas : ;e t a s i d e flrr t h e culture uf organisms,

p r o c e s s i n g , b l e n d i n g end f i l l i n g of Lin.1 v a c c i n e w s t be a p p r o a c h a b l e on ly t h r o u g h an anteroom equipped f o r washing. change nf Lootwear , and rhe donning of p r n t e c ~ i u e clo~hing such a s c l e a n gowns that d o n o t shed dunr o r h i b r e r . Yliere open

o p e r a t l u n a arc car r i ed o ~ ~ t , e i t h e r a cap and mask o r a total

1 head hood s h o u l d be worn. A c c e s s t n t h i s a n t c r o o n must be from other p a r t s n i the p r o d u ~ t i o n area and n o r d i r e c t l y from the F e x t e r i o r .

l A p r o d u c t i o n u n i t and it. s t a l l can be used for the produc- tion of more t han one v a c c i n e Lb a s e q u m t i a l p r o d u c t i o n prnprammc. Tetanus v a c c i n e , however, r e q u i r e s a s e p a r a t '

l D2.4 Premises f o r breeding and keep in^ an imals

I I t i s a c o r n o n l y m i s t a k e n view that a n i m a l s call bc kep t any- where and under any conditions.

I f r e l i a n c e i r LO bc placed

upon rhe r r s u l t r of an imal e x p e r i m e n t s , the a n i m l s must be housed under cond i t i on5 i n which t h e y w i l l r e w i n heal thy and w i l l n o t bo s u b j e c t e d to c r o s s i n f e c ~ i o n .

I ~ h s y r e q u i r e :

(a) clean Living conditions with p l e n t y of I r p h t and goad v c n r l l a t i o n . Ani-ls E r n e r a t e 8 great deal oi

body heat as w e l l a s h u m l d i ~ y and uvercrowding can he

( b ) a good b a l a n c e d d i e t with some v a r i e t y . I t i s i,orLan t t h a t c o n ~ a m i n e ~ i on of £uod and bedding,

which i s u s u n l l y caused by wild r o d e n t s , r h r i l d he prevented;

BLG/'UN~P,! l 7 - 1 Rev page L5

I 1 is imporLant t v conLrol t h e b a c t e r i a l , f u n e a l and h e l - rnirlLh p o p u l a t i o n i n t he br rcd i t jg s t u c k .

Fatly f ~ l t e r c u r r e n t i n f r l c t i o n s a r e a b l c t o a f f e c t Lhe r e s t l l t s o i animal e x p e r i m e n t s . Such 1 n C e c t i o n s should bc k e p t t o a m i n l m u m by segregation of a n i m a l s from d i f f e r e n t s o u r c e s , s e l e c t i v e b r e e d i n g from t lea l thy s t o c k and as a Last r e s o r t by s u i t a b l e t r e a t m e n t with t h e r a p e u t i c a g e n t s .

' Ihe q u a l i t y can t n l of d i p : l t h c r i o toxoid r e q u i r e s a Large € s u p p l y of g u i n e a - p i g s , and s u c h c o n t r o l s s h o u l d n o t be arLvmpted

11n1e.s good a n i m a l s arc a v a i l a b l e e i t h e r from o n e ' s own f a c i l i i y o r f rom a r e g u l a r s u p p l i e r .

Such an an imal facility s h o u l d bn c n p a h l c C I E l luusing and i C n r c e s s n r y b r e e d i n g t h e sn in la l s u s e d f o r a number 01 v a c c i n c s s u c h as d i p h ~ h c r i a , p e r t u s s i s and t e t a n u s , a n d may be uscd a l s o f o r t h e t e s L i n g of c h o l e r a and t y p h o i d v a c c i n e s .

For thc t e s t i n g of d i p h L h e r i a t o x o i d , however, o n l y g u i n e a - p i g s a r e needed i n a p p r e c i a b l e n u m b e r s .

D i p h t h e r i a t o x i n p r o d u c t i o n can bc c a r r i e d i ,u t e i t i ~ c r i n s t a t i c bottle c u l t u r c o r i n i e r m e n t c r s .

( l n some l abora to r i e s a n i n t c r m e c i i a t e t y p e of p r o d u t 1 i : ~ n i s done i n b o t r l e s on shak.ir>g r a c k s , )

D . 1 F'roduction methuds

(a1 'Fhc a d v a n t a g e s of t h e b o t t l e method i n c l u d c s i m p l i c i t y and t h e low u u t l a y i n equ ipment .

1. The ( T A W Ilanclbook LHL t t ~ La).<: a n d h ~ r a ~ c m t . n t ; I ( AI, ima l r

: E d . C h u r c l ~ i l l L i v i ~ ~ ~ ! . c ~ b n t m , E d i r ~ h u t . ~ h , l ,c~nrlon, Knc: York, ? c h

P i l l L i f ln . l Q ? b j i s a ~ i i c ) - t L I ~ ~ L I ~ r s l c l ~ , l i , c f u r 1 1 i3!;,,c,ct:. i u v o l v c d iir ( h c carc crf ; i?l imals .

! About W O Lnoculaked Reus boLkler l i n c u b a t e d f o r about seven days a rc needed tu produce 100 litres of t o x i n w i c h 50-100 I f u n i t s p e t

ml.

l. F i l t r a t i o n c a n be e f f e c c e d through paper i n g l a s s funne l s and t h e o n l y o t h e r a p p a r a t u s r e q u i r e d is a n automatic i n o c u l a t i o n s y r i n g e .

(b) F e r m e n t e r p r o d u c t i o n o f f e r s c o n s i d e r a b l e economies i n medium, t i m e and space. A 300-

l i t re f e r m e n t e r w i l l y i e l d 2 5 0 l i t r e s oL t o x i n a t 150-300 L£ u n i t s p e r m l a f t e r 48 h o u r s ' i n c u - b a t i o n w i t h a great ly dirinlshed number of

s s c p c i c m n i p u l a t i o n s . A C e m e n t e t for d i p h t h e r i a p roducc ion does not h a v e t o be cumplex. b u t i t d o e s need t o be fabrlca~ed t o the h i g h e s t engineering s t a n d a r d s from t h e b e s t quaLiLy m a t e r i a l s . O u t Line s p e c i f i c a t i o n for such a

f e r m e n t e r , t h e c o s t of which need n o t exceed US$ 1 5 000, i s included i n Appendix D.2. F i l t r a t i o n of f e r m e n t e r c u l t u r e s r a i s e s g r e e t e r problems than are e n c o u n t e r e d wi th s t a t i c c u l t u r e s owing to the g r e a t e r d e n s i t y and d i s p e r s a l of t h e bacterial s u s p e n s l u n .

3 . 2 s e p a r a t i o n of t h e t o x i c f i l t r a t e

Use of a diatm#ce'ous e a r t h f i l l e r a i d i n a sysiem of ch 6 s a s e t a f i l i e r ( s e e Appendix D . 3 ) ha5

ve for s e p a r a t i n g t h e Loxic £Lltratcs ft- t h e b a c t e r i a l c e l l s . Anorher simple a p p a r a t u s Cor the

e m e n c e r h a r v e s t i s described in

3:3 P u r i f i c a t i o n of the a n t i g e n

P u r i f i c a t i o n i n v o l v e s f i l t r a t i o n nf t h e t o x o i d akter c i p i t a c l o n w i t h s m o o i ~ t r c s u l f a ~ e . T h i s can bp e f f c c ~ e d

n u s i n g s Rfic.hnr,r Cuntltl o r , ~ l ~ e r n a ~ i v ~ ~ l ~ u r i f l e d and l o t c r d e ~ d - l x i f i c d .

F

D3.4 Hlendinp of bulk

l l l end inp n f f i n a l b u l k vaccine s h u o l d he done i n r r i n g l e large conLYincr s i n c c i f b l e n d s are prepared i n a nuntbcr o f

b o t t l e s hey need t o be retcsLed t n r steri liry, s a f s ~ y and s u c l ~

a d l l i t l ~ ~ n a l l r 5 L s as may l>e nocdcd. A a u i r a h l e v c s s r l vljivh t a n be used b o r h a r a fcrme~~rer and blcndt-r i+ del icribed i n

Appendix 0.2. F u r a l i r i nf n e L e r r a r y c q u i ~ ~ m c ~ ~ t and r u g g e l t e l l sources of p r o c ~ ~ r i n y , i t , see A p p e n d i x D.23.

D4. STAFF

D4.1 Q u a l i f i c a t i o n s and numbers

The number o f s t a f f i n v o l v e d i n t h c p r o d u c t i o n of d i p h t h e r i a t n x o i d depends t o some e x t e n t upon t h e volume o f t o x o i d t o he produced and t h e number of b a t c h e s r e q u i r e d .

I t i s n o t a d v i s a b l e t o cmpLoy s t a L I w h o have no knowledge of m i c r u h i o l o g y b u t a w e l l - t r a i n e d nucleus of s t a f f can trafn o t h e r g t a f f q u i t e quickly. The ? r u d u c t i o n of thc m e d i a w o u l d r e q u i r e one y r a d u n t c s c i e n t i s t and une t e c h n i c i a n .

Main tenance o f s eed c u l t u r e , yrrpararion I I ~ i n o c u l ~ ~ m , g r o v t h of organisms. s e p a r a t i o r ~ , p u r i f i c a t i u ~ l and d e L u x i f l ( a L i o n O I t o x i n , can be c a r r i e d out by s gracluaie m i c r u b i n l o p i r c w i t h somc y e a r s ' c x p t . r i e n c c and t h r e c t c c h n i ~ i o n s .

Q u a l i t y c o n t r o l of Lhe 1)111k t o x v i d a n d ttle f i n a l p r o d u c t w i l l r c q u i r r a l s o g r a d u r i e r r i c n t i s t and one t r c l ~ ~ l i r i r n .

The animal f a c i l i t y s h o u l d ile s u p a r v i s e d by e s e n i u r t e c h n i c i a ~ l w i t h adeyuarc e x p e r i e n c e and t r a i n i n g ir, i h e nainLc[l- ar lcr of s m a l l a n i m a l s .

f'rovi8:ion s h o u l d bc- made Lor p r u - fr~iional v e t e r i n a r y c o n s u l t a l i o n wllcn r e q u i r e d .

HI.(,; Llh'!JF.' 77.1 Rev. 1 page 17

n,.: t c c h n i r r l s i n i f a h w l d liave i,ad a n , i d u c : < ~ i n n beyvi,d l rc l lou l i ~ l g .

1" l u r t l ~ r r e d u r a t i o r ~ t h e y s h < ~ u i d )l.ivc had

an oppor~unity of ~ t u d y i r l ~ r n i ~ ~ u h i o l ~ g y , and i i a v r a ork kin^ knOWledf , e $,l sterili t e c h n i q u e s and L l t e h a n d l i n h i > l e t u r i a .

Tt~e should have spent ar l e a s t i l l r ee t o s i x

mnntiir i n a p r o d u c t i o n u n i t a c t i v e l y c n p g e d i n L I I C l m n u f a c l u r e and c o n t r o l o f b a c t e r i a l v a c c i n c s .

A 1 1 s ta f f s h o u l d be i n good h e a l t h , and should be i m u n i r e d a g a i c s t 01 k n a n t o have n a t u r a l mumunity against a n y infectious a r g e n l s m i t h e t t h e y m y b e r e q u i r e d t o h a n d l e . They i h ~ u i d be immunized a g n i n r t t u b e r c u l o s i s , and n o t be suffering f rom any o t h e r r e s p i r a t o r y o r drarrhoeal d i s e a r e . Any staff members h a v i n g a s e p t i c wound should n o t e n t e r t h e

;;:, . :I

fie r e c h n i c a l s t a f f l o o k i n g a f t e r t h e b r e e d i n x co lony ? 'V . .. .:; !.

exper imenta l . n i m l a s h o u l d a l s o b e i n povd h e a l t l l and f r e e [ram

: any l a t e n t i n f e c t i o n .

D4.4 Organi E a t i o n of a c t i v i t y

q u a l i t y c o n ~ r n l and p r ~ d u ~ t i ~ n s h o u l d be a d r n i l l i s t r a t i v e l ~ : SepareLe b u t i~ i s i n p u r t a n ~ that q u a l i t y control i t .€ [ h a w

f i r s t -hsnd knavledge and e n p o r i e n r e U € p r o d u c t i o n Thero is

eed for a n i m l t r s r i n g Lo he d u p l i r d t o d , but L t i s imilor tanl the d e t a i l e d r r s u l L s oi a l l " i n process" a r d Eiilal t e s t 5

@ b ~ u i l l be auai l a b i e tu b o t h ~ r o d u c t i n n and q u a l i ry c u ~ ~ t r n l

BLG/'UMIP/ 77. 1 R P V . 1

page 18

Diphtheria t o x i n can be o b t a i n e d in consirtcntly good yie from m e d i a based [)n papain digests o f

b e e f . Deta i l s of the Eurmulat ion of a medium s u i c a h l e T u r s t a t i c c u l t u r e v f C , d iphther iae and an enriched medium capable u f s u p p o r t i n ~ m x l - mum yields of toxirk i n I c r m e n t f r cu l Lure a r e descri hcd i n Appendix 0.1. Wny lnhorarorirs grow C. d i p h t i ~ e r i a e in a s e m i s y n t h e t i c m e d i u m w i t h a bas is o f c a s e i n h y d r o l y s a t c .

F o r the f o r m u l a t i o n of such a medium, s e e the original literature as mentioned in Appendix D, l

I t i 6 important that Lhe meat used For the yrnduction o f t h e media s1,ould be 01 good q u a l i t y i l r . r l used w i t h i n e week oi s l a u g h t e r ; when r r o r e d i t s h o u l d he k e p t under proper co ld storage conditions. Batches o f papain differ c o n s i d e r a b l y i n e i f e c t i v e n e s s , and i t i s , t h e r e f o r e , advisable t o prepare a sample b a t c h D £ medium from a new baLch oi papain and dete.rnine

toxin yield. l € the t e s t b a t c h oI m e d l ~ ~ r n proves satisfactory, the papato should be t i l l e d i n t o s e a l c d conta iners and stored at 4 3 - 3 C u n t i l needed.

Tnc s tra ins which are in g e n e r a l use are ruhstraios ol 1.;- C . d i p h t h e r i c PW'-strain ( e , g s u b s t r a i n C N Z O O ~ ~ . such a ~ L r a i n should be obta ined £ion1 a l a b o r a t n r y experienced i n the

f production of d i p h t h c r ~ a LoxFn. 1

..:.rl :; ..A< .,g, Db.? Maintenance of the atrain

s t r a i n should be maintaii~ed i n the lyophilized s t a t e *

Meintr~rance r r l t h e strain for long p e r ~ o d s in culture is i n a d v i s a b l e . A new LyuphiLircd culture stlou1d be used LOT

each batch of t o x i n . F o r t h e lyaphiiira~lon i t is preferable

for the s t r a i r l to be grown on Locf f l er -agar , c o n t a i n i n g the medium ior rke f c r m m t r r cu ltures in s t ead of rhr u s u a l

beef Irorh c o n t a i n i n g on ly O . j % glucose of pH 7.6.

:3 ~ r c p a r a t i o ~ ~ for produc t iun

~h~ frccee-dried strain is inoculated on Loeffler-agar ates t ~ . y b glucu! ;g , 0.57. ~ ~ u e l l e r ' s g r o u ~ h f a c t o r s@1ution*

7.8, made w i t h n o r m a l beef b r o t h ) slopes contained in

~ ~ r . ~ - ~ ~ ~ ~ ~ d i l u k r i c r and fro. which the :;eed culture^ i n f lasks

l~ources 0s 5 u i t a b l e b s t r a + n 3 c n be obtained froni the Secretariat [Chief, K ~ o l o g l c J 1 3 7 .

BLG;UNDPj77.1 K c v . 1

page 20

a r e i n o c u l a t e d a f t e r 24-36 h o u r s ' growlb. A scrercapped b o t t l e s h o u l d 11avc t h e c a p left I O L I S C L U a i d a e r a t i o n .

6 . Thc s e e d c u l t u r u s uacil f u r static f l a s k y r u d u r t i o n a r e :ram i. Er lenmeyer f l a s k s which c o n t a i n t h e s m c mqdlum a s w l l l

v a n L u a l l y bc used i n t h e Rvux tlasks. The E r J c m e y e r flasks

.~ie placed i n t h e L n c u b a t o r a f r e r i n o c u l a t i o n from the L o c f f l e r p l a t e r o r s lopes and a r e used as soan ar growtll is suf f i ~ i r n t . ' e f o r r U S L L I ~ t h r s e e d c u l t u r e s f o r i n o c u l a t i o n

of t h e f i n a l tuedium they a r e checked r n i c r u s c o p i c ~ l l y (Cram a n Jor h - e i s s e r s t a i n ) f u r con tamina t ic ln and normal a p p e a r a n c e .

D . The s e e d c u l t u r e s f o r f e r m e n t e r p r o d u c t i o n are p r e p a r e d i n 500 m1 Er lenmeyer f l a s k s c o n t a i n i n g 2 0 0 m 1 of t h e medium w i t h 0.5 m1 M u e l l e r ' s growth f a c t o r s o l u t i o n {see Appendix Dl) and w i t h t h e a a d i t i o n of 0.5 m1 o f an a n t i f o a m i n g agent ( S i l i c o n e )(E a n t i f o a m "A" Hopkins and Willians, d r e d w i t h t h r e e p a r t s of l i q u i d p a r a f f i n i n a S e r v a l 1 high-speed omni- m i x e r ) . The f l a r k s a r e i n o c u l a t e d w i t h c o l o n i e s s e l e c t e d from t h e p l a t e s , which h a v e been grown f o r 24 o r 3L hours .

h e Er lenmeyer f l a s k s a r e c l o s e d w i t h cot ton wool plugs and p l a c e d i n a r o t a r y s h a k e r and r o t a t e d a t 1 6 0 rpm.

The shaking i s c o n t i n u e d f o r a b o u t 24 hours i n an i n c ~ b a t o r a t 35'~. T h i s e n s u r e s t h e p r o d u c t i o n of a n inocu lum o f o rgan isms i n t he

log . r i thn ic p h a s e o f g r o w t h ready fox i n v c u l n t i o n For p rnduc- t i o n i n a f e r m e n t e r . Abundant g rowth w i t h h i g h LE v a l u e s i n t h e r e e d c u l t u r e s d o e s not n e c e s s a r i l y g i v e t h e h e s t r r r u l t s i n t h e tank and m i c r o s c o p i c a p p e a r a n c c of t h e b a c t e r i a

i n a Cram . r a i n o f t e n gLves irnporLant i t l l o r m t i o n . 'fie s h a p e , s t a i n i n g p r o p e r t i e s a n d morphology of t h e b a c t e r i a a l s o g i v e u s e f u l i n f u r r m t i u n . The hest r c s u l t s have been o b t a i n e d w i t h c u l t u r e s s h o u i h ~ g m a l l , more or l e s s dl~mh-bel l -shaped b a c t e r i a . The s e l e c t i o n uf r r p d c u l L u r e r r e q u i r e s p r a c t i c a l experience a n d i t i s " d v l s a b i e t h a r i t s h o u l d a lways be done by a h i g h l y c x p o r i e n c r d s t a f f member who is in ~ h n r g e a£ t h c p roduc t ion .

BLG/IJKDF/ 7 7 . 1 R e v . 1

page 21

D 7 . 1 Growth O E C , d i p h t h a r i a e

The per iod o f i n c u b a t i o n deperlds on the method n f prnduitiun

cl losen and f o r e a c h method of c u l t i v a t i o n t h e o p t i m a l growth p e r i o d must be d e t e r m i n e d p x p e r ~ m e n t a l i ] . I t is a d v i s a b l e t o

I ~ a r v e s ~ 3~ a t i m e when Lhe p u r i t y ( ~ [ / m ~ PN) i s at its m a x i m . The h i z h e s t p u r i t y is a L t a i n e d b e f o r e t h e h i g h e s t t o x i n cuncen- - - -

t r a t i u n h a s been r e a c h e d .

D7. l l i ) In s t a t i c c u l t u r e u s i n g ibe medium d e s c r i b e d i n Appendix 0.1 a maximum p u r i t y o f a b o u t 800-LW0 ~ f / r n % PN i s l i k e l y t o be

D

r e a c h e d a f t e r s i x o r seven d a y s of i n c u b a t i o n a t 35-16 C .

DI.L(ii) I n f e r m e n t q r r g rowth i s much f a s t e r and the maximum p u r i t y w i l l u s u a l l y be r e a c h e d w i t h i n 48 h o u r s . S t a i n l e s s -

s t e e l f e r m e n t e r r c a n be of a n y size from 2 0 l i t r e s upwards, need not be highly i n s t r u m e n t e d s i n c e t h e growth of d i p h t h e r i a r e q u i r e s o n l y c o ~ t r o l on i m p e l l e r - s p e e d ) a i r - s u p p l y and tempera- t u r e , and may be p u r c h a s e d from commercial m a n u f a c t u r e r s o r made t o o r d e r by a good f a b r i c a t o r of o t a i n l e s a - s t e e l . Al though t h e

i n i t i a l c a p i t a l i n v e s t m e n t m a y seem h i g h t h e y have an a s su red work ing l i f e o l a t l e a s t 10 y e a r s and a r e l i k e l y Lo p r e s e n t t h e minimum i n d e w n d r un servicing when carefully d e s i g n e d . The

medium Ls S e i t z f i l t e r e d d i r e c t l y i n t o t h e t a n k which h a s keen & v t n c l a v e d , and h n t h the medium and t a n k are h e a t e d a t 100 C f o r h a l f m hour c i t h e r i n a Yoch steamer n r i t 1 an r u t o c l a v c . Shc f e r n e n t e r is cooled r a p i d l y by means of a J r L of cold w a t e r o r b y co ld vater I l o w i n X tbrou€,h t h e j a c k e t t u rrducr t h e 0

t c m p c r a t u r e t o l e r r ~ h a n 35 C . This t a k e s about 'U m i n u t e s ; <>

t h e v e s s e l i s then h e l d overn l s ,h r in a n i n c u b a t o r 3 L 35 C. 'The n e x t m o r n i n s L t ~ e t a n k i s rernoucd Irom rhp incu t ra to r and 0.7 I of ~ n t i f o a m - ~ / l i ~ u i d p a r e t fin m i x t u r e (23% Ant i r u m - A) i s added b e f o r e t h e whole suntent of une r e e d c u l t u r e flask i s i n u c u l s t e d i n t o the mcdium. Tempera tu re r e g u l a t i u n and v u r t e x -

s t i r r ing a r e ~ a r t e d and d u r i n g t h e g tuwth p e r i u d t h e a i r - f l u r

BLG/UNDP/~I. 1 Rev. I

page 22

BLG/UNDP/ 7 7 . l Rev. 1

page 2 3

~ ? . 2 ( i j S t a t i c c u l t u r e s frtw RCJUX b o t t l e s can be f i l t c ~ r d t h r o u g h f i l t e r p a p e r [ f l u t c d f i l t e r s ) i n g l a s s l u n n e l s .

B o t t l e s which show a t u r b i d i t y i n d i c a t i v e of coz:~arninaLicn fihould be d i s c a r d e d ( C . d i p h t h e r i a e g r a s as a p e l l i c l e ) .

The f i r s t f l u i d p a s s i n g t h r o u g h the p a p e r may n u t be q u i t e c l e a r and s h o u l d be r e t u r n e d i n t o t h e f i l t e r . T h i s p r o c e d u r e s h o u l d be r e p e a t e d u n t i l t h e f l u i d p a s s i n g t h r o u g h i s q u i t e c l e n r .

l

1 O f is controlled a t r e g u l a r i n t e r v a l s And ! The nLAximum p u r i t y t10(30-2000 t.f/mg PNI i s reached abo,lt

~?.Z(ii) The d e n s e b a c t e r i a l s u s p e n s i o n i n t h e f e r n e n t e r c a n be

k i l l e d by t h e a d d i t i o n of 0 . 5 x t o l u e n e . The c o n t e n t s a r e t h e n s t i r r e d for 15-30 m i n u t e s and h e l d a t 3 5 O ~ f o r a t l e a s t one hour by which time t h e c u l t u r e is u s u a l l y dead .

E x p e r i e n c e s h o u l d be b u i l t up i n t h e t i m e t h i s t a k e s .

more of saline t h r o u g h the t i l t e r ca*e(s)+ y i e l d i n g a

furL her o f 11 a s t e r i l e f i l t r a t i o n 0~ toxin

F i l t r a t i o n of t h e dense b a c t e r i a l s u s p e n s i o n f rom f e r m e n t e r c u l t u r e s i s more d i f f i c u l t t o ach ieve .

The f i r s t phase of t h i s p r o c e s s i s t h e removal of t h e d e n s e b a c t e r l a l msr and o b t a i n i n g a e le s r , b u t ~zut n c c e s s a r i ly S , l t r a t T h i s can be a c h i e v e d i n v a r i o u s a y p m p r i a t e pieces o f eqtiiprncnl, s u c h a s : (a) o r d i n a r y m u l t i s h e e t filters ($ell; , C r r l d o n F u r d , e t c . ( s e e Appendix D . 2 3 , l t e m l&) ) , w i r h r e l a t i v e l y hick h o l l o u frames to a c c o m o d a t e l a r g e quantities

oL b a c t e r i a l mass mixcd wilh f i l t e r a i d ; (b) MetafilLer ( s e e Appendix 0.23, iLem 16) ; o f [ c ) "hune-n~adc" f i l t r a ~ i o n vesse l

u i r h fi1Le.r c a n d l e s , as dascri l led i n A p p e n d i x D . 4 . Tn a l l cases t h e b a c t e r i a l s ludge i s t h o r o u g h l y mixed r L t h F-3% uE

f i l t e r a i d ( l i y f l a Super C e l l ) . Afcrr f i l l r a t i o n , 2 0 l i t r e s n r

j

I, r h i s wasfound e x p e r i m e n t a l l y i o r a 7 5 - l i t r e t a n k

c o n t a i n i n g S5 l i t r e s of medium.

36-48 hours r i t h a t o x i n ltivc-l 150-300 and rhc msxirnvnl

i The second phase i :; the f i l t r a ~ i o n i n t e n d e d to s t e r i l i z e h i t r r e and is per formed i n t h e u s u a l wnnsr rhrough prvyvrl l rtcrliized f i l r e r s .

Hvuevcr, t h i s phase o t 5 t er i l e

f i l t r a t i o n i s n o t a b s o l u t e l y n e c e s s a r y i f t h e f i r s t p h a s e i s

l ' pe r fo rmed under s t e r i l e i o n d i ~ i o n r I n t h i s case i t is enough

CO add l : l0 000 o ~ e r t h l o l a L e t o t h e c l e a r t o x i n f i l t r a t e , s h a k e i t w i t h enough t o l u e n e s u c h t h a t a t h i n l a y e r o f t o 0 1 0 uene forms 1.. . ' on t o p , and t h e t o x i n c a n b e k e p t many weeks a t 5 C-3 C for

l a ~ t ~ P ~ r i f i c a t i o n " w i t h o u t d e t e r i o r a t i o n .

cnr,~,es, =hen t h e r e W a r h i n g ~ err passed t h r o u g h t h e s t e r i l e

i ,i D 7 . 3 S t o r a g e o f t o x i n

l , . . L If crude toxin i s kept s t a n d i n g Eur some months t h e r e i s

4 a s l i g h t d e c r e a s e i n Lf v a l u e , a n d a l a r g e d e c r e a s e i n p r o l e i n - ..,- ,

2 N . r e r u l t i n g i n a form of " a u ~ o p u r i € i c a t i o n " . I t may be u s e i u l ,

. .... C +:S;

t h e r e f o ~ e , to keep t l te toxin at l e a s t f o u r weeks i n s to rage

.. , ,- F . b e f o r e f u r t h e r processing. I f t h e t o x i n i s n o t s t c r i l e - f i l t e r e d , t h e n i t r a n b e p r o t e c t e d a g a i n s t b a c t e r i a l ~ r o c t h

. - . during t h i s period by i i w r t h i o l a t e and t o l u e n e , a s d e s c r i b e d ;:S.. ;

.:<:c': above. T h i s storage procedure a l s o makes i t p o s n i h l e t o C.'. ' > S ..:.(;cu, combine a ,>umber o f Latches i n t o one l a r e e hatr;h llefdre

Lr'xin C o n c e n t r a t i o n i s r e a c h e d a f t c r hours , as a ,.Wishing LoXic b e f o r e t h e m a i n b u l k oi f u r i n ,

f u r t h e r prucessing .

! ! U7.2 H a r v e s t i ~ r ~ r,f the toxin l ,

A t t h i s stage i t i s p o s s i h l c t u T o l l o ~ ' e r e n ~ procrrlurc:; L u r t h e d c t ~ > x i L icnr . ion

Live rhc f i l t s r arc disrard~d. n ~ i s i)revrl'tr

l o s s e s by a d w c p c i o n 01 t o x i n oil t h e f i l t e r pads .

A. To puri fy the t.clxin a n d t h e r c a r t c r t h e p u r i f i e d t o x i n , o r

R. To d e t o x i f y the crude Loxin and t h p u r i f y t h e t o x o i d .

B L ~ U N D P / ~ ~ . I Rev. 1

P a w 24

The advautages of A a r e t h a t a h i k h c r p l ~ r i t y r,f toxoid LS a c h i c v e d b u t t h e r e i s a r e a l r i s k hat r e v e r s a l t u t o x i n may occbr. There are a d e q u a t e chccks t o t e ~ t f o r t h i s I ~ c c Appendi X n . 9).

The advantage o l 1; is t h a t r e v e r s a l i s l e s s l i k e l y but crrt, n o t i m p o s s i b i e . T h e p r o d u c t i s no t q u i t e s o pure anJ t h e r e i s a p o s s i b i l i t y t h a t t h e r e may b e more f r e q u r n ~ a d v e r s e r e a c t i o n s whr t h i s p r o d u c t 1 s i n j e c t e d but t h e s e a r e n o t s e r i o u s .

Method A - p u r i f i c a t i o n of t u x i q -L

To thc f i l t r a c e add 5 g/l of sodium b i c a r b o n a t e , s u Z f i c i e n t anrmonium sulfate t o g i v e a f i n a l c u n c e t ~ t r a c i o n of 23;: ~ n d 0.257,

c h a r c o a l (Darco G-60 or o t h c r s u i t a b l e q u a l i ~ ~ ) . A sample s h o u l d hc removed aL t h i s s t a g e , c e n t r i f u g e d , and t h e LE c o n t e n t

oi t h e s u p e r n a t a n t measured. I t s Lf c o n t e n t s h o u l d be about

70% of t h a t of t h e p a r e n t f i l t r a t e ; t h i s i s a check on t h e

q u a l i t y of t h e c h a r c c a l . I f 70% i s r o u t i n e l y a c h i e v e d t h e t e s t may be d i s c o n t i n u e d on s u b s e q u e n t l o t s .

I f a r e c o v e r y of g r e a t e r or l e s s than 70"A i s a c h i e v e d t h e o p t i m a l c h a r c o a l l e v e l s h o u l d be d e t e r m i n e d by t i t r a t i n g t h c c o n c e n t r a r i on of c h a r c o a l

a g a i n s t t h e p e r c e n t a g e r e c c v e r y as measured by rhe I . £ : wherc recovery exceeds 7 0 7 the c h o r c ~ i n l l a v c l s l > o u l d be i n c r e a s e d ,

w h e w i t i s less than 7f l% i t s h o u l d he d e c r e a s e d . Each new l o t of c h a r c o a l s h o u l d bc s o t i t t a t c d .

The m i x t u r e i s s t i r r e d f o r 30-60 minutes and f i l t e r e d e i t h e r t h r o u g l ~ a BUchner f u n n e l , t h e pads of wh ich

are p r e c o a t e d w i t h d ia tumaceous e a r t h , o r th rough a p r e p a r e d meta- f i l t e r . L t t h e m e t a f i l t e r i s u s e d , i t i s advantageous t o add 3-h?. of diaLomaceous e a r t h t o t h e s u s p e n s i o n to he f i l t e r e d . When f i l t r a t i o n i s comple te the f i l t e r s a r e washed v i t h a 23% s o l u t i u n of a tnmonlum sul f a t e t o r e c o v e r r e s i d u a l Toxin. The volume r e q u i r e d fo r t h i s r a n g c s from about 2 l i t r e s for a HUchncr f u n n e l t o 1 5 l i t r e s f o r a m e L a f i l i c r .

BL(:/UWP~ 7 7 . 1 Kev . I

page 2 5

To L ~ I C lilt rat^!, min b u l k combined w i t h wash ing \ , 1 5 added a f u r t h e r 23% uf ammonium s u l i a t e a n d the miatur r : i s l e r t i n the c o l d o v e r n i g h t . Becore f i l ~ r a t i o n 1-22 of d i a t m c e o ~ v e a r t h

i s added t o the f i l t r a t e , which now c o n t a i n s p r e c i p i t a t e d t o x i n . The s u s p e n s i o n i s t h e n f i l r e r e d th rough BUchner f u n n e l s , f i t t e d w i t h f i l t e r p a p e r s p r e c o a t e d w i t h d ia tomaceous e a r t h . The t o x i n

is p r e c i p i t a t e d on t h e pape r s , sucked d r y on t h e vacuum l i n e , washed i n s i t u w i t h a 44% s o l u t i o n of amnonium s u l f a t e and a g a i n d r i e d . The c a k e s are c a r e f u l l y s c r a p e d uLf t h e f i l t e r payer i n ~ u a s u i t a b l e c o n t a i n e r . Thc f i l t e r p a p e r s a r e washed

r e p l a t e l y w i t h s a l i n e ur 0.1% s d i u b i c a r h o n a r e s o l u t i o n . Lhe wrshinlr bulked w i t 1 1 t h e - in r e s i d u e and the t o x i n dissolved with gmrle stirring. The d i a ~ o ~ c e o u r ear lh which remains i n

s u r p c n s i o n 13 remuved by f i l t r a t i o n a n d the n m o n i u n s u l l a t r by e x h a u s t i v e d i a l y s i s , A t t h i s point the p u r i t y and Lf corjtent

I i f n o t a l r e a d y d e t e r m ~ n e d to asbess r e c o v e r y ) a r e measured and r e c o r d e d .

T o x o i d i n g o f p u r i f i e d t o x i n

The t o x i n i s d i l u t e d t o 500 ~ ~ / m l , a d j u s t e d t o pH 7 . 6 5 N sodium hydroxide and f o r m a l i n a n d sodium b i c a r b o n a t e

added, each t o e f i n a l E u n c e n r r a t i u n of 0.5% and r e a d j u s t e d H 7 . 6 i f necessary. o The solut.lun i s s t e r i l i z e d by f i l t r a -

, h e l d a t 20-25 Lor t h r e e LO f o u r rrceks, a sample

removed, dialysed and ~ e s t c d fo r frecdmn from s p e c i f i c ~oxiciry he skin of g u i n e a - p i a s ( s e e A~rrendix D . 1 2 ) +

I f there is

ac t ion indicating t h e prcscnce of d i p h i h c r i a t o x i n , the ~'

b o t t l e s a r e h e l d for a f u r t h e r 7-14 d a y s a t t h e same tempera- and r e t e s t e d . I f t ~ x o i d i n g i s i n c o m p l e t e a t Lhis p o i n t

rther 0.1% of formalin s h o u l d be added t o t h e buolk and i n g r e p e a t e d a f t e r a f u r t h e r two weeks a t 20-25 C.

d e t o x i f i c a t i o n i s complete the bulk i s h e l d a t 35 C

i f i c a l i o n of c o x i z

Toxins irom b o t t l e c u l t u r e s a s d e s c r i b e d are u s u a l l y by i n c u b a t i n g t h e m f o r i o ~ l r t o ! ; iX weeks a t

f l r rmal in C c o c ~ a i n i n g 40% formaldehyde) ; t h i s

B L C / W L ' / ~ ~ . ~ R e v , 1

page 215

has t o be checked ( s e e Apprndix U.17). I f t h e concenCraLlon i s lower, more has t o be added. A f t e r d c t o x i f i c a t i o n c o n t t l m i n a t i o n c a n be p r e v e n t e d by rbe a d d i t i o n o f P.014? t h i o m e r s a l t o t h e

0 0 t o x o i d and s t o r a g e i n t h e c o l d (5 C - 3 C).

The d e t o x i f i c a t i o n of t o x i n from f e r m e n t e r s needs a d d i t i o n a l p r e c a u t i o n s . U s u a l l y 0.6' of f o r m a l i n (40% formaldehyde s o l u - t i o n ) i s s u f f i c i e n t ; t h i s q u a n t i t y i s added s t e p - w i s e . F i r s t t h e t o l u e n e i s f i l t e r e d o f f by p a s s i n g Llie t o x i n th rough wet f i l t e r paper and 0.1X C ~ r m s l i n i s addcd v e r y c a r e f u l l y under cont lnuocls s h a k i n g t o e n s u r e comple te mixing.

The same proce- d u r e i s fo l lowed f o r the rtext f i v c d a y s w i t h the a d j u s t m e n t of pll on day 3 t o pH 7.3 and on days 6 and 7 t o pH 7.1.

The b o t t l e s a r e p l a c e d i n an i ~ ~ c u b a t o r f a r s i x w e e k s ( b o t t l e s a r e c l o s e d w i t h c o t t o n v001 p l u g s ) , a f t e r which t ime 0.131% t h omersal

0 t o Is added and t h e t o x o i d p l a c e d i n a r e f r i g e r a t o r a t +5 C - 3 C.

S o - c a l l e d " r e v e r s a l oE t o x i c i t y " has n o t been observed by f o l l o w i n g t h i s procedure.

P u r i f i c a t i o n of t o x o i d

Many p u r i f i c a t i o n p r o c e d u r e s have been d e s c r i b e d . F r a c - t i o n a l s a l t i n g - o u t w i t h ammonium s u l f a t e i s a s i m p l e and s a t i s - f a c t o r y p r o c e d u r e . It i s advisable t o c r m c e n t r a r e the t o x o i d f i r s t i n o r d e r t o remnvn thc hblk of nurr-protein r t i t roge l l (gee Appendix ~ ~ 1 4 1 , and reduce t h e volume c o be h a n d l e d , e . g . by same u l L r a f i l t r a t i o n procedure (see Appetidix D . S ) , L f no u l t r a f i l t r a t i o n a p p a r a t u s i s a v a i l a b l e a n i n i c i a l c o n c e n t r a t i o n can a l s o be performed by a t o t a l s a l t i n g - o u t with amnoniurn s u l f a t e , fo l lowed by c e n t r i f u g a t i o n and d i a l y s i s .

A f t e r t h e c o n c e n t r a t i o n procedure t h e s a l t i n g - o u t s h o u l d be per formed, p r e f e r a b l y a t a t o x o i d c o n c e n t r a t i o n o f 750- 1500 ~ f / r n l . A s t o x o i d s d i f f e r f rom baLoh t o ha tch i t i s c r i t i c a l tn determine Lhc vpt imal a~mirnnlu~n s u l f a t e c o n c e n t r a - t i o n s ~ l e c e ~ s a r y f o r each b a t c h vf t o x o i d hy c o n s t r u c ~ - i n g a s a l ~ i n g - o u t c u r v e a s shown

i l l Appendix D . 6 ,

BI.G/UNIIY/~~.~ Rev. 1

page 27

D7.5 " l n p rocess" c o n t t u l

I C a r e f u l m o n i t o r i n g o f samples from s e e d and p r o d u c t i o n c u l t u r e s t h r o u g h o u t growth i s FmportartL i n

o r d e r t o e n s u r e t h a t

c o n t a m i n a t e d o r a b e r r a n t c u l t u r e s a r e re-cognized a s s o o n a s p o s s i b l e . T e s t s f o r b a c t e r i a l p u r i t y , o p a c i t y , pH a n d t o x i n

I p r o d u c t i o n s h o u l d be i n c l u d e d . Where a number o f t e s t s a r e

i a p p l i e d a t a g i v e n p o i n t i n t h e p r o d u c t i o n a n d c o n t r o l of t h e v a c c i n e i t is advisable t o c a r r y o u t t h e t e s t s i n such an o r d e r that the ones l cas t demanding of r e s o u r c e s and g i v i n g t h e quickesL results be p ~ r f o r m c d f i r s t and shown t o bc s a t i s i a c t o r y b e f u r r t h e more d e w n d i n g and time-consuming t e s t s a r c under- t a k e n .

? 1 B a c t e r i a l p u r i t j E

B a c t e r i a l p u r i t y i s m o n i t o r e d by e x a m i n a t i o n of a l o o p f u l

o f c u l t u r e which h a s been t r a n s f e r r e d t o a s l i d e and s t a i n e d by Gram's method. A t l e a s t 2 0 m i c r o s c o p e f l e l d s s h o u l d be c a r e -

fully examined f o r s i g n s of c o n t a m i n a n t o r a b e r r a n t o rgan isms . I n a d d i t i o n , seed l o t s s h o u l d be inocu) la ted o n blood-agar s l o p e s , i n c u b a t e d f o r t h r e e days a t 35 C , and smears Erom t h e r e s u l t i n g c u l t u r e s e ~ a r n i n e d a s a l r e a d y d e s c r i b e d .

The rat0 of i n c r e a s e of o p a c i t y and t h e changes of pk of p r o d u c t i o n c u l t l ~ r e s a r e characteristic of the s t r a i n , medium and ct11tur.e c o n d i t i ~ n s . D e v i a t i o n from normal b e h a v i o u r of

the strain d u r i n g growth m a y i n d i c a t e Che p r e s e n c e of a n u n d e t e c t e d c o n t a m i n a n t . The r a t e of t o x i n p r o d u c t i o n s h o u l d

be m o n i t o r e d by Rarnon f l o c c u l a t i o n ( s e e Appendix 0.7) i n o r d e r t o v e r i f y t h a t t h e time chosen t o h a r v e s t the c u l t u r e i s o p t i - mal , b o t h i n t e r m of a c h i e v i n g t h e g r e a t e s t y i e l d and p u r i t y . I t i s , t h e r e f o r e , i m p o r t a n t t o measure t h e c o n c e n t r a t i o n o f PN ( n o n - d i a l y s a b l e n i t r o g e n ) a t t h e t ime of h a r v e s t of t h e c u l - t u r e a n d a f t e r d e t o x i f i c a L i u ~ l and purification of t h e a n t i g e n .

' --;.S . ;! :.

; : .. *.g+ . Af t c r thc f i r s t stage of d e t o x i f i c a t i o n the t o x o i d mus t

' L * > .,. . . be examined f o r r e s i d u a l t o x i c i t y (see Appendix D - 1 2 ) . T h i s

',.-:C . , . .d .

G;,?. m y be durle by i n j e c t i n g 5 m1 of t o x r ~ i d s u b c u t a n e o u s l y i n t o

. A .

a normal g u i n e a - p i g and o b s e r v i n g f a r symptoms oi d i p h t h e r i a

B L C / I : M I P . ; ~ ~ . ~ Rev. 1 page 28

i n t o x i c a t i o n o v e r a per iod of 30 d a y s , vr by i n j e c t i n g 0 . 2 m1 of a 1/10 d i l u t i o n i n t r a d c r m a l l y i n t o t h e d e p i l a t e d s k i n of a normal g u i n e a - p i g and e x a m i n i n g for s i g n s o f d i p h t h e r i a e ry thema 48 h o u r s a f c e r i n o c u l a t i o n . I f the t o x o i d is found t o he t o x i c , more f o r m a l i n m u s t b c added and t h e tuxold shown t o be n o n - t o x i c before removal from t h e incubator.

L£ c o n t e n t should be checked by Ramon f l a c c u l a t i o n ( s r c Appendix D.7) t h r o u g h o u t t he detoxification and p u r i f i c a t i o n s t a g e s t o e n s u r e t h a t the t o x o i d i s r e ~ o v e r c d i n adequate y i e l d and is undamaged.

The quality n f the f i n a l bulk t o x o i d abould be checked a s follows;

S L e r i l i t e . Inoculate n o t less t h a n 5 m l l of sample inLo cach 0 of two 300 m1 of t h l o g l y c o l a t c b ~ u t h . I n c u b a t e aL 25 C and 0

32 C For 14 days rsspcctively and observe daily f o r s i g n s of corlLarnination. I T buch growth occurs t h e roxoid f a i l s t h e Les t u n l e s s t h e Lcst is shuw~l by r c t e s c i n g LI) have been L n v a l ~ b . roxoid f a i l i n g t h e t e s t can be r c f i l c e r e d a n d r c L c s t e d pruv ided Lhat t h e l e v e l uF c o d t a m i n a r i o n i s lnw and t h e t o x o i d uun- p y r o g e n i c .

be volume of samplc i n b c u l a t e d must be a t l c a s t sufficient t o e n s u r e n l eve l o f a s s u r a n c e e q u a l t n t h a t provLdcd by thc p o s t - f i l l i ~ r g s t e r i l i t y test.. To a c h i e v e this B voluurc of a r l c a s t :

2 0 x t h e number n f f i l l i n g l o ~ s t o be p r o p a r e d for the b a t c h nt bulk coxoid

m1 The d i l u t i o n f a c t o r between b u l k t o x n i d and t h e f i n a l p r o d u c t volume

B L G / u ~ ~ ) P / ~ ? . I R e v . 1 page 24

- ( spec i f i c t o x i c i t y ) . I n o c u l a t e no r Leas t h i n 2 0 0 Lf o f

b u l k t o x o i d s u b c u t a n e o u s l y i n t u a t l e a s t . f i v c gu inea-p i gs weigh ing 250-350 g . Observe f o r 30 days. I f any of the g u i n e a - p i g s

show s i g n s uf d i p h t h e r i a i l l t o x i c a t i o n t h e b u l k t u x o i d must i ~ e r e j e c t e d or f u r t h e r d e t o x i f i e d . I f a n y of the g u i n e a - p i g s d i e

f rom causes o t h e r t h a n d i p h t h e r i a i n t o x i c a t i o n , t h e t e s t m y be

I repeated.

O t h e r , mnre s t r i n g e n t , t c s t s £or res idua l toxicity which may be used La supple men^ L ! I ~ mandatory t c s t described above a r e des- c r i b e d i n Appendix D.12 t o g e t h e r w i t h t e s t s for r eve r s ion t o

t u x i c i t y a n d e t a b i l i t y ( s e e Appcndix D.9) which shou!d be a p p l i e d t o a l l batches of toxoid .

l T o t a l c o m b i ~ r i i l ~ power tcst

The q u a l i r y o f d i p h t h e r i a toxoids can be measured by de ter - mi n a t i o n c ~ f the r a t i o o f L Q ~ B ~ combining power ( b i n d i n g u n i t s ) t o Lf units i n t h e bulk t o x o i d p r e p a r a t i o n (see Appendix ~ ~ 8 ) . For good t o x v i d s t h e r A t i n uL T.C.P. t o 1.f should a p p r 0 x i ~ t e t o , o r s l l g l l t l y exceed, o n c but since the value found f o r t h e r a t i o with a given t o x o i d will v a t y from one laboratl>ry t o a n o t h e r , Lhe minimum a c c e p t a b l e v a l u e w i l l have t o be d c t c r m i n e d by c a c h p r o d u c e r i n the l i g h t oi t h e i r uun e x p e ~ i e n c e . In

h o s t l a b o r s t o r i e s t h i s t e s ~ i s no t doac b u t i t is a d v i s a b l e t n

i n c l u d e i t .

Chcmica l p u r i t y

The number of Lf u n i t s p e r milligram of ptotcin nitrosen

(set Appcndix 0.7) s h o u l d bc noL less t h a n 1500.

s h o u l d he t e s t e d . I f the volume ~o b r i n u c u l a t e d exceeds 1 0 m 1 i c stlouLd be d i v i d e d among two u r more b o t t l r s of medium. Mcabrane f i l ~ m t i o n , i f a v a i l a b l e , o f f e r s some a d v a n t a g e s a t t h i s staRe.

I BLC; UNDP/ 7 7 . 1 Rev. 1

BLG/UNDP/77.1 Rev. 1 p a g e 31 I' !

page 30 is d i f f j c u l ~ t o i n a s a t i s f a c t o r y f o r m , and a Prepara-

D8. POOLlNG AND STORAGE OF ~IJW ~ O X O ~ D tion as "a lhydroga l" i s commerc ia l ly a v e i l a h l e . Two methnds

f u r t h e preparation of a lumin ium p h o s p h a t e a r e given in I D . 2 L . phospha te is a l s o used a s an - adsorhwl l - .

! D5.1 P o o l i n g I me c o n c c n ~ r a t i o n o f a lutninium muat h a t exceed W per i I The v o l ~ t l l e o f a n i n d i v i d u a I h a t c h of c o x i n or l u x a i d is s i n g l e human dose.

u s u a l l y a b o u t 50-lm l i t r e s . b e f o r e p r o ~ e c d i r l g t rr l t r a f i 1- c r a t i ~ n and/or p u r i f i c a t i o n i t i s c o n v a n i p a ~ to pool several n9.2 p r e y g r a t i o n and Lest5 on f i n a l 3 I , b a t c h e s t o make a b o u t 500 litres ( o r if facilities pernit). I f by u l t r a f i l t r a t i ~ ~ ~ u r s a ~ t i n g - o ~ c t h e volume is ln of the final b u l k t h e tOxoids are added . .

i reduced a ~ ~ r o x i m t e l y 1:50, t h e n t h e result an^ concen t ra ted t o the a l u m ~ n i u m phosphate, rile pH of the mixturf is adj' 'sted

! toxoid would be a b o u t 10 l i t r e s , a c o n v e n i e n t q u a n t i t y to handle to ~h~ i s h e l d i n t h i s p ~ for 48 h o u r z j t l len

f o r P u r i f i c a t i o n by f r a c t i o n a l s a l t i n g - o u t . ~ f t ~ ~ purification, the p~ is r e a d j u s t e d C O 6.6-7.0. A d j u v a n t is t o I. the of t h e c o n c e n t r a t e d and p u r i f i e d bulk toxo id would be perrussis component and t h e pH a d j u s t e d t o 6.b-7-0- The I i ' less ( s i x t o e i g h t l i t r e s ) , a g a i n a c o n v e n i e n t volume f o r

requisite q u a n t i t i e s of a d s o r b e d t o x o i d s and p e r t u s s i s are

t h e P r e p a r a t i o n of final v a c c i n e . : pooled , a n d t h e p ~ checked . The v a c c i n e i s m e r t h i o l a t c d

1: 10 000, t a k i n g i n t o a c c o u n t t h e quantity of merth io la te~ I D8 .2 S t o r a g e which e n t e r e d th rough t h e v a r i o u s comP@nents-

Co-rcial of a lumin ium h y d r o x i d e a d s o r b t o x o i d The c o n c e n t r a t e d , p u r i f i e d t o x o i g s which arc very t a b l e

0 0 can be s t o r e d i n a r e f r i g e r a t o r ( + 5 C-3 C) fo r ve ry long and d o n o t r e q u i r e a p e r i o d e t low P H to adsorp- i I ' periods ( U P to several y e a r s ) b e f o r e f u r t h e r p r o c e s s i n g . I L l is however, t o check t h e ~f value of = t o r e d t u x o i d s

be f o r e f u r t h e r p r o c e s s i n g . 1 .

Where only ~ ~ ~ - g r a d c ( t y p e 1 2 o r 111) g l a s s is a v a i l a b l e

I a d v i s a b l e to add a b u f f e r i o the f i n a l r u prevent l: rising a b o u t 7 .0 d u r i n g t h e s t o r a g e period of

j l f i n a l c o n t a i n e r s . . DILLrTINC: AND F!LI,TNc

~ h ~ ~ ~ are B number of f o r m u l a t i o n s of v a c c i v c s . The

n e s t i n use arc as L ~ l l o w s ;

i In c o u n t r i e s d i p h t h e r i a ~ o x o i d is used as an adsorbed

1. ~ i ~ h t h ~ ~ i a t o x o i d - a t least 2 5 Lf per d o s e f o r LOxo id ; l'laiil Loxoid u n s u i t a b l e f o r primary imun~zation ! except whell sornhined with p e r L u v s i s v a c c i n e , has an

ndsnrbed qr combined v a c c i n e . I t 1 5 advisable

a d j u v a n t e f f e c t . t o lrse p l a i n ( n o n a d s o r b e d ) d i p h t h e r i a t o w i d

a l o n e .

b9.1 P r e p a r a t i o n o f adsorbed coxoid I

Adjuvants s u c h a5 aluminium p h o s p h a t e o r hydroxide u s e d t o p r e p a r e a d s o r b e d t o x o i d s , ~ d j ~ ~ ~ ~ ~ my be prepared l o c a l l y o r o b t a i n e d from a comercial source, The latter is e s p e c i a l l y a d v i s a b l e for aluminium hydroxide gel "hieh

! I

page 32

2 . T e t a n u s Enxold - a t l e a s t 5 Lf p e r dose in p l a i n o r adsorbed combined v a c c i n e , but at least 1C 1,f p e r d o s e i s r e q u i r e d f o r uncombined t e t a n u s vacc ine whether p l a i n or a d s o r b e d .

3. P e r t u s s i s v a c c i n e c o n t a i n s no t more t h a n ?O I n t e r n e t i o n a l Opecicy U n i t s (0.u.) w i t h a pu t ency of not l e s s than 4 I .U . p e r s i n g l e human d a s e .

m e msjority of w n t r o l r e s l s a r e c a r r i e d out a t t h i s s t a g e whe11 a l l r h e i n g r e d i e n t s o f t h e v a c c i n e , t n x o i d s ( d i p h r h P r l a and tetanus), p e r t u s s i s suspension, a d j u v a n t and d i l u e n t have b e e n b l e n d e d . These t e s t s arc :

~9(1) Fotency t e s t Appendix D . l l

D91II: S p e c i f i c t u n i c i t y t e s t Appendix D . 1 2

D P ( I 1 I ) I n n o c u i t y t e s t Appendix U . 20

D9(1V) S t e r i l i t y t e s t Appendices D . 19

and D . 2 7

l ' D ~ ( V ] I 'h iomersal c o n t e n t Appendix U. 15 \ ' !

D q ( V 1 ) pll Appendix D , 16

D ~ I v I I ) Aluminium c o t r t e n ~ Appendix 11. L8

DS(VIIZ) Free ( r e s i d u a l ) f u r m a l i n

c o n t e n t Append ix D . L7

D9(IX1 T e s t f o r a d s o r p t i o n Append ix D.7

The v o l u m e of sample used i t 1 t h e s t e r i l i t y t e s t s h o u l d r ~ u t be Less khan 20 r ( t h e number o f f i l l i n g l o t s C O br. produced from t h e b u l k ) ml. I f t h e volume t o be i n o c u l t l t ~ d e x c e e d s 10 m1 h u t is 20 m 1 or l ess t h a n 2 0 m 1 t h e ~ r one more 300 a 1 b a t t l e ol t h i o g l y c o l a t e medium should be used and t h e i n o c u l u n e q u a l l y d i v i d e d between t h e t w o bottles.

Apar t frm t h i s , t h e media and p e r i o d s a n d temperatures of incubation a r e a 5 d e s c r i b e d i n Appendix D . 1 9 .

B L G / u N D P / ~ ~ . ~ Rev. 1

page 33

I f t h e vaccine is unadsorbed t h e antigen conrent p i

d i p h t h e r i a a n d teLanus s h o u l d be checked by f l o c c u l a t i o n .

If the v a c c i n e i s adsorbed, a sample s h o u l d be c e n t r i f u g e d

and t h e c o n c e n t r a t i o n of free t o x o i d i n t h e eupernatant d e t e r - mined by b l e n d f l o c c u l a t i ~ n (see Appendix D . 7 ) .

Vaccine is

I acceptable if n o more t h a n 2M o t rhe toxoid originally added is f r e e i n t h e s u p e r n a t a n t .

There are several methods Q£ rpeasuring the po tency o f d i p h t h e r i a t o x o i d , but f o r e a c h the principle i s the same. A group of g u i n e a - p i g e 16 immunized wLth 8raded d o s e s of t h e toxoid and their responses measured e i t h e r by d e t e m i n i n g the a n t i b o d y titre i n t h e sera or by chal lenging che ani lpala by t h e s u b f u t a n e o u s o r i n t r a d e r m l route .

I n order t o rule out the v a r F a b i l i t i e s that are i n e v i t a b l y : involved in an a n i m a l test, a s i m i l a r group of animals is

'. Lrpmunized w i t h A r e f e r e n c e preparation t h a t has been c a l i b r a t e d > d , n Ln l d t e r n a r i p n a l U n i ~ e A me potency of the s h o u l d - -

I be expressed i n i n t c r n a t i a n * L U n i t s . . +: *'! :., .,'h .!.$L . >.:P : Methods for measuring the potency of d i p h t h e r i e toxoid are

described i n Appendix D . 11 . L W : ' '

B(. 3 Filling i n t o final c o n t a i n e r s

The r e q u i r e m e n t s for f i l l i ~ l g t h e f i n a l c o n t a i ~ r e r s shall be d e s c r i b e d i n the General Requi rements f a r Manufac tur ing tab1 ishrnents a n d control l a b o r a t o r i e s (UHU Technical R e p o r t

1966, No. 323) {see Appendix D.28). The vulume p r e s e n t

n g l e b u l k c o n t a i n e r a t any t i m e shall determine the i z e , However, t h e s i z e o t t h e f i l l i n g l o t will be

d by t h e c a p a c i t y of t h e f i l l i n g equipment t o be used he p r o c e d u r e a d o p t e d Ln f i l l i n g . I f the p a r e n t f i n a l

d i : ; l r i b h t e d in c o n t a i n e r s e t smiler size, each s u c h

BLG/UNDP/~~, 1 Rev. 1

page 36

B L G / U N D P / ~ ~ , ~ Rev. 1

page 35

c o n t a i n e r s h a l l form a f i l l i n g l o t and be i n d i v i d u a l l y lab~lled a s such . If, however, r h e f l l l i n g i s c a r r i e d o u t d i r e c t l y from t h e f i n a l bu lk c o n L a i n e r , the number of f l n a l c o n t a i n e r s f i l l e d a t one s e s s i o n w i r h o u i a stoppage or b r e a k s h a l l b e deemed as a f i n a l l o t o r I i I I i n g Zat f o r a l l t h e t e s t s on L i n a l c o n t a i n e r s .

For s i n g l e - d o s e c o n t a i n e r s ampoules o f a p p r o p r i a t e c a p a c i t y c a n be u s e d , and f o r t h i s purpose a l s o c o m p l e t e l y a u t o m a t i c ampoule f i l l i n g and s e a l i n g e q u i p m n t is a v a i l a b l e .

The f i l l i n g o f b i o l o g i c a l s must be c a r r i e d o u t u n d e r s t r i c r a s e p t i c c o n d i t i o n s a s t h e p r o d u c t o n c e c o n t a m i n a t e d c a n n e i t h e r be r e c o v e r e d n o r t e r m i n a l l y r e s t e r i l i z e d .

I t i s , t h e r e f o r e , p r e f e r a b l e t h a t f i l l i n g s h o l ~ l d be c a r r i e d o u t iiy a corrrpletely au tonmted p r o c e s s w l t h l e a s t i n t e r f e r e n c e by t h e f i l l i n g s t a f f . Such c o m p l e t e l y a u t o m e t i c f i l l i n g , s t o p p e r i n g and s e a l i n g equipment f u r m u l t i d o s e containers is c o m e r c i a l l y a v a i l a b l e ( see Appendix 0 . 2 3 ) . S i m i l a r l y . a u t o m a t i c a n p u u l e f i l l i n g and s e a l i n g eqtlipmelrt i s a l s o a v a i l a b l e ( s e e Apper~dix U.21) f o r f i l l i n g s i n g l e - d o s e ampaules . S e m l - a u t m a t i c machines f o r f i l l i l l g v i a l s f u l l o w e d by m n u a L s t o p p e r i n g a n d c a p p i n g o r manual s t o p p e r i n g and m e c h a n i c a l c a p p i n g c a n a l s o he u s e d .

i t is p r e f e r a b l e , however, to use c m p l e t e l y a u t o m a t i c e q u i p m e l ~ i s o a s t o m a i n t a i n a s e p t i c c u n d i t i o n s t h r o u g h o u t t he o p e r a t i o n . Such machines n e c e s s a r i l y r e q u i r e the u s e o f v i a l s p r o p e r l y s t a n d a r d i z e d w i t h r e s p e c t t o budy d i a m e t e r , h e i g h t a n d d i m e n s i o n s o f t h e mouth s o a s t o be c o m p a t i b l e w i t h s t a n d a r d r u b b e r c l c s u r e s . S i m i l a r p r e c a u t i o n s a r e a l s o r e q u i r e d for t h e dimer:s ions of ampoules t o be used i n a u t o m a t i c f i l l i n g .

I The q u a l i t y of g l a s s c n c t a i n e r s i n f l u e n c e s t h e k e e p i n g q u a l i t y o f t h e f i n a l p r o d u c t . It- i s n e c e s s a r y , therefore,

i! t o usc n e u t r a l b n r o s i l i c a t e b.!..? type 1 c o n t a i n e r s i n i h e i n t c r e s L of q u a l i t y end saLcLy o f t h e p r o d u c t thoufitt ( 1 . 5 . P , rype 2 g l a s s may b e p e r t ~ ~ i s s l l ~ l c i n same c o u n t r i e s .

'L'h<s q r l a l i t y of rubtrer c l o s u r c s i s a l s o i m p o r t a n t . Nilbber i s knuwrl i u a l l s o r b ph ( :na l s and m e r c u r i a l p r e s e r v a t i v e s a n d

good qualLry or cosfed rubber i s n r r d e d C V prevr!nt t h i s . S o u r c e s of rubt ier s h a ~ ~ l d be checked, t h e r e f u r c , f o r c o m p a ~ l - b i l i t y w i t h Lhr v a c c i l ~ e and i t s p r e s e r v a t i v e .

i i u ~ y l r u b b e r

SLappers t r e a t e d w i t h gush p r e s e r v a t i v e s a s t h i 0 0 l ~ a l or p h e n o l p r i o r t o uae i n the f i n a l product a r e found t o a b s o r b a minlmum g t i r n t i t y o f p r e s e r v a t i v e du r ing s t o r a g e of t h e v a c c i n e . The

u s e ot b u t y l o r o t h e c r u b b e r s toppers which have been s h a m t o be s a t i s f a c t o r y is recommended.

The a d v a n t a g e of f u l l y automatic f i l l i n g a n d c a p p i n g equipment is not that i t E a v e s l a b o u r b u t that i t r e d u c e s con- t a m i n a t i o n and t h e r e b y p r e v e n t 8 u n n e c e s s a r y d i s c a r d i n g of v a l u a b l e v a c c i n e . S t r e s s s h o u l d be p l a c e d on t h e need f o r . -~ -

r e g u l a r m a i n t e n a n c e of such e q u i p m e n t .

P r i o r t o f i l l i n g , the v i a l s and s t o p p e r s must be washed and s r e r i l i z e d . PUny types of equipment a r e a v a i l a b l e f o r t h i s

~ u r p o s e , and a l l s t of some m a n u f a c t u r e r s i g given i n Appendix . - D.23, item 10.

The f i l l i n g mus t be done i n s p e c l r l l ~ designed rooms o r done u s i n g a v e r t i c a l l aminer - f la a y s t e m which g i v e s good I " ~ l a s s 1 0 0 ~ ' ) c o n d i t i o n s f o r a t e r i l e work ,

TLle P a n e s of some

M n u t a c t u r e r s o f such equipment a r e gLven i n Appendix 3 . 2 3 ,

i t e m 11. Such l a m i n a r - f l o w systems s h o u l d b e t e s t e d f rom t ime t o t i m e t o e n e u r e t h a t they a r e d e l i v e r i n g s t e r i l e a i r .

T h i s

m y be done by e x p o s i n g n u t r i e n r m d i a i n P e t r i - d i s h e s and t h e r e a f t e r i n c u b a t i n g them t o t e s t f o r s t e r i l i t y . A l t e r n a -

t i v e l y , t h e c o n t e n t s of t h e p e r r i - d i s h e s m y be t r a n s f e r r e d t o more e a s i l y t r a n s p o r t a b l e containers f o r f u r t h e r i n c u b a t i ~ n -

It i s i m p o r t a n t to check t h e f i l l i n g p r o c e s s r e g u l a r l y by f i l l i n g a t least 500 e m p o ~ l e s o r v i a l s w i t h a s u i t a b l e n u t r i e n t r d i u m . p r e f e r a b l y a t t h e end of t h e w o r k i n g day.

The f ~ l l e d

.mpoules s h o u l d be i ncuba t ed and i f more t h a n 1% of them are 8hOun t o be c o n t a m i n a t e d , t h e f i l l i n g a r e a , equipment and o p e r a - t i n g p r o c e d u r e must be i n v c s r l g a t e d a n d improved.

.h I n s p e c t i o n

Every s i n g l e o r m u l t i d o s e c o r ~ t a i n e r s h n u l d be insF'ectcd SuaLly under p r o p e r i l l u a i n s t i o n t o d e t e c t t h e p r e s e n c e o f

e i g n p a r t i c l e s . A simple a r r a n g e m e n t f o r t h i s p u r p o s e i s

-..-- - 4 ,

BLGi urnpi 71 .1 Rev. 1 B L G ~ U N D P C ~ ~ . 1 Rev. l ! page 3 6 page 37

l a t u b e f i t l e d a t the t o p of a black and back- by a

e m b e r o f staff. A p m r ~ from Likese proto-

i ground and i L l u m i n . r i n g t h e b a ~ k ~ ~ . ~ ~ d . The f luor rscent cols, careful records e h a l l be k e p t i n t h e l a b 0 r a t o r Y indicating

I is 'Overed p r o c e r s i n g , r e s t i n g , t i l l i n g and d i s t r i b u t i o n . 'l1 shade i n o r d e r t o p r o t e c t the eyes of the

s t a f f from a n y d i r e c t glare. v i a l n are examined records s h a l l be retained throughout t h e period

I . 8 .

against b o t h white 5I.ck background , and vialJ ampoules v a c c i n e , ~h~ record shs11 b e a v a i l a b l e fo r i n s p e c t i o n a t

showing foreign P a r t i c l e s and defec t s in closures shou ld be times by the n g ~ i o n a l c o n t r o l a u t h o r i t i e s .

r e j e c t e d .

D1O. TESTS Oh' FINAL ~RODLJCT

~ 1 1 . 1 L a b e l l i n g D1O.L T e s c s --

~h~ l a b e l l i n e of d i p h t h e r i a t o x o i d on t h e container and The product a c ~ e r filling s h a l l bc suh jecred

Lkle . , package l l cmp ly w i t h t he RequlremenLS of S e c t i o n a Or

f3110wing t e s t s : part A of the l e q u i r e m e n t s f o r Diphtheria a n d Tetaf iu$ ?moid l.

( s e e Appendlx D.26). DlO( I 1 t e sL ( i f not done ~~~~~d~~ 11

l on f i n a l b u l k )

DIVIII ) I n n o c u L ~ ~ t e s t The size and t y p e uf c o n r a i n e r end package s h a l l bc that Appendix D.20 t h e condiciul l* at u s e - I t is

D l O i l l l ) Idcr.Lity t e s t , but under some c i rc l l rns tances In which A p p e n d i x n, 10 l d r e n m y be assembled a t a c l i n i c , Or

Dlfi(ri') S t e r i l i r y test be mure a p p r o p r i a ~ e t s p ' c i a l l y i f g j e t - Appendices D, 19 !. sed. E x p e r i e n c e s w i t h j c t - i t l ? e c I l o n guns,

a n d U.21 a d s o r b e d v a c c i n e s are used have given

D ~ v I v ) rh iamprsa l C O , L , , ~ Appendix n . 15

nlO{VT) l,.$ e r a t i o n m u s t be g i v e n period O v e r Appendix I ) . l h

f p vial m y be u s e d . 1 f a physicial ' i s

"D('JTI A l u n l i n l u m cont r r , t i n d i v i d u a l c h i l d r e n i n h i s and Appendix 11. 18

n a number of separate. n~casians D L L ) - 2 - Reporting "t dxa ellt present in t h e v a c c i n e must be shown tn

e s s i n g t h e growth o f any orgsnisrns ir'tro- F o r each "L f i n a l vaccine a w r i t t e l l p r t , t a c t , l s h n , , l c l br

tine d u r i n g the removal of a sing1e of

c o p t a i ! ~ i n g l l t h e e s s e r l t i s ~ inforlIlarlon n n Lhc I,ro- t h a t a v i a l o n c e opened shouLd

and CeSLing nf Lhe vacc ine . J c st,c,uld Lhe daLa Of terr i h a ~ havv been p e r i u m e d he l rLilr lY

B L C / U N D P / ~ ~ . ~ Rev. 1

page 38

used on t h e same day and n o t be r e u s e d a n s u b s e q u e n t d a y s . ~f s t o r a g e and reuse a r e a b s o l u t e J y u n a v o i d a b l e , the v i a l m u s ~ be

O I 0 k e p t i n a r c f r i g e r a ~ o r a t 5 - 3 C and never f u r mgre than one working week.

111 2. H E IBASE

The mechanism for t h e r e l e a s e of v a c c i n e s sllould be d e r e r - mined by t h e n a t i o n a l c o n t r o l a u t h o r i t y . Where t h e r e i s n o n a t i o n a l c o n t r o l a u t h o r i t y , p e r m i s s i o n t o m a n u f a c t u r e v a c c i n e s and t h e mechanism f o r r e l e a s e s h o u l d be s p e c i f i e d by t h e n a t i o n a l h e a l t h a u t h o r i t y .

I t i s i m p o r t a n t t h a t o n l y t h o s e v a c c i n e s t h a t f u l f i l P a r t A oE t h e WtlO Requi rements f o r D i p h t h e r i a Toxuid ( s e e Appendix ~ . 2 6 ) are released f o r u s e .

Where t h e p r o d u c e r i s s o l e l y r e s p o n s i b l e Eor q u a l i t y c o n t r o l and t h e r e i s no n a t i o n a l c o n t r o l a u t h n r i t y , t h e n i t i s e s s e n t i a l t h a t t h e c h i e f o f t h e q u a l i t y c o n t r o l l a b o r a t o r y of t h e p r o d u c e r reports t u t h e d i r e c t o r o f t h e i n s t i t u t e i o d c p e n d e n t l y o f t h o s e i n v o l v e d i n t h e p r o d u c t i o n of t h e vacc i r l e . 'The d e c i s i o n con- c e r n i n g t h e r e l e a s e of a l u l oL vaccine must be made by the c h i e f o f Lhr c o n t r o l 1 a h ~ ) r a t o r ~ .

- . . --

TJ 1 3 . RE'J'AI NED S A W U S

A t t h e time of release uf a l ac of vaccille, t he m a n u f a c t u r e r must s e t a s i d e and t - e f r i g e r a t c enough s a m p l e s t o s e r v e two e s s e n t i a l p u r p o s e s :

BLG/UM)P/ 7 7 . 1 R e v . 1

page 39

( i ) To e n a b l e a n i n v e s t i g a t i o n t o be c a r r i e d out I n t h e even^ of B c o m p l a i n t f rom a u s e r c o n c e r n i n g t h e p a r t i c u l a r l o t . I t i s advirable, t h e r e f o r e ,

t o s e t a s i d e sufficient samples t o a l l o w f o r a f u l l

test Q £ t h e f i n a l vaccine to be r e p e a t e d .

(11) Tu ensure that t h e pr0ducL h a s been s i o r e d correctly and t h a t t h e puLency has been malntaincd t h r u u g h o u t Lhe storage p e r i o d .

c

The storaxe of r e t a i n e d s a m p l e s o c c u p i e s r e f r i g e r a t i o n s p a c e that may be l i m i t e d , L l i t only i f i t i s known t h a t t h e comple te l o t ha s been d i s t r i b u t e d , u s e d o r d e s t r o y e d a t t h e end of t h e e x p i r y p e r i o d s h o u l d such s a m p l e s be d i s c a r d e d . I t w i l l b e u s e f u l from time t o trrne t o k e e p some s a m p l e s

beyond t h e e x p i r y d a t e , and t o c h e c k on t h e k e e p i n g p r o p e r t l e s , p a r t i c u l a r l y f o r p o t e n c y .

D14. SURVE11,lANCE OF VACCI NES

The surveillance of t h e use of v a c c i n c s i s the responsi- b i l i t y of t h e N a r i o n a l t :overnmer~t H e a l t h A u t h o r i t y . Such srlr-

v e i l l a n c e f a l l s into two c a t e g o r i e s :

D14.1 S a f e t y

A l t h o u X h e v e r y attempt i:; made in t h e l a b o r a t o r y t o ensu re t vaccines are s a f e hy s u b j r c t i d g e t 1 t o t s t o t e x t s f u r f r e e -

from abnormal t o x i c i t y , a c u n s t a ~ ~ t w a t c h s h o u l d n e v e r t h e - a be kept far a d v e r s e reactions i n children.

There a r e c o n t r a i n d i c a t i o n s to t h e u s e of a l l v a c c i n e s ,

-.md an i n v e s : i g a t i o n of a l l a d v e r s e reac tiuns s h o u l d be made , @ r t i c u l a r l r i f they appear t o bc l u t ~ r e l a t e d .

Dl4.2 Efficacy

DiEferent populat ions d i f f e r i n t h e i r antibody responses t o v a c c i n e s , and i t i s e s s e n t i a l to ensure t h a t t h e cornuni ty i s g i v i n g adequate antibody respnnses to a11 components o f the v8ccine.s u s e d . Many factors such as nllt.ri t i o n a l s L a t u s , inter- c u r r e n t i n f e c t i o n s and the age 01 Che c h i l d r c n havc a n e f r e c t upan the responsrs. A paired b lond sarnplc heLtlrc! and a i ~ e r v a c e i n n t l u n s h r > i l l d be takcn from a numhcr of the gaccitlees end t i i r a r e d tu measure the a n c i h o d y responses i h nrdcr to have gome idea a t t h e e f f e c t i v e n e s s of the v a c c i n a i i o n campaign. Furthermore, i t i s advisable to obtain blood s a m p l e s from the same c h i l d r e n two t o f i v e years l a ter t o determine how much ant ibody (and thereby p r o t e c t i o n ) has been mainta ined .

T h r o u g t ~ r ~ ~ ~ t the manual r e t e r c n c s has been made to t h e reg l~ irements s p e c i f i e d i n ~ l t c R e q u i r e ~ ~ t c ~ ~ t s f o r D l p h t l ~ c r i a Toxuid, P e r r u s s i s v a c c i n e , Tetanus 'roxoid and combined v a c c i n c s ( 1 9 7 8 ) . Thc part of these Requirements w h i c h r e f er s L W Diphtheria ' I 'oxoid i s reproduced i n A p p e n d i x D.2b of t h i s manus l .

U L G ~ U N I , P / ~ ~ . L R ~ ! v . I

page 6 1

h rurmnaiy protocol ( s e e Appendix U.241 f o r recording the r e s u l ~ i of Leris on a p a r t i c u l a r f i n a l l o t ul vacc ine i s included i n the milnoal. lllr t e s t $ s p e c i f i e d l c > l l o w clascly those

inrlt lded i n thr UbO Reqnireuents, nod tile p r n t u c o l i s i n ~ e n d e d 11.1 the rcpurtinp "I data t o L ~ E n a t i o n a l c v ~ > t r n l a u t h o r i t y or ~ C I chu a ~ i t h o r i t i e r i n o t h e r c i ~ u n t r i e s t o wlhon (hi : vsrclnc n ~ d y he pxporiecl. llic r e s t s s p c ~ i f i e d are In sucli d e t a i l t h s l i n s p e r -

f i o n of ~ l l c p r o t o c o l s , l ~ o l l d p2ive a11 indiratiun o f w l ~ c i l ~ e r lie b a t c h m c c t s the W H O Requiremenis i n every p a r t i c u l a r .

LIS'I' OF At'L:E,NDlCES

Media . . . . . . . , , . . . . .

Prnduction i n ferrncnters . . . ,

Assembly of a n ~ e t a l i l t e r . . , . . . , . . . . . . 5 2

F i l t r a t i o n o f fe r rncn te r trilrvcst . . . . . - . , . . 5 3

I f l ~ r a f i l t r a t i u n r ) f toxoirls , . . . . . . . . . , 5 >

Purilication 01 Loxoid . . , , . . . . . . . . , 58

D e t e r m i l ~ a i i o n of L f / m l . . , , , . , . . . . . . . 60

~ o t a l Combining Yvwer T e s t (TCP) . . . . . . . . . h>

S t a b i l i t y a n d r e v e r s a l t c b t . . . . . . . . . . . . 66

I d e n t i t y t e s t . . . . . . . . . . . . . . . , , . . 68

Potency t e s t i n g o f d i p h t h e r i a t o x o i d . . . . . . . 69

T e s t s f o r s p e c i f i c toxicity and r e s i d u a l 7 9

t o x i c i t y i n bulk t o x o i d . . . . . . . . , , . . . T e s t f o r i r o n . . . . . . . . , , . . . , . . . , B0

D e t e r m i n a t i o n o f T o t a l N i t r o g e n ITN) and 82

on-Dialysable P r o t e i n N i t r o g e n (PN) , . , . , .

Tests f o r t h i u m e r s a l c o n t e n t . . . . . , . . , . . 84

Test f o r p H . . . . . . . . , . . . . . . . . . . . 86

T e s t f u r f r e e fo rmaldehyde . . . . , , , . , , , . 8 6

H ~ ( ; / u N I ) I ' / 7 7 . 1 ltev . 1

pagr 43

'Tebt fur alulllllliurn con1enL . . . . . , . , . . . H l

TesL tor s l ~ ~ . i ! i r . y . , . . , . , , . . . . . . Uk

T e s t [ o r innucuilp , . . . . . . a . . . . 8')

F ~ u l ' a r a l on #.>l n l u m i ~ \ i f ~ ~ n I I io: .p l~: ! t e s u s p e ~ i f . I ('11 . llil

M i x l n ~ , u e s c e ! ; o r v a c c i i l ~ ~ . . . . . . , , . , . 51

' ; u p g e s ~ ~ d 5 . 1 1 r c c ~ 01 c q u i p r n , ~ . l ~ rhewirals . , 9 4

5 u m m r y p r r ~ c v c o l f o r d i p h t l ~ e i l a t o x o i d . . . . . 1('3

Flow c h a r r ' i and dara r e c o r c l i r ~ & , . . . . . . . . L08 WHO Requi rements T G K D i p l l t h e r i a Toxo~d . . . . . 17R

VHD Requi rement9 t o r S l e r i l i t y

WHO Requ;rerr.ents f o r M a n u f a c t ~ l r i n g E s t a b l i s i u n e r ~ ~ s and C o n t r o l L a b o r a t o r i e s

RLt; j l I t iDi ' . l : . l Rev. 1

PaR'' $ 4

APPENDIX I ) . 1

A . For pror luct iur l i n flasks ( s t a t i c c u l t l ~ r ~ ) - ~- -

3eei i s d i y f s t e d f o r ah~111t ~ W O t ~ o u r h ~ t $0-50°C w i t h a p a n c r e a t i c t r y p s i n suspension a t a pll k e p t c o n s t n l ~ t l y a t 8 .0 . A f t e r t h i s d i ~ ~ s t i o n t h e i n i x t u r c ' i s b u i l e d with L % g l a c i a l a c e t i c a c i d and t he r e m a l n i n g b u e f i s f i l t ~ l r e d o f f .

The ncxt d a y b a k e r ' s y e a s t i s a d d e d (one I ~ u u r , 3 0 - 3 5 ' ~ ) . h e a t e d t o $ 0 ' ~

and 0. % Z s u d i u ~ u l a c t a t e a d d e d . A f t e r f i l t r a L i o n s m a l l a rnounts ( d e t e r m i n e d c x p c r i r n e n ~ s l l y ) o f t c r r o u s s u l f a L c o n d svdiunl c i t r a t e are added t o g i v r an u p t i m a l i r o n c o n c e n t r a t i n n ( see

A p p e n d i x D. 13). The medium i s s t e r i l i z e d by ~ u t n c l a v i r l g and 0 . 3 % s t e r i l e rna l tnse added a n d i l \ o c u l a L e d .

I n t h i s medium maxinial t o x i n y i e l d i s u s l l a l l y r e a c h e d a f t e r about 10 days a~ o 3 5 - 3 6 C , b u t it i s a d v i s a b l e t o h a r v e s t wlieri t h e ratlo ~ f / r n ~

protein nitrogen is o p t i m a l , u s u a l l y a f t - sin t o s c v e o days . T h i s ratio i s i m p o r t a n t Lur the f i n a l p u r i t y of t h e t o x l n o r

tox0id a f t e r p u r i f i c a t i o n . F u r t h e r details of t h e prepara- t i o n of ? h i s medium c a n be found i n J . P a t h . Lac!. (L9'171, 35, 3 7 3 .

H. For p r o d u c t i o n - i n fgrmenters

1 8 . 2 kg o f good qualiLy hecf musc le i s g r o u n d and w a t e r added L n l 2 0 l i t r e s . F l o a t i n g f a t is r a r f f u l l y removed h p means of a s k i r m e r and a f i l t e r p a p e r .

Temp~rature 1 s 0 a d j u s t e d to ?O C and t h e fat is remvved once more, W l ~ e n a l l ttre f a t h a s been r-emoved t h e pH i s adjusted to 6 . 9 5 by t h e

a d d i t i o n of 5m% NaOH. 'The t e m p e r a t u r e i s then r a i s e d t o 0 50 C and t h e mass i s s t i r r e d s l o w l y w i t h a n electric s t i r r e r , pH i s r e a d j u s t e d tn 6 .95 ,

Thc f l e s h i s d i g e s t e d f o r s e v e n p e r i o d s each of 30 m i n u t e s a t 5 0 ' ~ .

Is: d i g e s t i o n . 2 0 - 2 5 R of papain, Zirnmerman U.P .11 , the

amnunt depending on the q u a i i Ly of ~ b o yrpai n, i s suspended in water b y means O K a h i g h - s p e e d mixer and added t o t h e m e a t . After l i v e minu te s , the m i x t u r e i s a d j u s t e d tn pH 6.95 w i t h SO%

I EaOH and checked evcry LO m i n u t e s .

2nd d i g e s t i o n . 2 0 . 1 g 0 6 papain i s suspended a s b e f o r e --

and digesLion c a r r i e d on tor a f u r t h e r 30 m i n u t e s a t pH b . 9 5 .

I The m i x t u r e i s t h e n adjusted to pH 7 . 0 .

3rd digestion. 1 0 . 1 o f p a p a i n 1, suspended as . b o a and

diBestion c a r r i e d uut for 10 m i n u t e s a t p H 7.0. A t t h e end O f

this t h i r d d i g e s t i o n , t h e q u a n t i t i e s oE NaOll added are checked . If a t each s t a g e they a r e about the same, the pH i u i the 4th 1 . d l y e s t l o n can remain a t 7.0. 1 f t h e q u a n t i t i e s o f NaOH have

decressed the L t h d i g e s t i o n is adjusted t o pH 7 . 1 .

5th d i g c s t i u n . 2 0 . 1 g of p a p n i n , ' 3 0 m i n u ~ e s a t PR 7 . L .

6th d i e r s t i u n . 2 O . i g of paprin, 30 m i n u t e s a t pH 7 . 2 .

7 t h d i g e s t i o n . 20.1 g of p a p a i n . 30 minutes a t pH 7 . 2 .

A t t h e end of Lhe seventh 30-minute ~ e r i o d t h e pH is eb t o 5 . 1 by t h e s d d i i i o n o f c o n c e n t r a t e d NC1. The

mix ture is heated to b ~ i l i o ~ and b o i l e d f ~ r 10 m i n u t e s . c e t i c a c i d i s added t o b r i n g the pH t n 4 . 1 The

The m i x t c r e i s c l a r i f i e d by f i l t r a t i o n through a S e i t a c d y i e l d s a c l e ~ r , p a l e - y e l l o w f l u i d .

The volume

e d t o 1 1 ~ 1 l i t r e s . A s a m p l e i s t a k e n tor determina-

amino-x, t o t ~ l - ~ and p r o t e i n - h ' . $The ptl i s a d j u s t e d

D L G / ' U N D P / ~ ~ 7 . l Re v . 1

page 4 b

Appendix D . 1

BLG/UNDP/ 77.1 Rev . 1

page 4 i

Appendix D. l

to 7.65 wit11 5UX NaOB. E i g h t e e n grams oE yeasL e x t r a c L i s added i n suspension f o l l o w e d by O , l % sodium l s c t a t e {sec below) end

180 m1 of h e l l e r ' s growth f a c t o r s s o l u t i o n . The pH i s t h e n raised t o 7 . B . A second sample i s t aken f o r anaLysLs and a 50% solution of d e f e r r a t e d m a l t o s e is. added i n such a q u a n t i t y Lhat the final c o n c e n t r a t i o n i n t h e medium i s 2 .5%. The volume i s a d j u s t e d t o 120 l i t r e s and pH t o 7,8. A f u r t h e r sample i s t a k e n for a n a l y s i s . 3 3 , 9 g o f MgSO .7H 0 i s added t o t h e c l e a r f l u i d . The medium i s s t e r i l i z e d by f i l t r a f i o n t h r o u g h a S e i t z f i l t e r - press c o n t a i n i n g t e n 2 0 x 20 cm s t e r i l i z i n g pads . The f i r s t 2 0 l i t r e s p a y s i n g t h r o u g h hhe f i l t e r a r e d i s c a r d e d . The s t e r i l e medium is h e a t e d t o 100 C in a s t e a m e r f o r 30 m i n u t e s .

1DO g l a c t i c acid (Be'%)

47 g w a t e r

The NaOH i s d i s s o l v e d i n the w a t e r and the s o l u t i o n a f t e r coo l i t rg added c a r e f u l l y t o t h e l a c t i c ac id . The r e s u l t i n g s o l u t i o n (187 g ) c o n t a i n s 602 sodium lactate.

Other media

There a r e many o t h e r media s u i t a b l e for d i p h t h e r i a t o x i n production. The c l a s s i c a l s e m i - s y n t h e t i c medium on t h e b a s i s of casein h y d r o l y s a t e i s d e s c r i b e d i n M u e l l e r , J . I f . & Miller P . A . , P r o d u c t i o n of d i p h t h e r i c t o x i n of h i g h po tency (100 1.f) on a r e p r o d u c i b l e medium, J, I m u n o l . (1911) g, 21.

For a m o d i f i c a t i o n o f t h i s mcdium, s u i ~ s b l e for sullmzrged c u I ~ i v a - t i o n i n f e t m e n t e r s , see: S t a i n e r , D, W , & S c h o l t e , M. J . , The p r a d u c ~ i o n of h i g h p o t e n c y d i p h t h e r i a toxin i a submerged c u l t u r e i n r e l a t i v e l y s i m p l e e q u i p m e n t u s i n g a s e m i - s y n t h e t i c medium, Ricltechnol._atld H i o e n g . , ( 1 9 7 3 ) No. 4 , p p . 283- 2 4 3 .

1 P e r l i t r e :

225 g %S04.7H20 p . a .

1.15 g b e t a - a l a n i n e

1 .15 g n i c o t i n i c a c i d

75 mg p i m e l i c a c i d

50 m1 1% CuSO,. 5H,O p . a . s o l u t i o n (10 g r / l i

4 0 nl lX ZnSOq.7H20 p . a . s o l u n ~ n ( 1 0 & r / l )

15 m1 1% MC1 2 4H 2 0 p.a . s o l u t i o n (10 K r / l )

30 m1 c o n c . HC1 p . a .

Ref.: W e l l e r 6 Miller as shown abuve. 2. f

' < + B . , . .*- p .a . = t h e h i g h e s i grade of p u r i t y

S J:,

3; ;- C.. %*%.

h Ly,,irel ferlnenLci i s a c y l i ~ ~ d r i c o ~ v e s s e l c u n t t r u c t c d ~1 : ; ( d , n l C s s - ~ ~ I C I , ~ ~ r ~ / ~ . r a b l y A l S l I l b .

A 1 1 C ~ ~ l ~ a ~ t

i,,l~Y,j,l,)~ w e l d s M v r ~ he p l i s h e d . Thc d i m ~ ~ n s i * ~ ~ ~ ~ h ' y , , l t l l r i p l - m a n ~ , . ~ r dcpcnd on ~ h t : s i z e 1 1 1 the LoL which i s , , , l , , A hbslnrll ~ ~ ~ ~ ~ ~ n ( ~ . r ~ ~ i s u i r d Eur a b i i e v i

, . , , ir a I , I , ~ o X i n r ~ r ~ ~ i 1 O d 5 c m in diamt:l .rr alid

1 0 i-,l:iR[,I d i t e r ~ ~ ~ i n l ~ ~ g 1 hi! I ) i m * ! l l S l f i f l ~ 0 1 ~ h c . I , I , . , , ~ ~ ~ L,l11> nlll:,L I , ~ ~ ~ ~ i l l ~ i l \ ~ l illr dimcnsi trns 01 111t: 3v.i l;ib18'

C-'

B L C / U N D P / ~ ~ . 1 Rev. 1

page 48

Appendix D . 2

~ u ~ l ~ i l ~ v c * , i f t h e f e r m e n t e r L:, L O bc b t e ~ i l ~ z e d by a u t o ~ l a v l n ~ ; thadtrng u p t lme I J ~ h a l l a n Iruul- Lu l loucd by one h o u r a1 125- I J O ~ C iur an empty f e r m e n t e r ) .

L i l t . l c r ~ u e ~ ~ l e r i s jacketed, a t ~ d t a p Mater, t h e temperature LI; wlrich i > a u ~ o m a t l c a l l y r c g t l l a t e d , flows Lt~r t l l~gh i h e j a c k e t th ln rc l~y c , o n t r o l l i n ~ the t c i ~ l i r c r a t u r e of t h e c u l t u r e i t 1 t h c ! L ~ I I I L ~ I I L C ~ a s r e q u i r e d .

i r j r i lr lrt and a i r o u ~ l c ' t . media i n l e t and sampling tube 4rl. dbsi l :ncd f u r cnnnecLicln t u r u b t ~ e r o r , p r e f e r a b l y , h e a v y w a l l s i l i r . . un t u b i n g . 'The air f o r s c r a t l o r l of tlle Lcrm~nter n l u h ~ Ilv a t e r i l e . w h i c h i s a c l ~ l s l v e d by f l l c r a t i o r ~ t h r o u g h 8,rtitablc Ail t e r s , e.g. ''Line b'i l t e r l ; " rnenufac tu red by

.?l:(:X(li'UIW ( s c e Appertdix ~ . 2 3 , iter!, 2 2 ) . The n u t l e t a i r st10111d hc handlt!d ui tti c a r e ircr:ause I t i s c u a t a m i n a ~ c d :

i t s h u u l d pass through b u ~ t l u s c u r ~ t a i n r n b : d i s l n t e c t a r l ~ s a l~d/cir 0 i h r u u p h terminal i i l l ~ r s c 1 r : c t r i c s l ly heated t u about 2>CI C ,

'I-h(! i n t v r i l r r of t h e f c r m c t ~ ~ c r may Le viewcd t h r d ~ t ~ g h a v i e r i n ) : ] ~ u r t , a tuvghe~tud glass d i s c , 7 . 5 - 10 cm d i a l n e t e r , f i L L ~ L I i n a t h r e a d e d or h i ~ l L e d h u l d e r s e c u r e d w i L h 0 - r i n g s ; c d m t , n t t n u z ~ n u t h e used to SECIJKC' t h e v i e w i n g d i s c .

The inturivr 1 1 1 ' lhe fermenter car1 bc i l l u m i n a t e d t h r o u g h a tccund, s i m i l a r p o r t . 11 two pQrts are i n c l u d e d i n t h e d e s i g n L J I C ~ a r c usual Iy s i t e d L J I I O L I S ~ L L ' each o t h e r on a d i a m e t e r , t ' i ( 1 1 1 r on t h c l i d or s h o u l d e r uL the f e r m e n t e r .

'~ 'hc ~ I L W ul a i r i u r aerclticln m u s t be monitored alld a s i m p l e W-I? LJI d o i ~ r ) : t h i s i s t o i r ~ s e r t a dlEferent ia1 f low m e t e r i n thc a i r l tric. l l u t u r u Chc s t c r i l e f i l t u r . The d e s i g n of t h i s meter I , ! ; i i l~ l~ l r . ( s e e F i g 1 ) and i L c a n t ~ c c o n s t r u c t e d i n t h e Iabora tu rY

B E / U N D P / ~ ~ . 1 Rev. 1

page 49

Appendix D.2

F I G . l - DIFFERENTIAL F L U METER FOR AIR F L W

.. C.T.

-4

l e l \ g t h and bole o f the cap i l lary rubr are chosen

at t h e des i red l a t e o t flow ran be r e a d e s a l l y un the scale. s t r u r n e n ~ must he callbrared i n i i t r e s / m i t l u ~ e by b l o w i n g

nto a n i n v e r t e d LO-LiLre b n t l l , f i l l e d u i r h r a t e r , the neck i c h is imersed i n w a t e r .

$he remprature U L Lhe c u i t u r e i n t h e f e r n e n t e r is CO"-

trolled by r e g u l a t i n g t h e t c n p e r a t u r c of the Water ilowin$ &h he jacket . The s i m p l e way t o ach ieve this is to

L 111 the Mater i n l e t t o the iucke t an e l e c t r i c i m W r b i ~ ~

r , which i s a u t n r n a t i c a l l y switched off and on as coamnded c u n t a c ~ thermaneter or o t h e r s e n s i t i v e t h e r m o s t a t i c d e v i c e

m ) in ser l ed i n t o the thtrmomeier well of the fe rmente r .

page 50

Appendix D . 2

Mix ing and aeration i n rl fermenter is ext remely i m p o r t a n t in order to ub~ain good growth o f c e l l s . Speed o f mixing (rpm), r i z c and type uf impeller and rate o f aeration ( l i t r e / minute ) &real.ly a f f e c ~ growth and w s t be opcimised for each s i z e and design of fermenter.

"Vor tex mixing" as described by Chain e t a l . , BvLlec in u f the Wcrrld Hea l th Organization (19523. 3 , 7 3 Is o f t e n used. The impeller as sketched i n the accanpe~lyiirg Fig. 2 is located about 15 cm above the bot tan of the fermenfer, and i s d r i v e n by

d i r e c t d r i v e of a ithree-yhasu) e l e c t r i c m o t o r .

F I G , 2 . SKETCH OF '[WE IWELLER IN A PERMENTER

. . . plan

For pruduction of pertuns is i n fermentor% L L i s p o s s i b l e and ~ l . ~ n v e n i e n t t o use a mapnelic dr ive for s t i r r i n g ( sec for

Cameran 6- Gndfrcy , Magnetic Drives, Biocechnical and Bioeng. (1974) Symp. No 4 , 821-835) .

BLG/UNDP/~~.~ Rev. 1

page 5 1

* d i a g r a m t i ~ v i e w uf a t y p i c a l Eermencer, as used B L C u n n a u g h ~ Lali~)ratiries: L t d . , Toronto, Canada, is represented i n F i g . 3:

BLG/UPIDP/ 7 7.1 Rev. l

page 52

APPENDIX D. 3

ASSEMBLY OF A MTAFILTER

F u r u s e , the i n e t a f i l t e r i s pre-coated w i t h 1-2 kg of R y f l o S u p e r c e l . This is done by f i l l i n g the f i l t e r w i t h d i s t i l l e d water and pumping i n Lhe Hyf lo as a s l u r r y i n a f u r t h e r 40 litres of d i s t i l l e d w a t e r . The pump 011 t h e f i l t e t i s r un u n t i l Lhe f i l t r a t e i s c r y s t a l clear. The Hyf lo i s now t o t a l l y located on t h e f i l t r a t i o n s u r f a c e . The a i r - o u t l e t va lve on the f i l t e r i s then c losed and the f i l t r a t e allowed t u rtrn to waste . Once a l l t he s l u r r y i s i n t h e f i l t e r t he pump is t u r n e d off, care being t a k e n to e n s u r e t h a t no a i r i s drawn t h r o u g h the pump. The filter i s t hen blown d r y w i t h compressed a i r and 1s ready for u s e .

B U ; / ~ D P / ~ ~ .l. Rev. 1

page 53

APPENDIX D.&

FlLTRATION OF FERmNTER HARVEST

me manual has s u g g e s t e d f i l ~ r a t l o n rhrough m u l t i s h e e t f i l t e r s o r Meta f i l t e r s but the f o l l o w i n g method through f i l t e r candles under p r e s s u r e i s a l s o e f f e c ~ i v e .

1 A v e r t i c a l c y l i n d r i c a l pressure v e r s e 1 of 150-250 l i t res

. c a p a c i t y i s needed and an o l d t y p e a u ~ o c l a v e L, q u i t e suitable. h i n r e r t made of h i g h q u a l i t y b t a l n i e s s - s t e e l , p o l i s h e d un t h e inner s u r l d c e w i t h a l l welds a l s o h i g h l y polished, I S placed I n s i d e a n shown in the f i g u r e . The Iouer par t OI the i n s e r t

i s c o n s t r i c t e d and f o m s the a c t v a l L i l ~ r a t l o n chamber.

To clean t h e f i l t e r a f t e r u s e , t h e a i r s u p p l y is t u r n e d o f £ and water i n t r o d u c e d a t h i g h p r e s s u r e t h r o u g h t h e f i l t r a t e o u t - . . - - - - l e t until t h e f i l t e r F s full, i . e . t h e u n i t i s back-Elushed. Air a t 5-10 p s i i s t h e n bubbled th rough the f i l t r a t e i n o r d e r to d i s l o d g e the f i l t e r cake. A f t e r 10-15 minures t h e a i r s u p p l y i s t u r n e d off, t h e l i d of t he f i l t e r and t h e main d r a i n - v a i v c opened, and the d i s l o d g e d f i l t e r cake d i s c h a r g e d . The . . - body o f t h e f i l t e r i s c leaned w i t h a h i g h - p r e s s u r e v a t e r - s p r a y and w a t e r i s f i n a l l y c i r c u l a ~ e d th rough b o t h the pump and the f i l t e r . The pump is t h e n turned o f f , disman~led and t o g e t h e r with the filter body and t u b i n g c o n n e c t i o n s i s washed w i t h f r e s h , d i s t i l l e d water. b o t h t h e pump and f i l t e r a r e f i l l e d with 1% formal-saline and stored i n t h i s s o l u t i o n between Kun8.

The r i n g - p a c k s of t h e f i l t e r are removed from t h e f i l t e r and c l e a n e d annually: the r i n g s should be removed from t he r o d s d u r i n g c l e a n i n g . The r i n g - p a c k s a r e c l e a n e d by i m e r s i n g i n b o i l i n g water. Lor 3 0 m i n u t e s . Care should Le taken t o replace each r ing-pack s e c u r e l y a f t e r c l e a n i n g .

o u t L e t uf f i l t r a t e

pressure s a f e t y va lve

I n l e t of a i r u n d e r pressure

i n s e r t

h o l e for

s u p p o r t f o r c a n d l e s

f i l t e r c a n d l e s

~ u / m ~ / 7 7 . 1 Rev. 1

page 54

Appendix D.4

Four o r mnrP f i l t e r c a n d l e s . 8tLarh.d t o a rrosa-like s u p p o r t arc p l a c e d i n t h e chamber. S u i t a b l e c a n d l e 5 a r e Rerkeleld f i l t e r c a n d l e s , Type N (the s o u r c e of B e r k e l e l d f i l t e r c a n d l e s i s g i v e n i n Appendix D-23).

A p i e c e o i heavy wall r u b b e r t u b i n g i s u s e d t o c o n n e c t t h e c a n d l e s t o a m a n i f o I d ( g l a s s o r s t a i n l e s s - s t e e l ) , which i n t u r n i s connec ted by a s i n g l e p i e c e o f t u b i n g t o t h e o u t l e t .

This o u t l e t c a n a l s o be l o c a t e d on t h e s i d e w a l l o f t h e v e s s e l . I f t S l o c a t e d on t h e l i d (as i n the figure), t h e p i e c e o f t u b i n g c o n n e c t i n g i t t o t h e o u t l e t s h o u l d be l o n g e r ( a s r e p r e s e n t e d in t h e f i g u r e ) , so a s t o a l l o w f o r t h e o p e n i n g of t h e l i d of t h e ves se I -

Procedure: t h e b a c t e r i a l s l udge i s poured i n t o t h e i n n e r j a c k e t , some f i l t e r a i d i s added ( e . g .

2- .3 t of H y f l v Super C e l l from Johns h n v i l l e C o . ) , t h e contents mixed by stirring, the v e s a e l c o v e r e d and t h e a i r pressure a p p l i e d .

I n i t i a l l y a Low p r e a b u r e of 0 .1 -0 .2 bar (kp/cm2) s h o u l d be u s e d and t h e p r e s s u r e is s l o w l y i n c r e a s e d t o i .0-1.5 b a r (kp/cm2: o r mare .

The f i l t r a t e c a n be c o l l e c t e d s e p a r a ~ e l y , o r t h e o u t l e t f u r t h e f i l t r a t e on t h e p r e s s u r e v e s s e l can be c o n n e c t e d d i r e c t l y 10 a s t e r i l e f i l t r a t i o n u n i t , s o t h a t c l a r i f i c a t i o n a n d s t e r i l i z a t i o n a r e pe r fo rmed s i m u l t a n e o u s I y .

A f l o w r a t e of a b o u t 63 l i t r e s i n one t o two h o u r s can be a c h i e v e d .

The d imens ions o f t h e l o v e r parC of the i n s e r t ( f i l t r a t i o n chamber ) , t h e number of c a n d l e s and t h e amount o f f i l t e r a i d s h o u l d be a d j u s t e d so t h a t when ( h e f i l t r a t i o n i s f i n i s h e d , t h e chamber i s a l m o s t f i l l e d w i t h b a c t e r i a l mrr and H y l l o .

T t i s i m p o r t a n t t o ensure t h a t t h e whole i n n e r v e s s e l with i t 6 c o n t e n t s a r e 8 t e r i L e before b e i n g discarded.

l l l t r e f i l t r a t i o n is c a r r i e d o u t by mean3 of c n l l o d i o n mem- b r a n e s s u p p o r t e d on fF lLer c a n d l e s .

( a ) p r e p a r a t i o n o f p a r l o d i o n s o l u t i o n

Tuo thuusand f o u r hundred mlllili t r e s of g l a c i a l a c e L i c a c l d a r e poured i n t o a 10-Litre b o t t l e which is p l a c e d on a r o c k i n g

o

a p p a r t t u s ( o r rocked m a n u a l l y ) p r e f e r a b l y i n a n i n c u b a t o r a t 35 C t o 38 C t o speed up the d i a s o l u t i o n of p a r l o d i o n .

. . .. .

From t i m e t o t i m e p i e c e s of p a r l o d i o n ( M a l l i n c k r o d t p u r i f i e d

p y r o x y l i n ) ere rddcd t o 1be aretic a c i d m d r o c k i n g c o n t i n u e d u n t i l a b o u r 210 g has been added .

A r u b b e r s t o p p e r L r Lied i n t o

t h e neck uf the b n t f l e , m d rocking i s can~inued for s e v e r a l d a y s

5: until the p s r l o d l o n is dissolved t o y i e l d a c l e a r , v i s c o u s s o l u - t i o n .

( b ) P r e p a r a t i o n of c a n d l e s

Ceramic f i l t e r c a n d l e s ( e . g . BerkefeLd f i l t e r c and le s , t y p e a m e t e r and 2 5 cm long, f rom t h e f i r m B e r k e f e l d

e r , 3100 C e l l e , F e d e r a l R e p u b l i c of ~ e r r m n ~ ) are c l e a n e d w i t h v e r y f i n e s a n d p a p e r , a f t e r which t h e c e r r m i c p a r t s h o u l d

b e touc.hed by hend o t h e w i s e the membrane w i l l n o t stick e c t l y t o t h e g r e a s y s u r f a c e p roduced by c o n t a c t w i t h h a n d s .

' h e p a r l o d i o n s o l u t i o n i s poured i n t o c y l i n d e r s , and a f t e r

I.. waiting f o r a t a 30 m i n u t e s u n t i l t h e s m a l l e s t a i r b u b b l e s have d i s a p p e a r e d t h e c a n d l e i s dipped i n t o the p a r l o d i o n s o l u -

for one i o t w o m i n u t e s : s s ~ l L v a c u u m ~ £ 2 > - 3 0 c m H g i s

e m e c a n d l e i s t h e n taken o u t of the

ned upside down e n d by g e n t l y r o c k i n g w h i l e s t i l l the p a r l o d i o n s o l u r i o n i s d i s t r i b u t e d e v m l y Qn

f3urfacc of t h e c m d l e , The c a n d l e i s thecl ~LTIIWKSC~ i n cold

.' t*pvater and left u n t i l the whole b a ~ c h of candles , up t o 30 or n prepared. The immersion i n Water results i n

f o r I r @ ~ i ~ ~ O E a collodiao membrane with ~ ~ l t r a f i l t r a t i n n

BLG/UNDP/~~. 1 Rev. 1

page 36

Appendix D.5

Bs/~m~/77.1 Rev- l

page 57

Appendix Q.5 -

The c a n d l e s are now washed f r e e from a c e t i c a c i d . This l $ done in the same apparatus a s that used f o r t h e u l t r a f i l t r a t i o n except t h a t i n the washing cycle pure water i s used i n s t e a d of toxoid ( s e e f i g u r e ) . The washing is continued for a few days , d n ~ i l the u l t r a f i l t r a t e does n o t show any a c i d i t y ( a s measured by indicator paper or pH meter).

[ C ) U l t r a f i l t r a t i o n procedure

[ I n the d e s c r i p t i o n the numbers r e f e r to the numbers shown i n the f i g u r e , )

The u l t r a f i l t r a t i u n c e l l (1) i s a c o n t a i n e r oi any shape IA .square c r o s s - s e c t i o n of 2 0 x 20 cm, heighL 3 0 - 3 5 cm is su i cab le l made hf g las s wt.tich i s p r e f e r r e d t o s t a i n l e s s - s t e e l f o r observing ille concentrated coxoid. The c e l l i s covered w i t h a s t a i n l e s s - s t e e l support plate ( 2 1 , carrying he candles 3 . O n e c a n d l e is f i x e d i n r u e a c h hole i n t h e support p l a t e . For the c e l l of Lhe a b w r a i z e , the plate has n i n e l ~ o l e s su t h a t

. . the c e l l consists of e "battery" of nine c a n d l e s . I n the c e l l there i s a l s o a g lass or s t a i n l e s s - s t e e l tube thruugh r h i c h the roxaid f lows i n c c the c e l l from a l a rge " h r i 9 t t c flask" ( 5 1 through t h e t r ~ h e ( 6 ) which reaches t o the b ~ t t ~ m of the f lask. This f l a s k l a a l s o f i t t c d w i t h a ~ u b e ( 7 ) open t b t he atmosphere through which [he a i r bubbles i n t o the flask as the i a x o i d flows out. When stoppcred tightly (8) the lower end o f r t i i s tubc ( 7 1 d e t e r m i n e s t h e l e v e l o f Lhe t o x o i d i n t h e c e l l . From the c e l l ( 1 ) rhe u l t r a f i l t r a t e passes i l l to the c d u d l e s ( 3 ) and i s then c o l l e c t e d i n the c a l l e c ~ i n ~ f l a sk (10) <usuaLly a 1 0 - l i t r e b o t t l e ) v i a tile u m u i l o l d (9) and the callec~ing cube. Another t u b e ( 1 1 ) l c a d s from t h i s hottte i n t o a "vacuuo~ can-

t a i n e t " (12) which may b e of any kind of conta iner l a r g r ancl s trong enough ( 1 0 0 - 2 0 0 l i t r e s ) t o hold a p a r t i a l vacuum 0 1 25- 30 cm Hg Ior sume tirnc, e . g . o v e r n i g h t . From this conLainer (12 ) a second cube (13) leads t o a pump [ a n ordinary water j e t pump is s u i t a b l e ) w h i l s t a t h i r d ~ u b e 114) is connected to a Pcrcury manomecec ( 1 5 ) or similar d e v i c e t o measure the partial vacuum i n the s y s t e m .

B L G / ~ P / ~ ? . 1 ~ e v , 1 page 58

Appendix D.5

The Capacity f o r u l t r a f i l t r a t i o n i s dependent on t h e number Of candles ad the rate of f i l t r a t i o n which is approximately

litre per p e r 24 h o u r s . I t is u s u a l t o have several batteries wi th a C ~ ~ o t ~ M a r l o t t e f l a s k ( 5 ) and vacuum container (12) but w i t h = p e r a t e c o l l e c t i n g f l a s k s (10). I f a l l f l a s k s

container$ a r e a d e q u a t e l y S t o p p e r e d i t is to

R ! x ; ~ ' u N u P / ~ ~ - 1 Rev . page 59

Appendix D.6

few hoLlrs t h e p r e c i p i t a t e s a r e removed by centrifugation'

yedissolved in and t h e LE v a l u e s are determined by f loccu-

lation (Ramun r e a c t i o n ) a f t e r d i a l y s i s C a e e A ~ ~ e r l d i x D - 7 ) - A

curve i s dram { s e e f ~ ~ u r e ) , i n which t h e percentage toxoid a g a i n s t t h e amonium =On-

centration, hi^ c u r v e will be S-shaped , w i t h a small evacuate [he sys tem once i n 24 h o u r s , and t h e n t o close the ' l 6 ) ' this ray u l t r a f i l t r a t i o n w i l l p roceed a u r o o n t i c e ~ l y day and night- Once o r t w i c e i n e a c h 24 hours the

(l0) i s opened Without b r e a k i n g t h e vacuum in the reminder the system by c l o s i n g t h e v a l v e s or clamps (17) . =he ultra-

is t e s t e d f o r t r a c e s o f p r o t e i n w i t h t r i ch loroace t ic or sulfo-aal 'cyl ic a c i d and i f no p r o t e i n is p r e s e n t , the hl t ra- "ltrate is discarded. I f p r o t e i n i a found, sny defec t ive candlesmust b e l o c a t e d , r e p l a c e d a n d t h e u l r r a f f ~ t r a t e r e c y c l e d . A

is d e t e c t e d b y i s o l a t i n g e a c h c a n d l e , t.kilg a

,., . part, or a s l i g h t bend, usually i n t h e region 25-2RYn

.: 8mmonium eulfate. t h i s c u r v e t h e r a n g e O f ammonium "lfate

;. concent ra t ions p r e c i p i t a t i n g most of t h e t o x o i d can be deduced. l t has been found t h a t t h e t o r o i d which precipi tates a t a low

- , . ammonium s u l f a t e c o n c e n t r a t i o n M Y be p o o r l y immunogenic. I t is advisab le , ~ h ~ r e f o r e , t o p e r f o r m the f i r s t precipitation a t

. . . . .., an ammonium l p h a t e c o n c e n t r a t i o n which w i l l remove ahou 30% 'I..

, c , , the roroid, r a s u 1 . d i n terns O £ ~ f / " l . '1"" f i r s t preCiPi-

~ : , , _ ~ . t e i r c e n r r i i u g o d u f f * n d d i . c a r d e d . A s e c o n d q u a n t i t y o f , ., .!;. anmranium s u l f a t e i s then added t o t h e toxoid calc'* ..v< l i q u i d from i n s i d e and t e s t i n g for p r o t e i n , The <* l e t e d fro. the c u r v e i n o r d e r t o produce about precipita-

t"xoid i s r e c o v e r e d from t h e c e l l ( 1 ) in there tion. ~h~~~ C o n c e n t r a t i o n s of ammonium s u l f a t e be about i s a 5 0 - f o l d or g r e a t e r c o n c e n t r a t i o n , ? ? < 27% for t h e f i r s t p r e c i p i t a t e a n d 37% f o r t h e second precipitate'

. ..e. lhe Whole procedure i s d i f f i c u ~ t t o perform a s e p t i c a l l y . is a d v i s s b l e t o adjust t h e pH a f t e r tile addition the

To reduce c a n t a d n a t i o n , t h i o m e r s a l i s added ( l : 1 0 000) and the L U ~ s u l f a t ~ t o 6.5-7.0 on e a c h o c c a s i o n - Using t h i s p ro-

tOxOid mixed *nd c u v e r e d w i t h a layer of boluene, * final of p u r i f i e d t o x o i d of a b o u t 50-bUF/. be @ . *pparatus i s p laced i n a c o l d roam at -r 5 ::-.j C . I lsual ly 500 .:' obta ined . F i n a l l y , t h e m r i m n i u m s u l f a t e is removed by

litres of t i l t r a t e can be reduced t o a ,,f l0 l i t res .' d i a l y s i n g the r e d i s s o l v e d second p r e c i p i t a t e ln ce l lophane bags

a b a t t e r y of 27 c a n d l e s in 12 to 14 days. b e s against c h l o r i n a t e d p o t a b l e c o l d running tapwater Or

d i s t i l l e d w a t e r .

~ ~ ~ l l ~ ~ c i ~ ~ of t h i s p u r i f i c a t i o n procedure the A P P E N D I X D. 6 urcg gives r o x u i d r w i t h a p u r i t y of 12m-1400 Lf/mg protein

0pn, ~ . , i ~ i s n o t a v e r y h i g h p u r i t y , b u t s \ l f f ic ient f o r PIIKTFICATI ON ~p ~ 0 x 0 1 ~ ueing good vsccines. w i t h f e r m e n t e r c u l t u r e s , which have

gher i n i t i a l p u r i t y of t h e c r u d e t o x o i d , the ethod can d p r o d u c t s w i t h 2000-3000 l f G m g p r o t e i n n i t r o g e n *

Optimum c o n c e n t r a t i nn o f a m o n i u m su l fa te for precipi- t a t i n g

and s p e c i f i c t o x ~ i d s h o u l d be determined by a As t h i s p u r i f i c a t i o n p r o c e d u r e c a n n o t be p e r f v r m e d under p i t o t e x p e r i a e n l - Ten m i l l i l i t r e s of the t o x o i d are pipetied c t l y a s e p t i c c o n d i t i o n s i t i s e s s e n t i a l t h a t t h e f inal seven t u b e s and i n c r e a s i n g q u a n t i t i e s o f p u r e

oduct d i a l y s i s be passed ~ h r o u g h a f i L c e r f o r ammonium ~ u l f a t e in t h e r a n g e of 1 9 - ? ? - 2 5 - 2 1 ( - 3 ~ - ~ 4 - 37-40 grams

I t r a t i o n and a added a s used i n t h e f inal per IC)(I m 1 t u x u i d a r e addcd t o i n d i v i d l l e l Lut ,es , A f t e r a ccine . ; u s u a l l y 0.01'1~ t h i o ~ ~ e r s a i ) .

B L G / U ~ D P ~ ~ 7 . 1 Rev. 1

page 60

Appendix D. 6

B L G / U N D P / ~ ~ . ~ R e v . I

page 6 1

OF p R ~ c ~ ~ ~ ~ ~ ~ ~ ~ ~ OF mxoro mo.Im suLmre A laboratory reference p r e p a r a t i o n which h a s been c a l i b r a t e d

~ f / m l

.

a t the I n t e r n a t i o n a l F l o c c u l a t i o n S t a n d a r d f o r D i p h t h e r i a is

I ed t o a working c o n c e n t r a t i o n o f 100 L£ u n i t s p e r ml.

A range of v~lumes of t h i s s o l u t i o n is d i spensed inLa a o f 7 cm 1 cm flocculatiun cubes. S u f f i c i e n t S a l i n e

e t o g ive a c o n e t a n t t o t r l volume of 1 m]. Sccnnd

test sample i s d i l u t e d t o g ive a n expccco r l c o c c e n t r a ' . i o ~ ~ of p r e c i p i t a t e

r i m t e l y SO L f u n i t s per ml, and 1 m 1 a l i q u o t s of t h i s l u t i o n are dispensed i n t o e a c h of the tubes contairlitlb: a n t i -

sin. The t u b e s are p l a c e d i n a v a t e r bath a t 5oUc, and BBrved a t r e g u l a r i n t e r v a l s fo r the f i r a t appearance of occules. Tfiis w i l l r e q u i r e the use of a m a g d i f y i n ~ l e n s and

rong i l l u m l d a t i o n .

The f i r s t , second and t h i r d m i x t u r e s c 0 f l u c c u l a t c a rc thc time taken f o r Lhe f i r s t i l o c c u l a t i o n

The f i r s t t u b e t o f l o c ~ u l a c a i s t h e one w h i c h c a h t a i n s t h e

n t O f a n t i t o x i n c l o s e s t i n e q u i v a l e n c e t o clie amount of a n t i - i n the sample, and for most p u r p o s e s the l,£ c o n t e n t of t h i s

First e can be taken a s t h e 1,f value of t h e s a m p l e + p r e c i p i t a t e

-. C o n c e t l ~ r a t i ~ n of a m o n i u m Sul ' f a te The r i m taken fo r the f i r s t tube t o f l o c c u l a t e ( ~ f ) is a

i n d i c a t o r of the q u a l i t y o f t h e a n t i g e n . I f a t g i v e n t r a t u r e and c o n c e n t r a t i o n of toxoid and a n t i t o x i n the K f

is i n c r e a s e d , compared w i t h n o r w l , t h i s i n d i c a t e s that rutigen has been damaged and is less Likely t o produce good

APPENDIX D. 7

DETERH N A T I O N ~ i : ~ f / ~ ~

locc"at ion v a l u e ( ~ f ) of a toxin o r toxoid i s lined by the number o f u n i t s of a n t i t o x i n

when lit' the produces B n o p t i m l l y ~ ~ o c c u l a t i n g mixcur, , ,

, .*

R L C / ~ ~ N D P / ~ ~ . 1 Rev. 1

page 62

Appendix D.7

Tube

A n t i t o x i n added (tf u n i t s ) 45 50 5 5 60 65 7 0

A n t i t o x i n added ( m l ) 0.45 0 . 5 0.55 0 .6 0.65 0,7

S a l i n e added (ml) 0.55 0.5 0.45 0 . 4 0 .35 0 . 3

D i l u t e d sample added 1.0 1 , O 1.0 1.0 1.0 1.0

I f i n t h i s example the first tube t o f l o c c u l a t e i s cube D t h e n the Lf v a l u e of the d i l u t e d sample 1 s 60 LE u n i t s / m l . NuLe, however, t h a t if the f i rsL t u b e t o f l o c c u l a t e i s Lube A or i.ul)e F ihis d o e s [lot i n d i c a t e e q u i v a l e n c e a L tha t level. L t would be n e c e s s a r y t o perfurm a r e p e a t t e s t u s i n g e i t h e r a d i f f e r e n t d i l u t i o n of Lest sample o r s e l e c t i n g a d i f f e r e n t r a n g e of d o s e s of reference a n t i t o x i n .

More p r e c i s i o n c a n be o b t a i n e d by making a l l o w a n c e f o r t h e sequence of f l o c c u l a t i o n a f t e r t h e f i r s t t u b e . When t h e d o s e i n t e r v a l f o r r e f e r e n c e a n t i t o x i n i s 10% a s i n t h e example above, t h e t o x i n e q u i v a l e n c e v a l u e i s t a k e n t o l i e between t h e f i r s t a n d second t u b e s t o f l o c c u l a t e , s o t h a t i f t h e second t u b e r e p r e s e n t s a h i g h e r v a l u e t h a n t h e E i r s t 2 , 5 % is added t o t h e e s t i m t e of f l o c c u l a t i o n v a l u e , and i f i t has a lower v a l u e 2 . 5 % i s s u h l r a c t e d . Thus, i n Lhe example q u o t e d , i f t h e second Lubr. t o f l o c c u l a l e had been t c ~ h e E t h e f i n a l value For the diluted s a m p l e would be 61, whereas i f t h c second t u b e L u E f o c c u l a t e was tube C Lhe f i n a l v a l u e would be 59. The t e s t should be per formed Ln d u p l i c a t e . The r e s u l t s a r e a c c e p t a b l e p r o v l d c d thaL t he r e p l i c a t e t e s t s do n o 1 d i f f e r by mote t h a n one t u b e .

BLG/UNDP/~T.~ Rev. 1

page 63

Appendix D . 7 -

I f t h e r e is no indicaiion of t h e e x p e c t e d t iocculat ion v a l u e of thc sample a v a i l a b l e i t is advisable t o o b t a i n a rough e s t i a a L e by use of a w i d e r r a n g e o f antiloxin c o n t e n t i n t h e tube b e f o r e p r o c e e d i n g t o t h e I l ~ ~ a l t e s t .

Example

A n t i t o x i n con t en t I c l f l 20 30 45 7 0 100 150

The l e v e l of t o x o i d a n d a n t i t o x i n c o n c e n t r a t i o n i n t h e t e s t m y be v a r i e d b u i t h i s w i l l marked ly affect- t h e t ime of f l o c c u - Tation s o t hat a t v e r y IOU l e v e l s t h e t e s t w i l l take t o o long, whils : a t h igh c o n c e n t r a t i o n t h e onset o f f l o c c u l a t i o n a y be so rapid a s to make i t d i f f i c u l t t o d l a t i r l g u i s h tlre f i r s t and s e c o n d t u h e s t o f l o c c u l a t e .

L .

I F l o c c u l a t i o n t i m e s of 30 t u 1 2 0 m i n u t e s a r e g e n e r a l l y m o s t convenient.

i Blend f l o c c u l a t i o n

ii The measurement of v e r y Low c o n c e n t r a t i o n s of t o x o i d i s b e s t

b . c a r r i e d out by t h e method of b l e n d f l o c c u l a t i o n . T h i s can be

Performed i n many ways b u t a l w a y s i n v o l v e s compar i son o f t h e Lf v a l u e oi a known toxoid a n d t h a t of a m i x t u r e of t h e sample w i t h

. t h a t t oxo id . k ' : i .

ii :.- . _Examplc ,J.. ~.

. ,., , , , i . ,., The 1,1 c o n t e n t L f .g known t o x o i d 150 ~ l / r s ~ ) i s checked w i t h

. ah a d d i t i o n a l 1 tit s a l i n e added to cacih t u b e . , : 8 '

' i ;; i a . '";':; ..

BLG/UNDP/~~. 1 Rev. 1

page 64

Appendix D . 7

Anti t ox in added (LE u n i t s ) 45 50 55 ' 60 65 70

A n t i t o x i n a d d e d ( m l ) 0 .45 0 . 5 0 .55 0 .6 0.65 0 .7

S a l i n e added ( m l ) 1.55 1 .5 1.45 1 .4 1.35 1.3

Known toxoid added (ml) 1 .0 1.0 i .0 1.0 1 .0 1 . 0

F l o c c u l a t i o n sequence 2 1 3

Est imated value of known tox in 49 1.f uni ts /ml . , At the same time f l o c c u l a t e known toxoid 50 ~ f / rn l with 1 m1 of unknown toxoid added t o each tube.

Tube A - H - C !! E - F - Antitoxin added

(Lf u n i t s ) 45 50 55 60 6 5 70

Antitoxin added (ml) 0.45 0.5 0.55 0 . 6 0.65 0.7

S a l i n e added (ml) 0.55 0 .5 D.45 0.4 0 .35 0 . 3

Known toxold (m11 1.c 1 .0 1 .0 i . 0 1.0 1.0

Unknown toxoid ( m l ) 1 .0 1 .0 1.0 1.0 1 . 0 1 . 0

F l o c c u l a t i o n sequence sequence 3 I 2

Estimated valuc f o r known toxoid I unknovn toxoid 56 L f un i t s

Con t r ihu t ion of unknown toxuid 56 - 49 = 7 L I u n i t s

B L G / U N D P / ~ ~ . ~ Rev. 1

page 65

Appendix D. 7

When a k n m toxoid and an unknown toxoid a r e f l o c c u l a t e d toge the r t h e mixture w i l l f l o c c u l a t e a s t h e sum of t h e i r va lues i f they a r e h m o g e n e w e . If d i f f e r e n t zones a r e involved they

, w i l l produce a confused p a t t e r n wi th two f l o c c u l a t i o n maxima.

- . < ~ . * .L*. . ~

..: , .+ - APPENDIX D.8

..... >.~

TOTAL COMBINING PWER TEST (TCP)

A s u i t a b l e r e fe rence toxoid which i s known t o produce an t e response i n l a b o r a t o r y animals as measured by t h e

y t e s t when used i n vacc ine sbould'be s e l e c t e d f o r use

The t e s t sample i n d i l u t e d t o a concen t ra t ion of 2 ~ ~ / m l . Volumes of 0.3, 0.36, 0.45, 0.55, 0.65 and 0.8 m 1 r e s p e c t i v e l y

xoid a r e dispensed i n t o a s e r i e s of n ix l a r g e t e s t tubes. A s i m i l a r s e r i e s of doses of t h e l abora to ry r e fe rence toxoid Le dispensed i n t o a second s e r i e s of tubes . A volume

.'- of 0.5 m1 of a s o l u t i o n con ta in ing 4 ru /ml of d i p h t h e r i a a n t i - i s added t o ea-h tube and t h e tubes a r e incubated r 30 minutes. A volume o f 0.5 m1 of a s o l u t i o n

u n i t s equivalent /ml of d i p h t h e r i ~ Loxin i s added and these a r e incubated er 20-25 C f o r 60 minutes .

e n t s of each tube i s d i l u t e d t o a volume of 2 0 m 1 l y 0 . 2 m 1 of t h e d i l u t e d mixcure i s l n j e c t e d

t r a d e m a l l y i n t o t h e f l a n k o f a shaved and d e p i l a t e d normal inea-pig. S i m i l a r l y , 0 . 2 m1 doses from rhe o the r tubes

i n t o o t h e r s i t e s on t h e same o r s i m i l a r guinee-

1 ; , i I B L G / W P / ~ ~ . 1 Rev. 1 S l I/ , page 66

' 1 l i I l Appendix D . 8 1 / (

The s i t e s are examined a t 48 o r 72 h o u r s a f t e r i n j e c t i o n f o r t h e p r e s e n c e of diphtherla ery theme. A m i x t u r e which c a u s e s e ry thema c o n t a i n s more t h a n 1 u n i t e q u i v a l e n t o f d i p h t h e r i a t o x o i d . A m i x t u r e which f a i l s to cause erythema c o n t a i n s less t h a n 1 u n i t e q u i v a l e n t . The end-poin t r e p r e s e n t i n g e x a c t e q u i - v a l e n c e is marked by m i n i m 1 erythema a t the s i te of i n j e c t i o n .

The test i s repeated u s i n g volumes o f t h e same i n i t i a l d i l u t i o n s e l e c t e d from t h e range 0 . 2 7 , 0 . 3 3 , $ 0 . 4 , 0.5, 0,6, 0.7 and 0 . 9 h1 s o t h a t the end-poin t of t h e f i r s t t e s t f a l l s i n t h e middle o f t h e range s e l e c t e d for t h e second t e s t . I f the r e p e a t t e s t c o n f i r m s v i t h the L i r s t c e s t , t h e c o m b i n e d r e s u l t mey be accepted. I f t h e test disagrees, f u r t h e r r e p e a t s m u s t be c a r r i e d o u t . The r a t i o of g f o r t he t e s t material s h o u l d be

L f n o t l e s s t h a n 80% of t h a t for the l a b o r a t o r y r e f e r e n c e t o x o i d .

The TCP of t h e t o x o i d C i s t h e r e c i p r o c a l of t h e vo lume o f t o x o l d d i l u t i o n found t o be e q u i v a l e n t t o 1 LU.

STABL LT TY AND REVERSAL TEST

T h e r e a r e two t e s t s f o r d e t e c t i n g r e v e r s a l of t o x o i d .

A. I n t r a d e m l t e s t

A s u f f i c i e n t amount o f the bulk t o x o i d i s d i l u t e d s o a s t o

g i v e 260 m1 o f a s o l u t i o n c u n t a i n i n g 80 Lf u n i t s / m l . me s o l u - t i o n i s d i v i d e d into six lots each of 40 m1 and {he r e s idue t g s t e d hy f ~ o c c u 1 a r : o n . ?do of t h e 40 m1 l o t s a re stored a t

0 G C , t w o a t 20 C a n d two a t 3 5 ' ~ - One sample storcd a t each t e m p e r a E u r e i s removed a f t e r i u u r r e e k s and a n o ~ h e r a f t e r 1 2 weeks .

B U / I R J D P / ~ ~ . 1 Rev. 1 page 67

The f o l l o w i n g tests are a p p l i e d t o each sample:

(a) A volume of 0 . 2 m 1 of t h e sample i s i n j e c t e d i n t r a d e r m a l l y i n t o t h e s k i n of a shaved d e p i l a t e d g u i n e a - p i g , The s i t e of

i n j e c t i o n i i s o b s e r v e d 48 h o u r s l a t e r . I f erythelna i s s e e n i t s

s p e c i f i c i t y i s conf i rmed by i n j e c t i n g a f u r t h e r 0 . 2 ntl of sample i n t o a n o t h e r norm41 guinea-pig and i n j e c t i n % 0 . 2 m1 of a m i x t u r e p r e p a r e d by a d d i n g 80 u n i t s of d i p h t h e r i a antitoxin t o I m1 of eltmple. T f ery thema i a seen a t t h e s i t e o f i n j e c t i o n of t h e sample above b u t n o t e t t he s i t e of i n j e c t i o n of t h e m i x t u r e o f t h e sample and a n t i t o x i n , t h i s i s evidence of t e v e r s i o n of t o x i c i t y and t h e bulk t o x o i d mist be r e t u r n e d fat further d e t o x i f i c a t i o n and tesubmittod t o t h e above test p r o c e d u r e .

cb) Each sample i s t e s t e d f o r L f c o n t e n t by t h e f l o c c u l a t i o n

tea t . Toxoid i s s a t i s f a c t o r y p r o v i d e d :

{ l ) t h e L f c o n t e n t of t h e sample s t o r e d f o r 12 weeks a t 3 7 ' ~ i a n o t less t h a n 60X oE t h e Lf c o n t e n t of t h e i n i t i a l sample;

( 2 ) 6 h e L£ c o n t e n t of t h e sample s t o r e d for 12 weekn at 20 C i y n o t l e s s t h a n 80% of t h e i n i t i a l sample; end

(3) o t h e Lf c o n t e n t of t h e sample stored for 12 weeks a t 4 C is not s i g n i f i c a n t l y l e s s t h a n t h e i n i t i a l sample.

' 8 , Subcutaneous t e s t

A sample of p u r i f l e d t o x o i d i s d i l u t e d i n s a l i n e c o n t a i n i n g -01% t h i o m e r s a l t o g i v e 3 0 0 m 1 of 10 LE p e r m1 toxoid& 0

. &kiquots of 100 m1 of the d i l u t e d t o x o i d a r e h e l d a t 4 C , 20 C l _ P d 3 5 ' ~ for sln weeks. Five m i l l i l i ~ r e s of each sample a r e

njected subcuLeneously i n t o each o f five g u i n e a - p i g s weigh ing : b c t ~ c c n 280 and 300 g , 2 . 5 m1 fin e a c h s i d e of the abdomen.

BLC/UNI~P/~ 7 , l Rev. L

page 68

Appendix D.9

The p i g s a c e weighed on t h e first, t h i r d and s e v e n t h d a y f o l t o w i n g i n j e c t i o n , t h e n weekly f o r a further t h r e e weeks.

The g u i n e a - p i g s are examined d a i l y i o r a n y e v i d e n c e of d i p h t h e r i a i n t o x i c a - L i o n ,

C r i t e r i a fo r a c c e p t e n c e

The animaLs s h o u l d show no s i g n s of d i p h t h e r i a i n t o x i c a t i o n .

IDENTITY TEST

The i d e n t i t y t e s t s on a b a t c h of t o x o i d can be c a r r i e d o u t by b o t h i n vitro end i n v i v o methods . Although i t i s a s i m p l e p r o - c e d u r e t o e s t a b l i s h t h e i d e n t i t y of f l u i d t o x o i d by a f l o c c u l a t i o n t e s t , i t i s n e c e s s a r y , i n t h e case of e d s o r b e d t o x o i d s , t o d i s s o l v e t h e a d s o r b e n t ( a d j u v a n ~ ) by he a d d i t i o n of sodium c i t r s t e .

For f l u i d t o x o l d , a d i p h t h e r i a a n ~ i t o x i n i s d i l u t e d so as t o c o n t a i n 2 0 I U p e r ml . increasing volumes o f 0.3, 0 . 4 , 0.5, 0 . 6 , 0 . 7 , 0.8 m 1 a r e p i p e t t e d i n t o a s e r i e s o f t u b e s ;

t h e volumes a r e m d e u p with normal saline t o L m1 and 1 m 1 of f l u i d t o x o i d , d i l u t e d t o a p p r o x i w t e l y 1 0 ~f/ml 1 s added t o e a c h o f the tubes. The t u b e s are p l a c e d i n a w a t e r b a t h aL 50'~ and o b s e r v e d con- t i n u o u s l y . The tube c o n t a i n i n g 0.5 m 1 of antitoxin or t h e tube on e i t h e r s i d e of i t m y slbow f l o c c u l a ~ i o n and s u c h f l o c c ~ ~ l a t i o r ~ e s t a b l i s h e s t h e i d e n t i t y vf the producc .

F o r an a d s o r b e d t o x o i d a b o u t 0 . 5 g of sodium c i t r a t e i s added t o 10 m 1 a £ t h e a d s o r b e d t o x o i d . The m i x t u r e i s i n c u b a t e d 0 a t 37 C f o r one o r two d a y s t a d i s s o l v e .

A f t e r t h e aluminium s a l t h a s d i s s o l v e d t h e f l o c c u l a t i o n t e s t s i m i l a r LIJ t h e one

s t a t e d above i s u s e d . Tlre f l o c c u l a t i o n o c c u r s i n t h e same wdy though i~ may t a k e a l o n g e r t i m e .

B L G / U N D P ; ' ~ ~ . 1 R&. 1

p a g e 69

Appendix D . 10

I n t h e i n v i v o t e s t a s u i t a b l e d o s e of t o x o i d i s in j ec ted i n t o g u i n e a - p i g s and t he presence of a n t i t o x i n In the g u i n e a - p i g b lood d e m o n s t r a t e d by c a r r y i n g o u t n p a s s i r e - p r o L e c t i o n t e s t i n guinea- p i g s k i n or by i n t r a d e r m a l c h a l l e n g e o f chc t e s t a n i m a l .

The

g u i n e a - p i g u s e d i n the t e s t for f recdm from abau tmal t o x i c i t y w i l l have r e c e i v e d a s u i t a b l e d o s e of t o x o i d , b u t t h e response should not be a s s e s s e d u n t i l 14 d a y s a f t e r i n j e c t i o n .

POTENCY TESTING OF D1 PHTHERIA TUXOlD

I n p r o c e s s t e s t i n q

A f u l l pu tency t e s t o f d i p h t h e r i a toxoid r e q u i r e s r a t h e r a large number 01 g u i n e a - p i g s and i n many c o u n t r i e s i t w i l l be import an^ t o economize as f a r as laboratury a n i m a l s a r e concerned . For ihe f i n a l b u l k of a v a c c i n e i t i s n e c e s s a r y t o p e r f o m a f u l l t e s t a s d e s c r i b e d below b u t economy i n g u i n e a - p i g s i h p o s s i b l e by e i t h e r p r o d u c i n g l a r g e b a t c h e s of v a c c i n e o r by combialng the f i n a l p o t e n c y tes t f o r a number of b a t c h e s .

For t h e p o t e n c y t e s t s , r e q u i r e d as " i n p r o c e s s " tests, a s m a l l e r number of g u i n e a - p i g s may be u s e d which were j u s t s u £ E i c i e n t t o g ive a g e n e r a l i n d i c a t i o n t h a t t h e m a t e r l a l h a s a t least a r e a s o n a b l e p o t e n c y . Bulk f l u i d toxoids could be screened

u s i n g few g u i n e a - p i g s by a method such as Lhat used in t h e U n i t e d S t a t e s o f America: 10 guinea-pig$ of 250-300 g ere i n j e c -

t e d w i t h n o t more t h a n o n e - s i x t h o f t h e cotal human immunizing dose and n o t more than s i x weeks l a t e r the a n i m a l s a r e c h a l l e n g e d w i t h 10 l e t h a l c h a l l e n g e d o s e s of d i p h t h e r i a t o x i n . At l e a s t 80%

of t h e g u i n e a - p i g s s h o u l d s u r v i v e .

A r e a s o n a b l e a c c e p t a n c e l e v e l for a n a d s o r b e d d i p h t h e r i a v a c c i n e i s hat t h e vaccine z h o u l d con la in ahouL 30 1 u / r i n g l e human dose. Therefure, as a rough g u i d e i t would Le satis- f a c t o r y t o Ebov thmt a v a c c i n e i s not less poLent t h a n a r e i e r e n c e

BLG/UM)P/~~. 1 Rev. 1

page 70 B K / W P / ' ~ page 71 f - 1 Rev. 1

Appendix D. 11

m a t e r i a l of such a potency l e v e l This c o u l d be checked hr i n j e c t i n g a g roup of 10 g u i n e a - p i g s - a s ~ c q v i r e d by WHO - w i t h

Lhc v a c c i n e and a s i m i l a r g roup u f 10 g u i n e a - p i g $ w i t h rhc r e f e r e n c e vaterial fullowed f v v l weeks l a t e r by the t i t r a ~ i o l ~ of

u f t h e number u f a n t i t o x i n u n i t s i n Lhi. b lood of b o t h g roups of

g u i n e a - p i g $ . I f the w a n t i t r e i U r e 1 e r a b l y geurnctrir mean) f o r ~ h c vacc ine Ls no t l o v e r rhan t h e one f u r rile s t a n d a r d , onc can cwpect t h a t the v a c c i n e w i l l be satisfactory i n the f i e l d . noth her p o s s i b i l i t y would be t o inject b o t h g r o u p s of gu inea- p i g s w i t h a s u i t a b l y chosen d i : u r l o n o f v a c c i n e and s t a n d a r d ,

fo l lowed by t o x i n c h a l l e n g e a f t e r f a r weeks. The s u r v i v a l p e r - c e n t a g e of t h e p i g s f o r t h e vacc ine under tesr should b e no t

lower than that f a r t h e s tandard v a c c i n e .

I f is lef t t o t h e National C o n t r o l A u t h o r i t y to d e c i d e i n which way t he potency s h a l l be measured and to d e f i n e a minimum l e v e l of accep tancy .

Po tency t e s t s

The f i n a l hulk v a c c i n e o r t h e vacc ine i n i t s f i n a l c o n t a i n e r must be t e s t e d f o r i m u n i z i ~ l g potency.

Although the EH0 Requi re - ments do nnt s p e c i f y any p a r t i c u l a r method i t i s essential t h a t t h e p a t e n s y i s measured a g a i n s t a national standard which h a s been c a l i b r a t e d aga ins t t h e i n t e r n a t i o n a l B t a s d a r d fur D i p h t h e r i a Toxoid.

'.€he i n c l u s i o i l of 1 r e f e r e n c e p r e p a r a t i o n i s e s s e n t i a l because the m t i L o r l n resp011se of g u i n e a - p i g s i n a d i p h r h e r i a ~ o x o i d may be a f f e c t e d markedly by d i e t , r o n d i t i ~ n . of m i n c e n - s n r e , s t r a i n of g u i q e a ~ p i g s and s u c h variables mke i t diffi- c u l l t o i d e n t i f y r e a l d iFFerences between different t o x u i d s .

Tesls based upon t h e u s e o i a s ~ a n d a r d p r e p a r a t i o n g e n e r a l l y i n v o l v e v s c c i o a t i o n of animals w i t h r n ~ l t i p l e d i l u t i o n s af t h e t e s t and r e f e r e n c e v a c c i n e s , and comparison of Lhe log dose r r s p o n s e Cutves of Lhc p r e p a r a t i o n .

I f the r e s p o n s e C U ~ V , , a r e

both reasofiabl l l i n e a r and do l l o t d i f f e r s i g n i £ i c a n t l y i n s l o p e ( b v a l u e ) ~ h c d i f f e r e n c e i n l o g dose r e q u i r e d t o a c h i e v e A given i e s p o ~ s r c811 be c 8 L s u l a ~ e d t u g i v e a n rstimatr of the r e l a t i v e p v r r n r y p i che t e s t p r e p a r a t i o n , I... c h . ratio of the rolume OL rhr s t a n d a r d preparation r e q u i r e d r o elicit 4 g i v e n r e s p o n s e over t h e va lue o f he t e s t p r r p a r m t i a n r e q a i r e d t o e l i c i t the same response . Since the put20sy of Lhe n a t i o n a l s~andard

w i l l have been c a l i b r a l r d i n i n l e r n a t i u n a l U n i t s i~ i s pos8ibl~. t h e r e f o r e , to a s s i g n a po tency u I t h e t e s r p r e p a r e t i o n a l s o i n I n t e r n a t i o n a l U r i i r s . As a g u i d e i t i s c o n s i d e r e d s a ~ i s f a ~ t o r y

if L d i p h t h e r i a toxoid h a s a pDtency of 30 ~ ~ / s i n ~ l e hum" dose o r more.

Suggented method a t a i r a y -for a d s o r b e d v a c c i n e

P r e p a r e three d i l u t i o n s i n s a l i n e of t h e n a t i o n a l i e f e r e n ~ e p r e p e r a t i o n of d iph the r i a v a c c i n e ( a d s o r b e d ) and t h r e e s i m i l a r d i l u t i o n s of each of the t o n o i d s o r v a ; c i n e s under test . he

c h o i c e of dilution w i l l depend upon t h e s t r a i n of gu inea-p ig and t h e c o n d i t i o n o f t h e animals. S e r i a l d i l u t i o n s of each

p r e p a r a t i o * s b o v i d conform t o B two-fold r a n g e , e . g . 1/20 , 1 / 4 0 , 1/80. T h e particular d i l u t i o n s u s e d a r e s e l e c t e d i n t h e

l i g h t of e x y e r l e n r c of Local c o n d i t i u n s such t h a t t h e m e d i ~ n dose for each prepara t ion t e s t e d may be e r p r c t o d to produce a men. i n t r a d e m m 1 rhalleoge score of a p ~ r o x i w t e l y 2 when Lntraderma l chal lenge method is uqed or t o p r o t e c t n p p r o x i m t t l ~ h a l f the a n i m l s when a ruhcutaneour lethal chal lenge dose is

1 used. t

. .

! I n o c u l a t e I m1 of he d i l u t n d v a c c i n e s u k u t a n c o u ~ l y i n t o h each group of guinea-pigs, which s h o u l d i n c l u d e n o t l e ss t h a n

f elgkr a n i m a l s f u r rhe i n t m d e m a l c h a l l e n g e t . e b f and not l ess than 16 for t h e subcu~aneuur l e t h a l c h a l l c n p . F i v e un inOcu-

l a t e d ruinea-pigs a r c u s e d a6 c o n t r o l s . L

! It i s adyipable L O d i s t r ~ b v t e the g u i n e a - p i g s uf each r r e a merit group randomly r h r o ~ ~ 1 1 s e v e r a l cages s i n c e t h e l e v e l of response can be affected b y Loca l c o n d i t i o n s within d i f f e r e n t

B L C / ~ W P / ' T ~ . ~ Rev. 1

page 72

cages. When r a r ~ d o r n i r a t i o n i s c a r r i e d u , ~ t i t i s n e c e s s a r y t o i d e n t i f y e a c h a n i m a l i n d i v i d u a l l y . A scheme t o r t h e identifica tiod oE i n d i v i d u a l a n i m a l s by dye mark in^: is shown a t the end of t h i s a p p e n d i x .

The responses of t h e g u i n e a - p i g s t o t h e s i n g l e dose of vaccine a r e d e t e r m i n e d 28 d a y s a E t e r i n o c u l a t i o n .

1. B y t h e m u l t i p l e i n t r a d e r m l c h a l l e n g e t e s t

Shave and d e p i l a t e t h e f l a n k s of a l l t h e g u i n e a - p i g s . P r e p a r e a d i l u t i o n of a s ln i t ab le l t o x i n i n b o r a t e buLfered s a l i n s c o n t a i n i n g 0 . 0 5 1 2 L£ u n i t s of toxin pe r 0 . 2 a l .

P r e p a r e from t h i s a f u r t h e r s e r i e s o f t h r c c e i g h L - f o l d d i l i ~ t i u r ~ s t o contain

r * s p c c t i v e l y 0 .0064 , 0.0008 and 0 . 0 0 0 1 Lf unl i t5 i n 0 . 7 m l . I n j e c t f o u r d i l u t i o n s i n t r a d e r m l l y i n t o f o u r separa te s i t e s on e a c h of t he v a c c i n a t e d g l ~ l n e g - p i g s i n

such a way as t o minimize i n t e r f e r e n c e between a d j a c e n t s i t e s .

I i i s s u g g e s t e d that :he i n j e c t i o n sites s h o u l d be placed 2 . 5 cm f r o m t h e s p i n e and t h a t a d j a c e n t sites s h o u l d be a r leas^ 5 cm a p a r t i n t h e fullowing p a t t e r n :

Head of

X 0 .0001

X 0.0512

g u i n e a - p i g

LF

Lf

L~ r u l r a b l ~ Loxin c u n t a l n s 0 .b7-1.33 Lr i n 1 ~1 u n i t and 1 Lf u n i t c o n t a i r ~ s h e t v e e n Z S 000 and 50 000 m i n i m a l r e a c t l n f i d o s e s f o r g u i n e a - p i g s k i n .

I t is p r e f e r a b l e for the toxin CO be u n p u r i f i e d and to have been s t o r e d Par a c o n s i d e r a b l e tune b e f o r e u s e .

~ L G / u N U P / ~ ~ . ~ Rev. 1 page 1 3

Appendix D . L 1

l t n j r c r : t h e unvaccinaLed control a n i m l s w l t h d i l u t i o n s con- t a i n i n g 100, 50, 2 5 , 1 2 . 5 a?d b.2> l u i l l i o n t h s of a a Lf unit nf t h e challenge t o x i n i n b o r a t e b u f f e r e d s a l i n e .

Examine a l l i n j e c t i o n s i r e s 48 h o u r s a f t e r i n t r a d e m l i n j e c - t i o n and r e c o r d t h e i n c i d e n c e of s p e c i f i c d i p h t h e r i a e r y t h e m . Record a l s o the n u m b e ~ of s i t e s f r e e f rorn such r e a c t i o n s a s t h e i n t r a d e r m l c h a l l e n g e s c o r e . Tabulate t o g e t h e r t h e i n t r a d e t m a l

c h a l l e n g e s c o r e s f o r a l l t h e an imals receiving e a c h d i l u t i o n of v a c c i n e and u s e t h o s e d a t a w i t h o u t f u r t h e r t r a n s f o r a s a t i o n t o o b t a i n a n e s t i m a t e of t h e r e l a t i v e po tency for each of t h e t e s t 1 p r e p a r a t i o n s by p a r a l l e l l i n e q u a n t i t a t i v e a n a l y s i s (F inney 1 .

a s s a y i s a c c e p t a b l e provided t h a t ( i ) t h e d o s e r e s p o n s e regression of the t e s t and r e f e r e n c e p r e p a r e t i o n are l i n e a r and p a r a l l e l : (ii) the toxin d i l u t i o n xhich c o n l a i n s 50 d l l i o ~ l t h s of an Lf gives a p o s i t i v e e r y t h e m i n e t Leas t 80% of the c o n t r o l g u i n e a - p i g s ; and (iLi) the d i l u t i o o c o n t a i n i n g 20 m i l l i o n t h s o f

,', a n L5 g i v e s no r e a c t i o n i n a t leas^ 8070 ot the g u i ~ ~ e a - p i ~ a . I c

t h e s e c r i t e r i a a r e no t met a d i f f e r e n t t o x i n s h o u l d be s e l e c t e d .

,X 2 . By the s u b c ~ ~ a n e o u s c h o l l e n ~ e t e s t

T h i s t e s t i n v o l v e s t h e immuniza t ion of g u i n e a - p i g s i n t h e s t t e s t , b u t the c h a l l e n g e i s g i v e n by on w i t h I nI of a d i p h t h e r i a t o x i n c o n t a i n -

DiD. The g u i n e a - p i g s are o b s e r v e d twice

and t h e number of a n i m a l s ~ r o t e c t e d by e a c h t h a t p e r i o d a r e r e c o r d e d .

S i m i l a r l y , s c u r l t r o l Rrn.lp of u n v a c c i n a t e d a n i m e l s i s l u d r d , each which is given 1 m 1 of a L:99 d i l u t i o n of t h e

f n B o l u t i u n used to c h ~ l l c n g c t h e vacc ina t ed animals (1 DSO)*

hods i n B i ~ l o ~ i c a l Assay. ond don I ~ h a r l c s

hepterr b and b.

BL.JZ~UNDP/TI. 1 Rev. 1

page 74

The b e s t method of e s t i m a t i n g t h e r e l a t i v e p o t e n c i e s o f t h e p r e p a r a t i o n s from t h e praporlion of a n i m a l s s u r v i v i n g the

1 c h a l l e n g e i s by p a r a l l e l l i n e a s s a y ( ~ ~ n r ~ e y ) . S i n c e r h i s . method i s i o p r a c r i c a b l e w i t h o u t the a i d of a c m p u l e r 2 it i s suggested t h a t the f o l l a w i n g s i m p l i f i e d methods be more prac t lc - a b l e a l t h o u g h they make less e f f e c t i v e use of t he d a t a :

( a ) Reed & bench 11338) Amer. J . ttyg., 27 , 4 9 3 . - (b) Worces te r & Wilson (1943) Proc, nac . Acad. S c i . ,

2 9 , 207. -

( c ) P l o t t i n g percentage p r o t e c t i o n a g a i n s t Loglo dose e i t h e r on " p r o b a b l l i t y paper'' a t a f t e r c o n v e r s i o n w i t h prubi t t a b l e s on ordinary graph peper, and measuring t h e dis tance between t h e r e g r e s s i o n L i n e s d r a m for t h e t e s t and s t anda rd p r e p a r a - ticrns. This d i s t a l ~ c e r e p r e s e n t s t h e log of

0 che r c l s t l v e potency of t h e iesL preparation i n terms of t h e r e f e r e n c e .

1,. 1 i

1 1 , A Lest i s v a l i d p r o v i d e d that: I ! ! I I i ) some b u t not a l l of rhe c o n t r o l ~ u i n c a - p i g s

I: I c h a l l e n g e d ulch 1/100 of t h e c h a l l e n g e dose :I . a£ t o x i n d i e ; 11 .

( i i ) he lowes t d i l u L i u n of v a c c i n e has p r o t e c ~ e d mote t h a n h a l f of t h e animals;

. ! I .

I ' ( i i i ) t h e h i g h e s t d i l u t i o n h a s psoLected l e s s than ! I h a l f ; and j . I L - -

' I 'robi~ - Analysis, 3rd ed . L u n g ~ ~ > (1971) (Cambridge t l r ~ i v . Press) .

h

L ~ r c t ~ i ~ analysis (:on ~ I I > y ~ r l o r a r d w i t h an i n r x p r l , r i v c p l ~ r k c l

ca l cu ia tnr w i t h s u i t o n l y pfugra~nmed magnetic c a r d s . F u r t h e r l informati trn can I)e o b t a i n e d from the WHO S e c r e t a r i a t , ( C h i e f ,

3iologicnls).

B ~.G/uNDP/~ 7.1 Rev . 1

page 7 5

I Appendix D . 1 1

( i v ) t h e d o s e response curves of the t e s t and reference vaccine do not d e v l a c e s i g n i f i - c a n t l y from p a r a l l e l i s m and l i n e a r i t y . I f nu standardization t e s t of p a r a l l e l i s m is a v a i l a b l e a test m y be a c c e p t e d provi- ded chat the glope of Lhe steeper c u t v e i s not more chan 150% of the s h a l l o w e r cu rve .

3 , Method o f a c s a y of unadsorbed vaccine

The p r i n c i p l e s for assaying u n a d s o r b e d vaccine a r e s i m i l a r t o chose already d e s c r i b e d f o r t h e assay of adsorbed v a c c i n e . There a r e , houever, three major d l l f e r e n c e s :

i vaccines a r e t e s t ed a g a i n s t a n unadsorbed a tandard ;

(ii) the doge r e s p o n s e c u r v e i s less s t e e p as a result of which the i n t e r v a l between d o s e s should be t h ree - fo ld r a t h e r t h a n t w o - t o l d ;

( i i i j the p r e c i s i o n of t h e assay i s l v w e ~ n e c e s s i t a t i n g the use of l a rger numbers of enlmals f n the expcr imenca l groups.

*:- , 4 . Suage.Rted method f o r r n d l v l d u a l i d e n t i f i c a t i o n of

, idea-p igs i n an anl- l , room

f i e following scheme allovs for the Ind iv idua l i d e n t i f i c a - tLon of more than 800 guinea-pige. T h e i d e n t i f i c a t i u n caflsi sts

of dye mrkr on head, back and l e g s . F o r r e c o r d i n g p u r p o s e s

, head and bask c o l o u r s a r e designated as a capi ta l l e t t e r , t h u s : ,.-."*? . .- .y:

BII~/l1NLll'/'71.1 R e v . I page 7 6

Appe~ldix L). t 1 L

A Red head

H Red back

C Green head

Green hack

E Mauve h a d

F Mauve hack

H Red h e a d , g r e e n back

K Red head, mauve back

I. Green head, red b e c k

N Green h e a d , mauve b a c k

F' b u v c head, red ba~:k

R h u v e h e a d , g r e e n ha,:k

Leg c o l u u r ~ t h u s ;

a r e designated

r e d a l e r t

i r o n t

r e d b ~ e i t .

f r o n t + l e f t

rear

grccn C left f r o r ~ ~

r igh t f ran t

l e f t f r o n t

+ r i g h t r e a r

right f r o n t

by s m a l l

1 l e L t r e a r

t i g h t f run t

+ l e f t r e a r

left

r e a r

Red head , red back

Green head, green back

Mauve head, mauve back

Ye l law head

Yellow back

Yellow head, rcd back

Yellow head, gretln b a c k

Y e l l o w head, mauve back

Red head, yellow back

Green hcad, y e l l o w back

Mauve h e a d , yellnw back

letters, and number

r j g h L Loth rca r front

riglkL b a t h f r o n t rcar

I r i g h t r e a r

r i g h t b o t h r e a r f r o n t

RLC/L.NI)P~'~ 7 . l Rev. 1

page 77

Colour L e ~ ~ e r 1. 2 - 3 4 5 &

L

g r e e n d l e r t l c f t r ighL r i ~ h t both front front f r o n t f runt r e a r

I l e f t + r i g h t r l e f t + r i g h t

1 reat rear r e a r rear

I mauve e l e f t r i g h t l e i t r i g h t both

t r o n t f r o n t rear rear t r a r ~ i

m u v e f l e f t l e f t r i g h t r i g h t b n t h f r o n t front f r O 1 1 t f r n n r rear

+ l e f t . - r i g h t + l e f t + r i g h t r e a r reat rear rear

I yellow h l e f i r ight lekt r i g h ~ both

f r u a t front r tar reer Eront

y e l l o w k l e f C l e l t r i g h t r i g h c b o ~ h f r o n t f r o n t f r o n t f r o n t r e a r

t left + t i g h t + l e f t + r i g h t I ' .' rear T c a t fear r e a r

,. .

Example: Hk4 = red hcad, green back, r i g h t f ront altd .I,,. -- , - . ,:. .: .. . ,. . , .

r ighr : r ea r legs co lou red y e l l r w .

m: Muve = methyl v i o l ~ t 2%

Green = m l a c h i t e green 1%

Red = basic f v c h s i n 1 X

Roil 1 l i t r e d i s t i l l e d watet. Remove from heat. , s p r i n k l e

10 g of dye gn the surface and stir in.

Yellow - picric acid 0.5%

Boi l 1 l i t r e d i s ~ i l l e d w a t e r . g o f dye on he surface- Do not

The adhesion of dyes t o a ~ ~ ~ l

, .

Remove £tom h e a t , s p r i n k l e s t i r .

f u r s i s improved hy t h e LO lCU m1 n i solution,

B L G / ' u N D P / ' ~ ? . ~ Rev. 1

page 78

B L G / U N D P / ~ ~ . I Rev. 1

page 79

5 . D e t e r m i n a t i o n o f LD50 by t h e m e t l ~ o d of Reed 5 Nuench 1

EKample -

Suppose 20 a n i m a l s per d i l u t i o n a r e i n ~ e c t e d w i t h d o s e s ( e x p r e s s e d i n m i c r o l i t r e s o f v a c c i n e i n j e c t e d ) o f :

200, 100, 50 , 25. The a n i m a l s a r e c h a l l e n g e d w i t h t o x i n .

Dose

2 00

1 W

50

2 5

S u r v i v i n g

l8

1 3

7

l

Dead

7

e r c e n t a g e m o r t a l i t y

2/41 = 57:

9/30 = 307,

2 1/29 = 72%

40/41 = 98%

4 ! : .L

, " Suppose one a n i m a l d i e d i n t e r c u r r e n t l y b e f o r e c h a l l m g e . I

The 50% m o r t a l i t y l e v e l i s found between d o s e s 1 W and 50.

50-30 20 P r o p o r t i o n a l d i s t a n c e = - = - =

0.48 72-30 42

1 Dose j u s t below 50% m o r t a l i t y is 50.

I l o g 5 0 = 1 .699

0 . 4 0 x l o g 2 = 0 .144 (0 .48 = p r o b d i s t . : 2 = d i l . f a c t o r )

l -, i . 1 . 8 4 3

l ~ ~ t i l ~ ~ = 7 0 = ED v a c c i n e l 5 0

I suppose ED50 s t a n d a r d i n same t e s t = 4 0 and s t a n d a r d

i c o n t a i n s loo I U / ~ I 4 0 ! [ Vaccine c o n t a i n s - x 100 = 57 I U / ~ L

i 7 0

( C a l c u l a t i o n can he f u r t h e r s i m p l i f i e d by methods of ? ,ant ic-or T i n t and C i l l e n , P a t h . Microbial. g. 298 ( 1 9 6 3 ) ; J . a p p l . BacL., 2 4 , 8 3 ( 1 9 6 1 ) . )

'RST FOR SPEClFIC TOXICITY

The c e s t f o r s p e c i f i c t o x o c i t y s h a l l be c a r r i e d o u t a s s p e c i f i e d i n t h e WHO R e q u ~ r e r n e l c s f o r B i o l o g i c a l S u b s t a n c e s

: NO. 1 0 (Requi rements f o r D i p h t h e r i a T o x o ~ d and T e t a n u s Toxold) See Appendix D . 2 6 .

For b u l k t o x o i d - P a r t A , S e c t i o n 3 . 4 . 3 , page 35

P o r f i n a l hulk - P a r t A , S e c t i o n 3 . 5 . 7 , page 3 6 -

BLG/UNDP/~~.I Rev. 1 page 80

Appendix D. 1 2

A d d i t i o n a l t e s t s f o r t e s + u a l t o x i 3 i n bulk toxoid_ -

Residual t o x i c i t y i n d i p h t h e r i a t o x o i d c a n be t e s t c d f o r by e i t h e r i n j e c t i o g 0 . 2 m1 of a ?ample i n r r a d e r m a l l y i n t o nQrmal g u i n e a - p i g s ' s k i n and observ ing t h e i n i e c t i o n s i t e for s p e c i f i c

d i p h t h e r i a e ry thema 4 8 h o u r s a f t e r i n j e c t i o n , o r by i n j e c t i o n . of a 0 . 1 m1 sample i n t r a d e r m a l l y i n t o n o r m a l r a b b i t s k i n and o b s e r v i n g d i p h t h c r i a c r y thcmii /l' h o u r s a f t e r i n j ~ c c i o n .

The i n t r a d u c t i o r l of t h e sample inLu t i s s u e ~ u l ~ u r e i s a l s r ~ a t e s t that i s a b l e t o d ~ c e c t d i p t ~ t l ~ c r i n t o x i n a t c u n c e t ~ t r a t i o a r 2 0 tlmes lower t h a n call bi. d e t c c t e d hy tht, i n t r a d e r m a l t e s t ,

--P-- .- - --

APPENDIX D. 2 3

TEST t-OR IRON

The ContenL o f media can h e estiu~.Led by f o l l o i ~ i n g method-

REAGENTS

Stock s t a n d a r d - 100

A q u a n t i t y o f 0.8636 g Ana la r f e r r i c ammonium s u l f a t e i s d i s s o l v e d i n 50 m1 of Analar n i t r i c a c i d and t h e v o l u a e i s a d j u s t e d i n a one l i t r e v o l u m e t r i c f l a s k w i t h d e i o n i z e d d i s t i l l e d w a t e r .

work in^ s t a n d a r d - l pg ~e/rnl

The s t o c k s g l u t i o n i s d i l u t e d 1/100 w i t h d e i u n i z e d d i s - t i l l e d w a t e r .

BU/UNDP/~~ . 1 Rev, 1

page 81

I Appendix D. 12

dl s u l f u r i c ac id

A volume of 7 m1 A n a l a r s u l f u r i c a c i d is added La 250 m1 d e i o n i e e d d i s t i l l e d water.

Constant b o i l i n g - H C 1

T t ~ i u ~ l y c o l i ~ ac id B.D.H.

~ t / m l a t FO'C 1 . 3 2 t o 1.33.

Aqueous

0.021. 4 : ) J iphcnyl 1;LO p h e n a n t h r o l e n e in isopropyl alcohul

Aliquot . o f 5 m 1 of the sample ( c o n t a i n i n g between 0.1 and are p i p e t t e d in to each of threc marked. acid-washed,

c r u c i b l e s ; 1 m1 of t h e working standard i s ~ i p e t t e d

c r u c i b l e and 2.5 m1 of ~ / 1 s u l f u r i c acid added c 0 each + fie ramplea are d r i e d on ho t p l a t e s under infra-red

when the samples have chsgred t h e y are e irhcr i n c i n e r a t e d i n a f u r n a c e a t 6m-620 C , or they my be t r a n s f e r r e d

nace a l r e a d y at 600-650 C and i n c i n e r a t e d £ 0 ~ two h o u r s

me ashed samples areal lowed t o c o o l . Six drops of

b o i l i n g HC1 are added t o e a c h crucible ( u s i n g a Pasteur t o d i s s o l v e t h e ashes. The s o l u t i o n i s t r a n s f e r r e d t o

hed 10 m1 g r a d u a t e d c o n i c a l c e n t r i f u g e tubes with zed w a t e r ( u s i n g P a s t e u r pipettes ) t a k i n g care n o t to

4 ml. T e s t blanks o f a p p r o x i m a t e l y 1 m1 d e i o n i z e d d i s -

ater a r e prepared a d s t a n d a r d s c o n c i s t i n g o f r e p l i c a t e .nd 2 ol a l i q u o t ~ o f the working iron s t a n d a r d .

S i x drops

BLG/UNDP/~~.I Rev. 1

page 8 2

Appendix D . 13

of c o n s r a n t b a i l i n g HC1 a r e added to each tube. The volume i n a11 of t h e t ubes i s a d j u s t e d t o 4 m1 with d e l o n i z e d w a t e r .

One d r o p of t h i o g l y c o l i c a c i d ( P a s t e u r p i p e t t e ) i s added t o a l l t h e tubea. Ihe c o n t e n t s a r e mixed thoroughly and Left s t a n d i n g f o r h a l f a n h o u r . A volume o f 1.0 ml of 60% sodium acetate is added t o a l l t h e tubes. The c o n t e n t s a r e mixed, 2 . 0 m1 o f -0.021. 4:7 d i p h e n y l L:10 phenanthrolene i s added and mixing is repeared . The c o l r n r s a r e r ead a g a i n s t d e l m i z e d w a t e r on t h e Unicem S P &X at 525 m)l u s i n g 1 cm c e l l s , A c a l i b r a r l o n graph i s con- s t r u c t e d t o c o n v e r t t h e absorbance measuremenLs i n t o concen t r a - t ions. The f i n a l r e s u l t s are exp re s sed i n ppm F e .

N.B. 1, The p l a t i num c r u c i b l e s are a lways s t o o d on p o r c e l a i n r i n g s .

2 . When no t i n use t h e c r u c i b l e s are s t o r e d i n approxime t e l y 3N.HCl.

3 . The ssa l e p l u s s t a n d a r d shou ld g i v e a r ecove ry B of 100 - 5%.

APPENDIX D. llr

DEERMINATION OF TOTAL NI 'I'ROGEN (TN) AND NON-DIALYSABU PROTEIN NITROGEN (PN)

The n i t r o g e n content o f samples can be e s t i a v r e d by t h e fo l l owing method:

Es t ima t ions performed on the sample as r ece ived y i e l d t o t a l n i t r o g e n iTN) va lues .

Estimations performed on samples a l k e r c x h a u s t i v e ove rn igh t dialysis a g a i n s t running wa te r y i e l d n o n - d i a l y s a b l e nitrogen (PN) va Lues .

BLG/WP/ 7 7 .l Rev. 1 page 83

Appendix D . 14

I a ) Reagents :

( i ) I n d i c a t o r

1, ethyl red-methy l e n e blue

2 par ts o f 0.296 methyl r ed a r e mixed w i t h 1 p a r t of 0.2% methylene b l u e , b o t h i n 95% e t h a n o l .

or 2 , Eferhyl red-brorn cresol green.

5 par t s of 0.2% brm c r e s o l g r een a r e mixed w i t h 1 part of 0.2% me thy l r e d , bo th i n 95% e t h a n o l .

( ~ i ) Sodium hydroxide-aodium t h i o s u l f a t e 6 o l u t i o n - aqueous s o l u t i o n of 50 g of NaOH and 5 p of N ~ ~ S ~ O ~ .

(iii) Bor i c a c i d s o l u t i o n . 4 g of H B 0 is d i s s o l v e d i n 100 m1 d i s t i l l e d wa te r .

3 3

(b) DeterminatLon:

A l ml sample of d i a l y s e d t o x a i d i s measllxed i n t o a 30 m1 I j e l d a h l d i g e s t i o n I l a ~ k . To t h e a m p l e 1.30 - 0.05 g

potass ium s u l f a t e , 40 - 5 mg a e r c u r i c o x i d e and 2.0 m1 concen- t r a t e d s u l f u r i c a c i d are added.

P o r c e l a i n c h i p s o r g l a s s beads

are added and the m i x t u r e i s d i g e s t e d f o r four hours under vlgorova b v i l i n g w i t h the a c i d condens ing well up into t h e neck of the f l a s k . The f l a s k i s coo l ed , a minimum of d i s t i l l e d

water ( abou t 5 m l ) t o d i s s o l v e t h e s o l i d s is added and a t h i n f i l m of v a s e l i n e Is a p p l i e d t o t h e mouth of the flask. Thsl

d i g e s t i s t r a n s f e r r e d t o g e l h e r w i t h t h e p o r c e l a i n c h i p s C O a rnlcra or semi-micro s team d i s t i l l a t i o n a p p a r a t u s of s t a n d a r d d e s i g n . To c o n d i t i o n the a p p a r a t u s a f t e r p e r i o d s of d i s u s e s team i s passed through i t fo r s e v e r a l m inu t e s .

Completeness

B I . G / ~ ~ N [ ? F / ? ~ . 1 Rev. 1

Page 84

Appendix D,14

01 tl..nsker of rhe d i g e s t i o n mixture can be t e s t e d by t he addi t ion of a drup of methyl orange i n d i c a ~ o r to the f i n a l r inse .

The 125 m1 Erlenme~er r e c e i v e r m n f a i n i n g 5 m1 of 4% boric acid and four drops o f the i n d i c a t o r i s placed under the c o n d e n s e r d t h

the t i p extending b e l o w the s u r f a c e , A voltime of 8 ml of t h e 8 o d l u e hydroxide-sodium rhiosulfate reagent I r added to t h e s t i l l a n d steam d ~ s t i l l a t i o n i s c o n t i n u e d u n t i l a b o u t 15 ol of distill- ate i s c o l l e c t e d .

The conrents of the r e c e i v e r a r e d i l u t e d t o 50 m1 w i t h distilled water and the umunium h y d r o x i d e i s t i t r a t e d w i t h 0.02 N H C l .

As the e n d - p o i n t the grey colour i s chosen o r the cirsr appcarence of the red one.

The L 1 4 4 value is d e t e r - mined u s i n g the same amount Q£ r e a g e n ~ s r i n s i ~ l g the d i g e s t i o n f l a s k i n the samt? m r ' n e r s rd using the sane d i g e ~ t i o r period er for the d t t e m i n a t i o n .

I t i s ~ o s s i b l e to defermi2e PN a l s o by p r e r i p i r a t i n p t h e p r o t e i n s w i t h 5% tr ichlaracet ic a r i d , washing the prec ip i ta t e and d e t e n i n i n p , TN in i t .

in t h i s case t h e obtained v a l u e 1 s n o t "non-di8Lyeohle n i ~ r o g e n " , but " T C A - p r e r i p i t a h l e n i t r o g a n 1 I , b u r both v l l u e s are very close t o " p r o t e i n n i t r o ~ e n " .

TEST FOR TIfT DPKRSAL C0;irEN.I.

Hy che~itica l-mean, - Procedure

B L C / U N D P ~ ' ~ ~ . ~ Rev. 1

page R 5

t S e p a r a i o l - y funnels are uashad w i ~ h c a n c e n t r a ~ e d n i t r i c ac id

~ n d rinscd w i ~ h t a g and d i s t i i l e d weMr. Two a l i q c o t s of a

standad 0 . 0 1 % a c u e o v s s o l u t i o n of thinrerssl (0.5 and 1 .B m l ) and two rarples of t h e L e s t s o l u t i o n ( L G > ) a r e added t o > i n d i v i d u a l reparatory funnels and rtlr, volunlc sd jus ted Lo 10 m1 w i t h a 1% s a l v ~ i n n of anm~onium a c e ~ ~ l t e ar pH 6 . 0 . Ten m i l l i -

l i r r c s of a 1 i n 10 d i l r t i o n of ;i i 1 e 5 h soluiion of D . O l ? l i i h i z o o e in c h l o r o f a r ~ u i s a d d e d to each f u o n r l and Lhr c o 1 1 t m L 3

shaken v i g o r o u s l y for 45 seconds. The ~ t ~ l o r u l o m ~ Layer i s

! c a r e f u l l y s e p a r a t e d .

The Q and 100' kr;~,i 'mission p o i n t s o n the spectrophoto- i n e ~ e r are s e t a t 490 nm using the diluted d i t h i z o n e solution

and a scan i s taken u i rhr Lest i o l u t i v n r Lro;n 47U Lo 520 nm. Tllr Lransmi!i!;ion a t 520 r m i s p l o t ~ e d a g a ~ n s t he t h i o m e r i a l r o n t e n r r s t i u ~ ~ OL t h e !;rindards on semi-logarithmic p a p e r and tile concentration 01 thiomersal in the sample l ie ternlined.

I by microbiological means

l l X u l r i e n t agar is poured r o a depti-t of 4 mn on a 5ul tablc

reCtlegular g l n r r p l ~ t c s e t i n a mtai [idme (B Large ~ c ~ r i - d i sh m y be vrcd b u t can a e c o m ~ o d a t e o n l y 8 few saml) les l . 11 i s e s s e n t i a i that t h e p l a l e is a b s u L + t e l y h o r i r u n ~ d i when the

I a g a r i s k i n g p o u r e d . C i r c u l a r wells u l approrin~alcly 8 "1

diameter r u t i n t h e agar s o that adjacent wel:s ale a L

l e a s t 30 m a p a r t . The p l a ~ e i s sccdcd wi th s u r i i c 1 ~ 1 1 t

! s ~ a p h ~ l ~ ~ ~ . r ~ ~ .UT~YI organisms t o p,tve a r o n i i u e n t gruwrh . , a t 48 h o u r s , l o c o r o c t l well 100 micru! irre s a u l i . 1 ~ ~ oi

1:2.5, L:5, L : ~ o , i:2U, I : & 0 and 1 : 8 0 d i l u t i o n s OI a scanJrrd S ~ i u ~ i u n oi L p/liLre sodium thiomersalare are randumly d i s - persed as ill r a n y l ~ s o f the t e s t v a c c i n e n . The l f j r r r i o n of each preparation i s c a r e l u l l y recorded.

reCr.rc~lru d i 1 u t i o 1 . i of thionersal. The e f f e c t i v e o n c e n ~ r a t i i s d i n tertt~s U£ t h e d i l u t i o n of reference thiomrrr.l

i u l u t i o n L a whrrh i t mostly closely a ~ p r o x i m t e a .

C r i L ~ r i a ior accepConce

'I'llt: Ll l io l l ler~al content rhuu l d be between 0 . 0 0 5 and 0.0;1%,

TEST FOR pli

Praccduro

A pH meler w i t h g lass and cnlumvl reference electrodes is s t a n d a r d i z e d w i t h slnndard t u f l e r solurivns a t pH 5.0 and 7 .0 . The e l ~ ~ t r o d e s a r c r i n s e d w i t h d i s t i l l e d water, d r i e d and t h e pU o f Lhc tcsc solution recorded.

C u i L f r i a f u r a t c c p t a n c c

t pl! va lue should be 7.0 - 0 .3 .

TEST FOR FREE P ~ M A I J E H ~ E

I 'ru~c& -

I thc v a c c i n e or toroid i s d i l u t e d w i t h d i s t i l l e d W a l c r t o 10 lnl .

2 m 1 1 . f t l ~ e d i l u t e d sample i s t r a n s f e r r e d i l l ~ l ')cl 111L ~ . L . S L 1 1 - 1 tl1f.e ( w i t h a b o i ~ i 61 m1 ~apacity) alld d i l q l t f d w i J x ~ i 1it.d wiltrr L U about 11 diluted sulkuric

BLG/:IUNI)P/S~ . L R W , 1 page 87

on a r i d lunu v o l . H S 0 of sp. g'. 1.84; u ~ l e v o l . water) l5 added

. m d ihc L l u U i r 2 d i ? u t e d w i r l , d i s t f l l o d water t o the 50 a1 mark. . Similarly a s e r i e s u l tubes w i t h known amounts of f o m l d s h y d e ' 3 r ~ prepared LO s e r v e as f o r m a l d ~ h y d e standards f o r corpparison, - ' using a 0.01% d i l u t i o n of Analar s o l u t i o n U £ formhldehyde

( c u n ~ a i n i n ~ n o t less than 377- ot C H . 0 ) and s e l e c t i n g q u a n t i t i e s L . c lose to t h a t cxpecred i n the sample.

To t h e sample tube and

t u each 01 thc standard tubc.; 10 m1 of f u c h s i n - s u l f r l r o u s a c i d

l C s L solutiun i s added. The m i x l u r e s ere allowed t o

..:t s t d ~ i d one hour a~ room temperature and e col .urimetric comparison of the sample tube w i t h the standards is m d e .

U . 2 g of b a s i c iuchsin i s di9solvrd i n 120 m l of h o t dis~illed water and,the solution a l l o w e d to c o o l .

A s o l u t i o n

2 g of anhydrous sodium s u l f i ~ e i n 20 rnl of d i s t i l l e d water Is added and then 2 m1 o f hydrochloric a c i d .

The sol~tiull is

diluted with distilled water to 200 m1 and ~llowed tu stand a t lcasr one hour. his s ~ , L u t i o n must bc f r e s h l y prepared.

C r i tcr ia for acceptance

~ h ~ s h a l l be t o have not nore than 0-02%

TEST FUR A W M l N L I I N CONTENT

3-rnl samples of t h e product i n I ~ u i l i n g t u b ~ s

c n a ~ e n ~ r a t e d sulfuric ac id and s i x drops of u " c v n l r a ~ ~ d n i t r i c a c i d . ' F l i t . t ,~br .s arc l i ea tcd u n t i l dectst.

c . tlcal i r ~ g 1:; c o n t i n u e d , wl t h dropwise

d r l i ~ i u r ~ "L n i t r i c a c i d , ullr i l ~ h c conLents 01 the t u b e s are

B I ~ ~ U N D P / ~ ~ . l Rev. 1

page RE!

c u l u u r l c s s . 'I'llc tubes a r e uuolcd and 1 0 m1 of d l s t i l l e d w a t e r i s c a r e l u l l y a d d e d . 11 t h e s o l u t i o n i s c l o u d y the t u b e s a r e I l o i l c d u n t i l c l e a r . Sodium h y d r o x i d e (50% W/") i s added t o c a c h tube u n L i l a p i t tk -ye l low r n d p o i n t i s o b t a i n e d w i t h mcthyl oranKe i n d i c a l o r . Any p r r c i p a t c Lurmlnp is dissolved by t h e a d d I r i o n n t d i l u t e s u l i u r i c a c i d . I'hc c o n t e n t s o r Lhe t u b e s a r c L r a n s t c r r e d t o s e p a r a t e 2>0-m1 Er lemeyer f l a s k s . t h e t u b e s u a s h c d out w i t h 2 5 ntl oF d i s t i l l e d w a t e r and t h e w a s h i ~ i g s pooled w i t h t h e concer l t s o f i h r f l a s k s . 'TO each f l a s k a r e added ? > m1 01 M/100 d i s o d i u m e t h y l e n e d i a m i n r t e t r a a c e t a t e , and L0 rnl o l a c e t a t e b u f f e r ( c o n t a i n i n s b8 g of sodium a c e t a t e , 38.5 g of dnunur>i~mi a c e t a r e , L 2 5 m 1 of glacial a c c ~ i c a c i d i n a L o t a l volumc 01 500 ml). The l l a s k s a r c b o i l e d g e n t l y f o r t h r e e m l n u l e s and 1 m1 o f p y r i d y l a z o n n p h t h o l i n d i c a t o r ( 0 . l X s o l u t i o n i n 05". c t h a n o l ) is added . Each h o r s o l u t i o u i s t i t r a t e d w i t h i'l/l00 cul,per s u l f a t e s o l u l i o n u n t i l a purp lc -brown e n d p o i n t i s reached. S i m u l t a n e o u s l y a b l a n k d e t e r m i n a t i o n i s a l s o c a r r i e d out u s i n g d r s t i l l e d water i n t h e d i g e s r i o n p r o c e d u r e i n s L e a d o i ssnll'lc. t;ach m1 o f 4100 C u S 0 4 . 51i20 = 0.2698 mg A l + l + .

C a l c u l a t i o n o f t h e r e s u l ~ s :

mg o f A1 p e r m 1 of sample = Klank t i t r e - S a m p l e r i t r e Volume o f sample ( m l )

C r ~ L e r l a f o r a c c e p t a n c e

'The a l u m i n i u m c o n t e n t i n t h e f i n a l b u l k p r o d u c t s h u u l d n o t c x c c e d 1 . 2 5 mg AI p e r s i n g l e human d o s e .

APPENDIX D. 19

TEST FOR STERILITY

T e s t s f o r s t e r i l i t y j h a l l be c a r r i e d o s t by t h e t e s t s a s s p e c i f i e d i n t h e r e v i s e d X e q u i r r x e a t s Eor ~ i o l o g i c a l s u b s t a n c e s No. 6 ( G e n e r a l Requi rements f o r t h e S t e r i l i t y u f A i u l u g l c a I S u b s t a n c e s ) . See Appendix D . 2 1 .

l , I N l I 7 . l R 1

page 8 9

Appendix DLx

I FOE t e s t 3 f o r b u l k L O X ~ L , - I % r t A , S e c t i o n 5 . 1 . 1 s h a l l

" P P ~ Y .

F o r t e s t s on f i n a l l o t - p a r t A, s e c t i o n 5 .1 .2 s h a l l

,: .. " P P ~ Y .

Care must be t a k e n t o e n s u r e t h a t t h e i n o f u l u m o f t o x o i d o r

v a c c i n e does n o t , i n t h e c o n c e n t r a t i o : , used i n t h e i n o c u l a t e d y.::. medium. d e c r e a s e t h e a b i l i t y o f t h a t medium LO s u p p o r t t h e growth

- --- APPENDIX D.20

TEST FOR INNOCUITY

The f i n a l b u l k and f i n a l l o t s h a l l b e t e s t e d f o r i n n o c u i t y .

One human dose1 b u t n o t more t h a n 1.0 m1 i s i n j e c t e d i n t r a - r i t o n e a l l y i n t o e a c h of f i v e a d u l t mice weigh ing 17-22 g a n d e human d o s e , b u t n o t more t h a n 5 . 0 m 1 i n t o e a - h of two i n e a - p i g s w e i g h i n g 250-350 g .

Vacc ine i s a c c e p t a b l e i f none o f t h e a n i m a l s shows s i g n s he seven d a y s f o l l o w i n g t h e i n o c u l a t i o n . If

p of t h e e n i m a l s d i e s o r shows s i g n s of i l l - h e a l t h d u r i n g t h e s p e c i f i e d , r e p e a t t h e t e s t . The s u b s t a n c e p a s s e s t h e t e s t

f none of t h e a n i m a l s i n t h e second group d i e s o r shows s i g n s i l l - h e a l t h i n t h e t i m e i n t e r v a l s p e c i f i e d .

s e i s t h a t s t a t e d a n t h e l a b e l o f t h e r a t i o n t o 5 e i n j e c t e d o r i n t h e accompanying i n f o r m a t i o n

BLG/UNDP/~~.~ Rev. 1 page Y O

APPENDIX D.21

PREPARATION OF ALUMLNIUM PHOSPHATE SUSPENSION

There a r e two d i f f e r e n t ways of p r o d u c i n g s u i t a b l e s u s - p e n s i o n s o f a luminium p h o s p h a t e , s t a r t i n g e i t h e r from alum ur from aluminium c h l o r i d e .

A. Method s t a r t i n g from alum ( A 1 (SO4I3 . K2S04.12H20) 2

854 g alum ( p o t a s s i u m a l u m i n i u m s u l f a t e 12 eq) is d i s s o l v e d i n s i x l i t res of w a t e r a n d f i l t e r e d , and s i m i l a r l y 685 g of t r i s o d i u m p h o s p h a t e (lie PO i2H 0) a r e d i s s o l v e d i n

3 4' 2 s i x L i t r e s o f w a t e r . The a lum s o l u t i o n i s h e l d a t a b o u t 3 7 ' ~ , a s i t is s l i g h t l y o v e r s a t u r a t e d a t room t e m p e r a t u r e .

iicrlh s u l u t i o n s a r e poured a t t h e same t ime v i t h mixing i n t o ? I L i t r e s oE w a t e r . The p r e c i p i t a t e i s c e n t r i f u g e d , t h e hcdinlcnt resuspended i n 13 l i t r e s of wacer and t h e s u s p e n s i o n c c n i r i f u p e d a g a i n . T h e s e d i m e n t i s f i n a l l y rcsusGended i n a [ o t a l volume n € 8 . 1 l i t r e s i n s a l i n e , homogenized, pH a d j u s L e d w i L I c 5 N Na0H t o 6.0 and a u t u c l a v e d f o r one hour a t 1 2 0 ' ~ . 'l'lrii. q u a n t i L y i s s u t f i c i e n c £or p r e p a r i n g 66 l i t r e s of a v a ~ c i n u u i t h 3 mg Alp0 /m1 or 0 .66 nag ~ l / r n l .

/l

i kg ui purc .?luminium chloride (AIC1. bH 0) is d i s s o l v e d 3. In IlisLi l l c J w a t e r , and t h e vr>lume i s m d e u p f o 30 L i t r e s .

l'hc ~ u t u l i u n i s then f i l t e r e d Llrrough a s o f t (KL) Liltrr s h e e t . ' ~ i m l l a r l y 4.73 kg uf pblrr t r i i c r d i u n phospha te ( ~ a PO 12H20)

4 ' i s d i s h ~ l l v e d i r c d i u t i l l c f l u a t e r , made up t o 30 l i ? r e s a n d d l s u l i l t c r e d t h r o u g h K 1 s h e e t s .

l [ t aper can be u h l a i n e d t c u m S e i t z o r C a r i s o n Ford .

To r ~ a k e t h e phr,bpl,atr, L50 I i L r e s o i d i s t i l l e d w a t e r l> l i l t e r e d t h r o u g h K1 s lwrt s i n t o a c o n t a i n e r o f 300 l i L r e b capa-

' c i t y . 1 . l ~ 30 l i t r e s 01 A l C 1 , 3 s a l u t i u n i s t h e n s l o w l y added and w e l l mixed b e f o r e a d d i n g the Na 3 PO 4 v i t h good s t i r r i n g u n t i l a ptl of 5.0 is r e a c h e d ( n b o u t 27-29 litres a r e n e c e s s a r y ) . F i n a l l y >O l i t r e s of d i s ~ i l l e d water a r e a d d e d , t h e s u s p e n a i u n w e l l mixcd, and l e f t s t a n d i n g for s e v e n days.

A f t e r seven d a y s t h e c l e a r s u p e r n a t a n t is s i p h o c e d o f f

and t h e same volume o f s t e r i l e d i s t i l l e d w a t e r added. t h e s u s p e n s i o n w e l l mixed and a g a i n l e f c s t a n d i n g . A f t e r s e v e n days t h c c l e a r s u p e r n a t a n t i s a g a i n s i p h o n e d o f f , d i s t i l l e d u a t e r added t o b r i n g t h e volume t o 150 l i t res nod t h e n 100 L i t r e s o f U.367 NaCl i s added . The t o t a l volume i s nuw 250

l i t r e s .

The s u s p e n s i o ~ ~ oE ALP0 4 . i s d i s t r i b u t e d w i t h r o n l i n u o u s mixing i n s i x - l i t r e volumes r n 10-litre b o t t l e s A steamed f o r 30 m i n u t e s and a u t o ~ l a v c d f o r f+O m i n u t e s a t 1 2 1 C. A f t e r

,' two o r t h r e e d a y s t h e s t e r i l i t y o f t h e phospha te suspens ion

is t e s t e d . T h i s p r o c e d u r e yLelds hoLLles e a c h c o n t a i n i n g 2

s i x lirres of Alp0 4 s u s p e n s i o n w i t h 6 m1 of aluminium phos- , phete/ml o r 1 .32 mg 01 aluminium p c r m l .

-.p - -

APPENDIX 0 . 2 2

MIXING VESSEL FOR VACCINES (SEE FIGCRE)

M a t e r i a l o f c o n s t r c c t i o n : s t a i n l e s s - a l e e l , good s a n i t a r y

q u a l i t y , e . g . AlS1316, a l l c o n t a c t p o i n t s h i g h l y p o l i s h e d . T h e dimensions a r e chosen a c c o r d i n g t o t h e s i z e of t h e batch which is d e s i r a b l e , and to t h e size of he a u t o c l a v e a v a i l a b l e .

With s u c h s i m p l e a p p a r a t u s magne t ic s t i r r i n g i s p r e f e r a b l e t o o r d i n a r y mixers w i t h s h a f t s , p r o p e l l e r s , e t c . , because n o s p e c i a l s h a f t s e a l s , c o u p l i n g s , e r . [which ofLen p r e s r r t l prc,blems

B ~ , ~ / u N D P / ~ ~ . l Rev. I

page 92

Appendix D .22

for s t e r i l i t y maintenance) are n e c e s s a r y . The l i d i s f l a t , s e a l e d with an O-ring or toElor1 gasketed end held under pressure by e i g h t or more clamps. At the b ~ t t m o f the vessel thcre is an o u t l e t tube. Through the l i d there are aeveral i n l e t t u b e s . lt i s aJv i8ah le t o have one separate I n l e t cube for each compon- ent of the vaccine . The outside par t s of the inlet and outlet tubes are s l i g h t l y roughened to improve the connect ion with rubber, o r , p r e f e r a b l y , s i l i c o n tubing.

DIAGRAM OF A SIMPLE B X l N C VESSEL - ._.- - - - -

. - ,I S l g h t glass .S

- - - - 0-ring seal

- .. .. Entry p o i n ~ s

S t u b i r i

base o f

vessel t o

Crossbar t o p e l l e r i s which d r i v i n g secured magnct and

)' motor are at t a hecl

l ' a k r o f f va l t c

1:-

Appendix D.22

stub i n base of vccscl t o which i m p e l l e r i s secured

+.

. . . . - - Impel l ? r

Driver1 m a ~ n e t

:area

I l - 'f OK D r ~ v i ng magne L

~nounted on metal p l a t e attached

!* With we l l-dts~ gned uxli Ls ta d r l v i n ~ n~htor t h e gap I x t w e e n the magneLs m y be as much as 4 cm. lf t h e t h i c k n e s s U € the v e s s c l

is 6 m, this means gaps between the magnets and the v e s s e l base o f up t o 17 mm.

T y p e o f rnulLiplc ceramic magncl u s e d i n s i d e vessel

hi^ eql;lpn,ent is dcsr i I ~ e d i n d e t a i l i l l Cameran, 1.

b Godf r e y , E. I . , k&utecbuu;. and l i i o e n g . , S m . (1974) NO. 4 , t j : !L-815.

~ L C / U N D P , ' ~ I . 1 Rev. I page 94

B L G , L ! N D P : ~ ~ . ~ Rt,v . 1

page 9 5

APPEND1 X D . 23

SU<;I;ES?!:D S 0 I : K C E S <)I' kQUI PWNT AND

CIIEMI CA 1.S

I'hu l .;Iiilrr,n below is r e p r e s r n t n t l v e O F m a n u f a c t u r r r . i i lr i~m w h ~ l m r t i s p o s s i b l e ( v u b t v i l , c r r l a i n e a u i v m e n t . chemicals. . . , ~~ .. --, <:I.L. l t ~,IIUIIIL? DL. ! i t r t , s s c d t h a t 111 most c a s e s t h e r e are o r h c r s u ~ ~ ~ i l d c ~ % \ r c r i f r c m , whwu sirni lar m a t e r i a l s o i e q u a l qunl1Ly may

-. 1 L . l'hi.5 1 1 ~ 1 , <hcrcfurc,, due5 no t e x p r e s s a 2 ' c l . h compan ies r n r r l l i oned.

1

I ' l l r . -.arnn i l ! , r i n giver) i n t h e manuals f u r d l l i h r l i c r i a , - l , PE,,,,) l.\..,?, %!h, 1 '"a''J

LcLallu.. J I I < I ~ ~ v r l u s s i ! : v o u z i r c c p r v d u t t ~ ~ n and . l . l tes2 KIIIS.WI;KKl;, Cc,L I i I'cderni K " ~ " " l i ' ' ' I 1'crm3r'y

I i ' i L 5 , l l l l . t c L ~ ' r r , a r c n u t supplcn le r~rary . kny picces of -

1 equil'mv~lC cdn u s e d [or t h e p r u d u c t i o n andjor ot - O.i l '2 PI,.

H & l a b 5 e l ~ ~ c ~ r o ~ l ~ , d i l l c l ~ ~ t l V L I C C ; I I ~ ~ . A L t ! ~ ~ u g l ~ 1he . H--.' ! 5 c 1 , " r a t r equipmeut, t h e h u l k t o x n i d pr,,ven bc ::ZCkhfiU ",,, ! . u ~ I P ~ c ~ ~ I CA 92bJ4 ~ L c l - i l r . and n i m - t o x i c , can h-. curther i n t h e same sLaLcs America ' ' ~ L I ioxold and l p c r l u s s i s vaccine a , l ~ Lhe same i ; rp l rn ra lns call br. used . _ l , E U . r C " */h. ~ < ~ ~ > < " I ~ ~ & ~ - " I Denmark

I . / I l ! luclaves for s L e r . l i ~ i n r j undcr sccam p r e s s u r e . One 3 Idrcc a u l i ~ c l a v e (1.5-2 m ) ( L u r t h e fennrnret, mixing v e s s e l ,

c c c . i:. r e q ~ ~ r u d , a s well a s rjne o r LW" s m l l e r ones (100- 0 L . A s o u r c e i l f s t C . a m w i t h a p r e s s u r e of allorlt 2 . i - 1 . 0 Ibar i s r e q u i r e d . 1 r $Learn i s nut a v a i l a b l e Lhen a u t r , c l a v e a w i c h e l e c t r i c stcam generators a r e requ i red .

AUiCO c", , E,i,, Pcnnsy lvan i i l l h 5 1 ? , U n i t e d S l a t e r 06 A n r r i c s

. . k c L c L v c s b r l , s L a i n ~ c s ~ - ~ ~ r c l [or yrrparali:,n "'lturc

. 1 L L , l .- ,r ,aI l ; * , heall',j, , i l L l n g L Y I I ~ . I"" sizes a r e convenient, <,,,,, . I l i l r c . one abuuL 40 l i t c r .

Z l d n 2 i A L L u r e r s a s " " ~ c r 5 allour and :

- SUT.IESM, Beograd, y r l y u s l a v i a ,I.,, C . r - R O I ~ S . l . V ~ ~ l . ~ s ~ ~ ~ ~ ~ . ~ t d . , R i d d e r k ~ r K ~ NeLhcr la"ds .

2. ' l < ' t a i r a l c c t r i c s t e r i L i ~ e r s , for glassware, I : I I h !;ON, z ~ ) z - ? ? H ~ n r k Way. Kings CrL'sS' etc. ['nc l a r s ~ . ( e . g . IaeigI,t 150 cm, " i d L h eO cm, d e p t h

.

l.,,,,d<,,, , b:llr, 1 ~ ' " l W c'1' inLcr l la l d i m e n s i o n s ) and o n e 50 - 8 0 X 4 0 cnl) art: s u i r a b l c .

I? I,(;,' UNDry 7 7 . l Keu . 1 page 9 6

7 . - - C C # I > I A L I I C ~ ,. f U L I . I I I . , L ~ ~ ~ . ~ L ~ t ) n o f f t ' r n ~ c n t e r h a r v e s t . Manu- l r l \ t 1 1 1 . c t t. :arc t t ~ ~ !;:ilnc i l b i 141)dcr 3 and b.

1 1 1 ~ ~ l ' b ~ c L h i ; t R l N ( ; A ( : , ' l ' a l j nan~ t r 1 , 8630 RUii Z H , -- . > h i L.<L,I, 1<1tld

- . ; l ( 1 1 1 . : ~ . i i o x 1 l O / t > , 101 !O l % r ~ ~ m a , Sweden - .. I : . I:I:/\LIb; Eb:L'IIl.I(:EN AI:, 1508 $le l suz~gan, Fcderal Kepubl i c

. ) L ~ : ~ L l l ~ a l l y

- i:llt.I.til' A C , 8 / 0 8 Ma111>cdorf Z l l , S u i tzer land -p

- I : O N ' L ' r \ C ' L ' - K O E ' ~ ~ ' ~ L 1 ( 1 J b I'zMI L L d , R l d t l e e , Nc thcr land - LI.K(;'lHOLIIX, 1'Clii h'?, l I U S 4 ( ; e t i u ~ ~ , Sweden

- hl:W I l t l l lN6WI (:K S['lliNTiI:LC C O , , P . 0 , Box ,m(,, !;(,W I ; t - u ~ l : i w ~ v l < , I J . . I . , l l l ~ i ~ c d S ~ d t e s U F America - -.

9. ! : : ~ ~ L ~ I ~ I ~ ' I I L t , ) r L i l l i n ) ; t ~ d l k vacciric i n t o f i n a l conLainers .

- jiKEN(:O i m h l f , 7 0 2 3 Er,h t c r c l i n g ~ , Fedcra l Republic 1 8 i (.crnlany

- ~WNIIPACE, 20145 Milan, l t a l y

- S'IRIIERS , Copenhagen, Denmark

- STXfJNCK AND CO., 5 KUln 3 0 , Federal Republic o f Cc r m n y

I t ) . E c l ~ ~ i p n ~ c n t Fur w a s h i n g v i a l s . Msnufacturers same a s u r ~ d c : ~ ~ 0 .

- I :~ :LWLN I n ! ; r ri lrrrc~~r S P A , IUOOYO Oprrir (h), I t a l y --

BLG/UNDP/~~-I Rev. 1

page 97

Appendix D.23

- S1:HlRP K G , 6711 Bark, Pederal Republic of Germany

- nlE BABEER CO, Sanford, Maine 0 4 0 7 3 , United Gtaten of America

1 l?. Equipment for pr in t in9 v i a l s

I - ~ U P A AC, Zurich 8, ~witzerland

1 3 . Water bath for f l o c c u l a t i o n r e a c t i o n . S u i t a b l e type i s "UNITEMP" Water Bath, C a t . No. 304/1600, Baird 6 Tatlock,

i 4 Chadrell H e a t h , Essex. England.

11. F i l t r a t i o t ~ equipment. I t i s necessary t o have various kinds - one f i l t e r p r e s s , with about 20 f i l t e r p l a t e s 20 x 2 0 cm; one m u l t i s h e e t s t a i n l e s s - s t e e l f i l t er with about 12 pla tes 20 x 20 cm; three s i n g l e sheet s t a i n l e s s - s t e e l f i l ters

with r e s e r v o i r s of 10 l i t r e s , two l i t r e s , 250 m1 each.

- BRITISH FILTERS L t d . , Cot Green, Maidenhead, Berkshire S t 6 3AG, England

- CMLSON-FWD Ltd., Ashton-under-Lyne, Lancashire,

1 England

l - SEITZ Werke GmbH, 6550 Bad Kreurnach, ~ e d e r a l

Republic of Germany

15. kmbrane f i l t e r s are very useful f o r f i l t r a t i o n of slmll / volumes of valuable 11116s (e.g. cuncenhmied and pur i f i ed ; ..: t o x o i d s . e t = ) . one larger f i l t e r (dimeter about 30 cm), end j4,: . lone smiler (0.14 FP) a r e necessary. I 1 < ! " $ .

t T; . . - MILLIPOKE CORPORATLON, Bedford, Massachussettr 01730,

'a , United S t a t e s of America

! 2 - SARTORIUS-MEMMIAEIFILTER GmbH, Gpctingen, ~ e d e r a l

; $ ? . S , R e p u b l i c of Germ8ny

-, a='

B L G / W D P / ~ ~ . ~ Rev. l

page 98

16. H e t a f i l r e r f o r t h e f i l t r a t i o n of b a c t e r i a l s ludge.

- METAPILTRATION Co. Ltd . , Hounnlow, Middlesex, England

17. Pure water appara tus . Capacity approximately 100 l i t r e / hour. I t is convenient t o have deminera l iaed water f o r washing and g e n e r a l purposes, and pyrogen-free d i s t i l l e d water ("water for i n l r c t i o n s " ) f o r the p r e p a r a t i o n of i n j e c t a b l e a . Equipment f o r dcmlnera l ized (de ion ized) water i s produced by msny -nu- f e c t u r e r s . e . e .

B L G / ~ P / ~ J . I . R ~ V , i page 99

Appendix D.23

20. CenLrifuge, continuous fLow,is necessary f o r handl lng l a r g e q u a n t i t i e s of t h e p e r t u s s i s suspension i n fermenter c u l t u r e . It should bc e l e c t r i c a l l y d r i v e n , about 20 000 rpm, wi th t h r e e t o four o n e - l i t r e bowls

- A ~ A - ~ V A L A . B . , BOX 1008, S-22103 Lundl. Sweden ( ~ ~ ~ t i ~ c l a r i f i e r , type mH hO9-34H-11)

- C. PADBERG CmbH, 7630 Lahr, ~ e d e r a l Republ ic of . ~- Germany

- b n e End, ~ u c k s HP14 ~ J H , England S H ~ ~ p ~ ~ ~ - ~ m ~ ~ ~ Div., Pennwolt Corp.. Warminster, pa. 18974, United s t a t e s of America -

MILLIPOHE CORPORATION, Bedford, Hassachussets 01730, United S t a t e s of America

21. Preeze-dryinR smal l ( [o r d ry ing 20-M

f o r maintenance of s t r a i n s ) High q u a l i t y d i s t i l l e d water a t low c o s t (no cool ing by t h e m o c w p r e s s i o n p r i n c i p l e is by: - eDWARDS V ~ C ~ ~ ~ ~ t d . , nor ~ o y a l , Susser, '"gland

- STIIwS SPA, 20090 S e t t a l a , I t a l y - PhBRLCA INSTuUmNTAL CIENTIPICO, Dr Juan F. Arenauren 76/78.

1405 Buenos A i r e s , Argentina 18. shakers . A small shaker (e.g. for nine - U S I F R O I ~ , 92 ~ ~ ~ l o ~ n e - ~ i l l a n c ~ u r t 9 France Er lenme~er f l a s k s of one l i t r e capac i ty ) f o r making is required. I f product ion of p e r t u s s i s i n shaking f lasks is 2 . Pan filter ,,,,its, t o produce a c lean atmosphere.

then one o r s e v e r a l l a r g e shaker s are required. - H I C B O F L G W , F ~ e e t ~ l ~ , ~ n l e y R o a d , ~ ~ f i an t s~

- EmNVEE; Gul tekdi , e, I n d i a Filter candles fo r u l t r a f i l t r a t i o n and filtration of

- JANKE U . KUNKEL IKA, 7813 Staufen, Fede ra l Republic t e r i a l s ludge. A t l e a s t 2* Pieces. o f Germany

- B ~ R K E P ~ ~ FILTER, 3100 C e l l e , Fede ra l Republic - NE" BnUNSUICK SCIENTIFIC Co., P.O. BOX 606, New Brunswick, N . J . , United S t a t e s of h e r i f a of Germany

- SCw*Cm FABRIK, Bietigheim, m r t t , Federa'

lY. C e n l r i f u ~ e . r e f r i g e r a t e d , gene ra l purpose, w i t h m u l t i p l e Republic of erm many 'p t u 5CD0 rpm, With h igh speed attachment 50 000 rpm.

Maximum t o t a l capac i ty about 6 1 . 6 litres. ~ c r o s c o p e , binocular, " i t h in t e rchangeab le eyepieces ~ b j e c t i v e s , o i l immersion. -

BECKCIAN INSmUi'lENE Inc . , F u l l e r t o n , CA 92634 United S t a t e s of America

- Ltd. Crawley, -, England

'5. S p u c t r u p h o t m e t e r , for g e n e r a l a n a l y t i c a l p u r p o s e s , e . g . :I I i c c l i~~an b p . EY or i i m i l a i i n a t r u m n t ,

1 i

I I 2 h. K . J ~ ldah 1 oppara t u s f o r n i t r o g e n d e t e r a i m t f on, p r e f e r a b l y

m i c r o t y p e . O b t a i n a b l e from any l a b o r a t o r y s u p p l y house s .

l * B L G / U W P / ~ ~ . ~ Rev. l

page 1M) i

I I 1 , 27. Cold rooms ( t ~ O C ) and i n c u b a t o r roams (+ ? ~ O C I . I t is I' . I ! p u s s i h l c t o buy f i x e d ( b u i lt-it11 o r s e c t i o n a l "walk-in" incuba-

I j t o r s to be a s semb led on s i t e . The advaateges are t h a t they i j can hc en la rged i f n e c e s s a r y by a d d i n g s t a n d a r d s e c t i o n s .

11 !

- IllIURRE OK, 33541 Tampere 54, Finland

- I.TH, ~ L U F J A LOKA; Yugos lav ia

i - VOLTAS LTD., Bornba~, I n d i a ( c o l d roome + &'C, - ~ 0 ~ ~ 1

I l 2 8 . Washing cquipmen~ f o r lassw ware. G e n e r a l l y s s i m p l e

i " b o t t l e b r u s h e r " may be adequa t e .

- THE THOMAS HILL ENGINEERING CO. LTD,, M, Yorks , England

I f more r e f i n e d equipment i s r e q u i r e d , then t h e mtlnuflc- turers of auLomat ic washers and d r y e r s s h o u l d be c o n s u l t e d , e.g.

- BETTER itUILCI' WCHINERY CORP., S a d d l e Brook, N.J. 07662, Dnited States of America

- METZSCH CebH, 8 2 U Waldkraibur~, F e d e r a l Repub l i c of Germany

, S , 29 , Warlng o r s i m i l a r type of b l e n d e r / h o m o ~ e n l z e r , f o r horno-

. , g e ~ r i a a t i u n of p e r t u s s i s s u s p e n s i o n . To be o b t a i n e d Erum . .

; : g c n e r a l I a h a r a t o r y s u p p l y houses ( s e e N O L ~ ) .

l Ncjrc . Sonit. of t h e above-mentioned equipment , a s w e l l as a l l

I q m a l l equipment, g l a s s w a r e , p l a s t i c w e r e , e t c . , can be g I oI1taint.d from ge l l e r a l l a b o r a t o r y s u p p l y houses such

! as :

BU;/UWP/~~.~ Rev. 1 psgc 101

I Appendix D .23

- mTllUn H. TIIOHAS CO., P.O. box 7 7 9 , P h i l a d e l p h i a , P a 191U5, Uniced States of America

- MIRU AND TA.rWCK, F r e s h w a ~ e r Road, Chadwell Heath, Es sex , England

- FISHER SCIENTZFLC CO., 711 Forbes Ave, P i t t s b u r g h , Pa 15219, United S t a t e s of America

- G A L L E N W , P.O. b x 290, Technico House, C h r i s t o p h e r S t r e e t , London ECZP 2 E R , England

l, Chemica ls and cunsumables

30. (Uio) chemica l s

- A T U S CHEEUCAI. INDUSTRIES Inc., Wilmington 99# Delaware, U n i t e d S t a t e s QI h e r i c a (Darm c h a r c o a l )

I.' - THE BRITISH DRUG HOUSES LTD., P m l e , England

- CALBIOCNEM A G , 6000 Lucerne 5, S w i t z e r l a n d

- MALLlNCKRODT CHEMICAL WORKS, 223 We8 t s i d e Avenue, P.D. Box 304, Jersey C i t r , NJ 07303, u n i t e d States of America

- E . MERCK, 51 Darmstad t 2 , F e d e r a l Repub l i c of Ge-ny

- NOR l T SALES CORP. L td . , Amsterdam, Ne the r l ands

, . ( N u r i t C h a r c o a l )

- :;LGMA CtlEk[ICAL CO., P,O. Bax 14508, -1

Mis>otiri 63178, Uni t ed S t a t e s of America .F r : ..

: . . .. . l , ~ a c ~ I . r i u l o g ~ c a l s , c u l t u r e media, F I E . : S m e f i r m as (IboVe ,. , m < ,

3 % k -#:+ .; and s p e c i a l i s t s : , > ; : d . . 2- ... .;*h .gi: - I;ENCAL IMMUNITY CO. , C a l c u t t a , I n d i a ( t r y p t o n e s P

,I. L . . W casein digests f o r t e t a n u s medium) ;;:%g:. . id> - U ~ F C O L4WIRATORIP.S. D e t r o i t , Wchigan 18ZJ2, United

ELG/UNDP/~ 7 . l Rev. 1

page 102

Appendix 11.23

- IhSl'ITUZ' PASTEUR PRODUCTION, 36 rue au :)octeur Koux, 75725 Par i s , Cedex L5, France

- U X O U LTU., kade Road, Basingstoke, nants RC24 OPW, bngland

- bHEFFIELL) CKEI.ILUL, 2400 Morris Avenue, Union, hev Jersey 07003, United Scaces of America (N-2-Case TT, 14-2-Amine A)

- COFLUIhG CLASS WOIORCS, Corning, hY 14836, united States of America

- J W S A . JObLING & CO. LTD,, Ualtone , Stone, Staffordrl>ire, ST15 UBG, hngland

- JENAER GLASWERK SCHOTT U. GEN., 65 klainz, Federal Republic of Germany

- VLB JENAER GLASWERK, Jena, German Democratic Republic

33 . Filter-paper, f i l t e r materials

- JClHNb MANVILLE, Greenwood Plaza, aenver, C o 80217 , Lnited States of America (t lyf lu F i l t e r Aid)

- REPUBLIC-SEITZ FILTER CQWORATlON , P .U. Bux 229, Mil ldale , ConnecticutA United States of America ( R e p u b l i c F i l t e r Papers)

- SCtlLELCHER U. SCHULL Gmbtl, 3354 Uassel, Federal Republic of Germany

- MMl'MAN INC., 9 Bridewell P l a c e , C l i f t o n , Hew Jersey, U n i t e d States of America (Reeve Angel)

- UltlATtMN LTD. , S p r i n g f ield Mill, Maids tone, England

- See also manufacturers of f i l t r a t i o r ~ equipment , item 14.

3 4 . Antifoam

- UOW CHEMICAL C O . , Indianapolis , Ind. 46260, United hrates of America

DLG/UNDP/~~ .l Rev. 1 page 103 Appendix D.23

- V ~ U K N F A I I R ~ K E N EAYER AG , kverkusen . Federal Republic of Germany

- HOPKLNS AND W L U I A H S . England

Rubber and plastic-ware.

- DmAWH C ~ P O R A T L D N , BOX 111, Rochester 14W1 S

Upiied S t a t e s of America

- NA~X;E C O . , 7 5 Panoram Creek Drive, Rochester, NY 14625, United States of America

- V ~ , ~ I I A - I ; U ~ K k b ~ , 518 Eschweiler, Federal Republic of Cermny

- VERNERET, F-44201 ~ v r y a / ~ e i n e , France Appendix D. 24

su-y PROTOCOL FOR DIPHTHERU *XOID ~ ~ U C T Z O N AND TESTLNG

L D E ~ ~ ~ Y I C A T I O N OF FINAL LOT

address of manuEacturer

t e of manufacture of f i n a l lot -

inal product (plain or

o f r e c m e n d e d single hulnan

-

BLG/ITNDP/~~.~ R e v . l

page I&

Appendix D.24

1. S t r a i n

Identity of C. d iph . tber iae strain used i n vaccl-

2 . S i n g l e harveat included i n f i n a l bu Lk * - Medium ueed

Period of i n c u b a t i o n

Appendf X D . 24

5. Adjuvant added

Nature

mg/~l r>r Ca

6. P r e s e r v a t i v e

Ne ture

C a n c e n t z a t i m i n f i n a l product thy assay or ca lcu la t ion)

Date of e a r l i e s t harvest i n c l u d e d

" l 7 . Buffer used - Condit ions of storage

concent ra t ion 3 . Bulk pur i f i ed toxoid

Reault of t e s t for ant igen lc puri ty L,f/mg p r o t e i n N

BLC/UNDP/~?.I Rev. L page L W

4 . F i n a l bulk k . ' Date of prcpara tion

Lf per m 1

Result of t e s t f o r re s idua l free f armaldehyde

pH

1. S t e r i l i t y

Date oE t e s t and r e s u l t

URS a tepcet t e s t necessary?

TESTS ON FINAL BULK

BLG/UNDP/~~.~ Rev. 1

page 105

Appendix D . 2 4

l N F 6 ( W I O N ON SAFETY AM) POTENCY

bulk p u r i f i e d f i n a l bulk or toxoid f i n a l lot

2 , S p e c i f i s toxicity test

Numbers of animals

Date of injection

Dose of toxoid in jec ted

Route of inJection

Period of t e s t

Result of t e s t

3. Potency test

Weight of anima 1s

Number of animals per dose of toxoid

Date of immunization - Date of challenge or

bleed

Chsllenge dose of toxin

Date of end of t e s t

B L G / U N D P / ~ ~ . ~ Rev. l

page 106

Appendix D. 24

I WSULT for- l e tha l chal lenge t e s t ( i t done)

l Potency of t e s t toxoid I U single human dose

95% conkidence l i m i t s :

l RESULT for multiple intradermal test (if done)

D i l u t i o n i

Mean score

ED 5 0

.. . .. . .. m1 -

........ m1

Dilut ion

Reference toxoid 1.u./*1

Test toxoid

Survivors Total

I t I I

, - Potency of test t o x o l d 111 single human dose

I

!

-; P

, 95% confidence limits: , - . -..:

. . ; . . % ,,,.

TESTS ON FINAL LOT b l r-

X, ' . ;.$?I: 1. Identity test.

%- +'. . *,- . ..#.P. .. . \

'..;.~1:. Test for diphtheria toxoid and resu l t

Test for tetanus: toxoid and r e s u l t .&.

Test f o r oertussis and result

Reference toxoid 1.u./m1

Test toxoid

I - ,

B ~ / u M ) P / ~ ? . 1 Rev. 1 page 107 Append ix U.24

S t e r i l i t y t e s t

Number of containers examined

Method of t e s t

Date o f s t a r t of t e s t

D a t e of end of test

Result

3 . Potency tes t

I f not done on final bulk, report this under f i n a l bu lk .

4 . Innocuity test

Number o f animals

Route of i n j e c t i o n

Volume of i n j e c t i o n

Date of s t a r t o f t e s t

Date of end of t e s t

Result

5 . P r e s e r v a t i v e content

I ILG/UNDP/~ 7 . l Rev. l page 1 0 7 A

Appendix D . ? 4

ResuLt of pH Lest

S i g n a t u r e of head 3 1 laboratory

C e r t i f i c a t i o n by person tnking overa l l re spons ib i l i ty for produc t inn of the veccine.

I c e r t i f y that l o t No. of the vaccine sa t i s f i e s Part A o f

the wno Requirements f u r Diphtheria Toxoid.

I. Signature

Mice

I Name typed:

Guinea-pigs

-

Concentration of preservative I

me orotocol must be accompanied by a sample of the labe l and a

BI.G/UNDP/~ 7 . 1 Rev . 1

page 108

APPENDIX D.25

FLOW CHARTS AND DATA RECORDING SHEETS

I n t r o d u c t i o n --- I t is u s e f u l f o r a producer of vaccine~ t o make s o - c a l l e d

"flow cha r t s " of the ~ h o l e ' ~ r o c e s s . Sume examples o f such f l ow c h a r t s a re shown in t h i s appendix .

I n t h e test of this manual a l t e r n a t i v e s have been desc r ibed for many steps i n the product ion process, The spec i f i c flaw c h a r t , t h e r e f o r e , w i l l depend on which of the a l t e r n a t i v e s has been chosen f o r each indivLduaZ s t e p i n t h e procedure.

I c i s u s e f u l a l s o f o r t h e l a b o r a t o r y workers t o r e c o r d da ta on work s h e e t s t h a t can be photocopied and accompany the ba tch of vaccine a s i t proceeds through the p roduc t ion process . Examples of such d a t a s h e e t s a r e a l s o inc luded i n t h i s a p p e n d i x .

EXAMPLE OF FLOW CIIART FOR THE PRODIICTION OF DIPHTHERIA TnXOID (FERMENTER CULTURE)

Prepa ra t ion of tux i n -

C . d i p h t h e r i a e l y o p h i l e recovered i n NZ Amine medium and grown on 2 L o e f f l e r s l o p e s f o r 24 h a t 35'

Seed f l a s k inocu la t ed wi th g r ~ w t h from one ~ o e f f l e r slope, Crown f o r 24 h a t

35' on shaker

NZ Amine medium p repa red

i r o n content i' ,

purity

0.5 m l seeded on s e v e r a l I.ocf f l e r s l o p e s . Grown 0 far 2 4 h a- 35 . These can b$ s t o r e d a t 4 f o r 8 weeks

BLG/UNDP/~~ .l Rev. 1 page 109

Appendix D. 25

Ferment e r , c o 2 t a i n i n g an t i foam A i n light m i n e r a l o i l , s tori l i z e d ( au toc laved)

f 120 l i t r e s of medium s t e r i l i z e d by f i l t r a t i o n i n t o fermenter

Seed f l a s k inocu- l a t e d with growth 5 f r o m one L o e f f l e r S t e r i l i t y s l o p e . Crown f o r 26 h a t 35' on 8 haker

a C u l t u r e

p u r i t y Lf con ten t

. I

F e r m e n t e r seeded w i t h c o n t e n t s of one seed

. . f lask; , Grown f o r 4 6 h a t 35 w i t h secation and s t i r r i n g . Sampled a t 24 and h8 h.

v

BLG/UNDP/~~.~ Rev. L page L10

Appendix D . 2 5 .h

4 C u l t u r e pur i ty ( 2 4 and 48 h samples) (page 12)

Lf c o n t e n t (24 and 48 h samples)

4 0.4% to luene added

Contents ot 2 fermen- cers pumped into p o o l i n g tank

------- B --_C_

D - I n p u t or o u t p u t O - P T O C ~ S . stage U - sWpled and

h e l d f o r dec i s ion

EXAMPLE OF FLOW CHART FOR THE PRODUCTION OF

D1 PATHERIA M X O I D

Pur i f i ca t ion of t o x i n -

f C u l t u r e p l u s t o l u e n e

P NaHCO (0.5%), charcoal Metitfilter ( 0 . 2 5 2 ) , ammonium ~ " 1 - precoat ed face ( 2 3 % ) and l lyf lo w i t h Hylla guP@rcel (9 kg) adlied supercel

BLG/uM)P/~~.~ Rev. 1

page 111

f Suspens ion filtered ~ h r o u g h e t a f i l t e r and filtrate c o l l e c t e d

D Lf content

h o n i u m su l fa te (21%) and nyflo wpercel (400 g) added w i t h sc i rr ing and t h e suspension stored overnight #L kV

Precipitated toxin separated by f L l t ra t i o n

,I ... C," . .~.. . .;C: , .;- .. ,

' :r

Toxin dissolved i n saline, f i l t e r e d and d ia ly sed aga ins t s a l i n e

Toxin recovered from d i a l y s i s sacs and pooled

Lf cantent Puri ty ( N . D , N . )

purified, concentrated t o x i n

-- - D - Input or output 0 - proces. sragc 6 - s a m ~ 1 . d h e l d for and

d e c i s i o n

B M/UNDP//~ 7. L Rev, 1

page 112

8 1 , ~ / ~ ~ / 7 7 .l Rev. 1 paEe 113

9 6

I r r e v e r s i b i l i t y aL 50 if Per m1 Lf coilt err1

P o t f ~ i r y Thionersai c o u t f u t P u r i t y iN.D.N. 1" P y r n g e n i c i t ~ S t e r i l i t y

EXAMPLE OF FLOW CHART FOR THE mODUCTION

OF DIPHTHERIA TOXOID

Preparation o f t o x ~ i d -

f P u r i f i e d , concentrated t o x i n

h NaHCO (0.5%), lysine 10.05h, and formalin (0 .5%) added t o toxin

0 S t u r t d a t 2 - 8 a s hulk concen t r a t E I at 500 L£ per m1

I S o l u t i o n f i l t e r e d through EKS pads i n t o 4 x 20- l i tre pyrex bot t les . B o t t l e s incu- EXAMPLE CIF FLOW CHMT FOR THE PR@\)I CTlON

01: LllPHTHERlA TOXOID baked for 3 weeks ac e 25 Preparat i on of f i n a l p roduc t - "diphtheria - toxoid" Toxicity (guinea-pig skin)

0 Concentrated ~ ~ ~ - p y ~ o g e n i c , diphtheria s t e r i l e

phosphate - b u t fered s a l i n e P Rott Ies i n c u b a t e d f o r

4 weeks e t 34' U

T o x i c i t y (guinea-pig skin)

U l t r a f i l t e r lq prepared l- Thiomersal added as

preservative to f i n a l . concentration o t C.OLL

F i l t e r e d Lhrough a 0 . 2 2 m i c r o n rr.~?rnbra~~e f i l t e r P Bottle c o n t e n t s pooled

and u l l r a f i l t e r e d

0.375X 2-phenoxyethanol added

P S t e r i l i z e d (filtered t h r o u g h EKS pada hacked with Whatman 50 f i l t e r p a p e r s )

B L C / U N D P / ~ ~ . 1 Rev. 1

Page 114

Appendix D . 2 5

Innocui ty Lf c o n t e n t

Potency

h To Filling Deparcrnent

EXAlIPLE OP FLOW CHART FOR TtiE PROD'JCTION OF FINAL PRODUCT OP DPT VACCIIGL

S t e r i l i z e d ( a u ~ o c l a v e d )

I- .' -1 Required a m o u n ~ Reyulred amount of c e ~ e n ~ l s o f d i p h t h e r i a Loxold d i s p e n s e d t o x o i d d i s p e n s e d

l Added LO A11'0 Added t n AlPO

s u s p e n s i o n an8

A 1 tored t o stand I

A l l w e d to s t a ~ t d Required arnrJunt Tor 2b h o u r s at fo r 2 4 hours a t f of yet t u s s i s

v a c c l n r d l - - l - --- - - - - - -

1 pl.~nse.t

n l , ~ / U N t ~ ~ / 7 7 . 1 Rev. 1 page 1 1 5

T Adsorbecl tetanus and d i p h - theria tuxoids blcnded i n f i l l i n g tank and p e r t u s s i s v a c c i n e co-streamed i n t o

Required rrn~ount a £ sodium c h l o r i d e added as a stcrile 0.5VL s o l u t i o n

'fiinmersal added to e £irnal c o n c E n r r a r j url of 0.01X

[,;,nit cnnients s t i r r e d , a l l ~ w e d t o s tand 26 h n u r j , s t i r r e d ngaln and sarn~llcd

A l u n i r ~ i u m cotltenL General safcLy

Mouse t o x i c i t y

Potency: U i p h ~ l ~ ~ r i f l 're t a w s l ' e r ~ u s s i s

e s e r v a t i v e conLent S S t e r i l i t y

Tank s e n t tu F i l l i n g Depar tmerlt

.D - - , _P____ -- --

D- TnPt lL o r u u ~ p . ~ m - p r o c e s s s a t '3 - ~ ; a . ~ l ~ d ~ W J

h e l d f o r decision

BI ,G/UNDQ 7 7 . 1 Rev. l page 116

SUGGESTED FORM FOR REPORTING DATA ON PRODUCT1 ON OF DIP11 F H E K I A TOXOID

DIPHTHERIA TOXIK PR1)DLlCTI ON

N o . A t h e crl l ture:

S t r a i n of C . d i p h t h e r i a e :

S e e d c u l t u r e -- b d i urn : DaLe o f preparat ion: Date of i n o c u l a t i o n : Microscopic c o n t r u l :

Production i n b o t t l e s

Medi urn: )ate of preparat ion: 'otal vo lume: ,~rnber of b o t t l e s : ' mtents per buttle: : re of i n o c u l a t ~ o n : hate of harvest : Number of battles harv

harvested: ~ f / n r l : N: ............. ,mg/ml PN: ............. .rng/ml ~ f / m ~ PN :

S p e c i a l remarks:

or P r o d u c t i o n i n fermenter

Medium: Volume : Date of prepara-

t i o n : Dxte of i n n c u l a -

t i o n : Growth p e r i o d : ...... hrs Sample dur ing growtli:

... ~ f / m ~ , ...... . ~ f / m ~ I'N; I t I ,

F i n a l t o x i n : ~ f / m l l

: . . . . . . . . . . . . . . .mg/ml PN: . . . . . . . . . . . . . , . rng/ml l.f/rng PN:

S p e c i a l r e m a r k s :

Volume o f t o x i n . 'X formalin a d d e d : cmc of f o r m . u s e d : date of incubation: end oi i n c u b a t i o n : L , i / m l :

1 T o x l c i t y contro l - Number of g u i n e a - p i g s . Volume ~ n j e c t e d : Date of i n j e c t i o n . Period of observa t iua . Results:

No, of the purified b a t c h :

D a t e o f s t a r t i n g : . NO. ( S ) of t h ~ t o x ~ i d batch(es) u s e d :

Tota l volume : . . . . . . . . . . . . . l iLr"s . b t h o d n f p u r i f i c a t i o n :

. . . . . . . . . . . . . . . . . ,Lf/rnl

. . . . . . . . . . . . . . . . . .rngjml

. . . . . . . . . . . . . . . . . . mg/m L

. . . . . . . . . . . . . . . . . ~ f / m a

mary conccfitra t ion procedure

a f t e r concentraLion: . . . . . . . . . . . . . . . . . . , , , , . . . . . . . . . .

RIG,/ LIN111'/77. 1 Kcv. 1 psgc 118

HL(;/'IJNUF, 7 i . 1 RC'". I page 119

Experimental curve % A . S . % 1.f

( R A s S . added per LW m? precipitated

L i m i t s uf s a l t i n g - u t chcl3cn:

% A . S . - . . . . . . . . .S-. . X A.S.

....... Volume before s a l t i n g - n u t : l i t r e s First prec ip i ta t ion: , . . g A . S . p:[! . , . . . ... Second p r e c i p i t a t i o n : . g A.S. pH:

. . . . . . . . . . . Volume a f t e r d i a l y s i s : l i t r e s Control of absence of amonirlm: Ana lys i s : . . . . . . . . . . . . . . Lf/ml

. m g / m l TN . . . . . . . . . . . . . . . . . . . . . . . . . . . mg/ml PN

l ~ f / r n ~ S . . . . . . . . . . . . . . ~ f / r n ~ PI . . . . . . . . . . . . . .

Recovery: . . . . . . . . . . X !

S t e r i l e f i l t r a t i o n : Preservative added: Conc. of p r e s c r v a t i v c :

I Method:

l D a t e : Result:

'roxici~y t e s t c i f d ~ m ) I - QuantiLy in . i ec ted per K . P . : Number u t g . ~ . i n j e c ~ e d : PaLe of i n j e c ~ i n n : Observation p e r i o d : uesult:

Appendi X D. 2 5

f Volune vi p u r . m x g i d ( d useJ ( L S more than ane b a t r h ha3 bt'cn .4 used s t a t e y o l u n ~ e s t o r cacti h a r c h se~ahately):

nal volume vaccine batch: . . . . . . . . . -li"es

( i f a p p l i c a b l e ) :

Conc, of adjuvant in v a : c i n e : Me~hoa of preparat ion:

S t e r i l i t y c o n t r o l d a t a :

~ I . o ~ \ ~ c ~ . I I s i . > r

BLC;Uh3P/77 . L R e v . 1 page 110

Appendix 3 . 5

Compos<tion of v a c c i n e

Diph. toxoid: . . . . . . . . . . . . . . . . . . .Lf/ml T e t . t o x o i d : . . . . . . . . . . . . . . . . . . . L f / d P e r t . v a c c i n e : . . . . . . . . . . . . . . . . . . . O u / m l b f a p p l i c a b l e . ~. Adjuvant : . . . . . . . . . . . . . . . . . . . / m& . . . . . . . . . . . . / mL P r e s e r v a t i v e : Con'. o f p r e s e r v a t i v e : S i n g l e human dose: . . . . . . . . . . . . . . . m 1

P r e p a r a t i o n of v a c c i n e

m 1 d i p h . t o x o i d . .Lf/ml ..... ............ m1 t e t . t o x o i d . L f / ! ~ l ..... . . . . . . . . . . . . . m 1 pert. v a c c i n e .OC/m1 {if a p p L i c a b l c . . . . . . . . . . . . . . . . . . m1 s a l i n e ..... m1 c ~ ~ ~ . a d j u v e n t .mg/ml - i f a p p l ~ c a h l e ..... . . . . . . . . . . . m 1 p r e s e r v a t i v e s n l . .mg!ml ..... . . . . . . . . . .

F i n a l pH . . . . . . . . . . . . . . . . D a t e :

C o n t r o l s of f i n a l bu lk

STERJLITY TEST

Performance of t e s t :

R e s u l t s :

Date :

S p e c i i , i c t o x i c i t y c e s

Number of g.p. i n j e c t e d : Dose i n j e c t e d p e r g . p . : P e r i o d o f o b s e r v a t i o n : Kesu lL:

BLG}UNDP/~?.~ Rev. 1 page 121

l Date. Number o f g . p . u s e d f o r v a c c i n e liumher o f g , p used f o r r e f e r e n c e : h t e on r e f e r e n c e v a c c i n e :

I ' ~ ~ ~ ~ l t s of L e s t :

C a l c u l a t e d (minimuml Potency: lu /ml

i ' . ~ r e e formaldehyde ( r e s i d u a l )

et hod used:

R e s u l L :

.. FILLINI: OF FINAL CONTAINERS:

~ u m b e ~ o~ fina: cunr. f i l l e d : volume p e r c o n t a i n e r : . . . . . . . . . . . . .m1 D a t e ( $ ) o f f i l l i n g : Number of lots:

.B. more than one f i n 2 1 Lot h a s been Cil1e.d ( i . e . in more than one working s e s s i o n ) t h e r e s u l t s o f the following s h a l l be s ~ a L e d f o r o f t h e s e

l o t s .

B L G / U N D P / ~ ~ . ~ Rev. 1 page 122

T o x i c i t y t e s t

Method:

R e s u l t :

Da t e :

S t e r i l i t y t c s r

Method:

R e s u l t :

Date:.

Potency t e s t - Only t o be d e s c r i b e d ( a s above f o r " f i n a l b u l k " ) i f no t

done on t h e f i n a l b u l k .

I n n o c u i t y t e s t

Number of g .p . i n j e c t e d : Dose p e r g . p . : O b s e r v a t i o n p e r i o d : R e s u l t : Da t e :

Number OF mice i n j e c t e d : Dose p e r mouse: O b s e r v a t i o n p e r i o d : R e s u l t : Date :

I n s p e c t i o n o f f i n a l c o n t a i n e r s

I n s p e c t i o n p e r f o r m e d : Number of f i n a l c o n L a i n r r s d i s c a r d e d : Reason:

BLG/'UNDP/ 7 7 . 1 Rev. 1

page 123

Appendix 0 . 2 5 --

Y Z S

I1 I, II

m m m D U W l. C Y I ) U L1 .r( .A .d 4 4 d

X X *

U U U Y Y L U Y Y 4 .3 .A 4 - 4

C Y Y U D a 'L P P

* u w a D i r l O C - 0 0 0

5 8 8 8 D C * - N m - " U U .. ..

g U

o

2 W

L

m u D

2.: .. .2 C I c x - u ' , D O . * O

0 - 0 m 4 E X > 3 0 m o

U . r l € - V)

& U - ( O U D S u l

C L a m z . r l u c 3 O S m u U 'L" U

C r n . r l O D z > e * =

2

S , m

C r Z f I!

W

W L* -

2

< D a t e -

C . 10% Thiomersa l r e q u i r e d 1 m 1 p e r l i t r e o f v a c c i n e = m1

Vslume of v a c c i n e - - m 1 L e s s : Volume of c o n c e n t r a t e = m 1 L e s s : Volume of p r e s e r v s -

10% Thiomersa l added

STIR DURING ADDITIOK OF COEIPONFNTS AND BEFORE SAMPLING

1 C . D i l u e n t r e q u i r e d

-

S i g . Lot N o . m 1

4ig. S i g . S i g .

m1 m1 m 1

Aluminium Phospha te Susp. S a l i n e s o l a t i o n ist tilled w a t e r

- Volume

30 m1

40 m 1

25 m 1

Lot No.

COMXXTS

T e s t

S a f e t y

R e f e r e n c e

S t e r i l i t y

T e s t

~ l ~ ~ i n i ~ m c o n t e n t

O s m a l a r i t y

PH & T h i o m e r s a l

Po tency D i p . & T e r .

Potency P e r t .

Volume

15 m1

15 m 1

15 m1

30 m 1

20 m1

B L C / U N D P / ~ ~ . ~ Rev. 1

pap,c 126

Appendix D.25

SUGGESTED FORM FOR RECORDING S U W Y DATA CLIECK 1.1 ST FOR DXPHTHERIA TOXOII)

Sample Test R e q u i s i t i o n Form

P r o c e d u r e As per WHO or appropriate control a u t h o r i t y

,' --- - - - --

----P-_

B L G ~ ' ~ P / ~ 7 . L Rev. 1 page 127

Appendix D. 25

P r o d u c t Lot No.

l Signa Lure Date Approval

_ _ - p - I

Desi rablc Limits

R e s u l t

C m e n L

Conclusion

AS specified

-L

._

-

- ---

R e q u e s t From R e q u e s t l 1 Da :- F

Check Test R e q u i r e d --

Aluminium Content

Culture Purity

CombinLng Power

Free-formerin Contenr

General Safety

Iron Content

I r t e ~ e r s i b i l i t ~

Lethality

t + / ~ r ~ o s s

Lf C o n t e n t

-

I

Mouse-toxoc~iy

Ni trogen Con- tent

(Non-dia 19- s a b l e / ~ o t a l )

Pll

Pntency

Preservative C o n t e n t

Sterility

Toxicity

[Systemic)

MRD Guinea- p1g S k i n

-

BLG/LJUDP/ '~~ . 1 Rev. 1

p a g e 128

Appendix D.26

REQLIiREIIENTS FOR D I P H T H E R I A TOXOlD

GENERAL CONSIDERATIONS

D i p h t h e r i a t o x o i d was one o f the e a r l i e s t . v a c c i r l e s a v a i l a b l e f a r p r o t e c t i o n a g a i n s t a b a c t e r i a l d i s e a s e a n d i c s use vhen o t p r o v e n e f f i c a c y i n i m u n i z a t i o n s c h e d u l e s krloun t a g i v e good s n t i t o x ~ n r e s p o n s e s has m a r k e d l y r e d u c e d t h e i n c i d e n c e a t t h e d i s e a s e .

Tbe e a r l y d e v e l o p m e n t s l e a d i n g t v ihe f n n u l s c i o n n f t h e L i r s t R e q u i r e m e n t s t o r D i p h t h e t l a Toxaid e r e d e s c r i b e d i n d e t a i l i n t h e I n t r o d u c t i ~ n end G e n e r a l C o ~ l s i d e r a t i o n s t o t h e R e q u i r e m e n t s f o r B L o l o g i ~ a l S u b s t a n c e s No. l0 . l 'IIle p u r p n s c of t h e s e G e n e r a l C o n s i d e r d l i o n s is t o d r a w a t r e n t i o n tu t he s i g n i f i c a n t developments t h a t have t a k e n p l a c e c l n c e t h o s e R e q u i r e m e l > t s w e r c f u r m u l n t e d .

One of t h e mos t i m p o r t a n t c o h s i d c r a l i u n s i s t h e a g r e e m e n t r e a c h e d o n che r o r m u l a ~ l u n o f r e q u i r e m e n t s f o r t h e assay of p o l c n c y . I n 1964 a l m o s t a l l c o u n t r i e s a d o p t e d t h e i r own r e q u i r e m e n t s and t h e r e was l I t t l e u n l I o r m i t y be tween c o u n t r l c s . A l t h o u g h many countries c o n t i n u e t o r e L a i n the i r i n d i v l d u a l i c y ill t h i s r e s p e c t there i s a g r e a r e r u n i - f o r m i t y of o p i n i o n c o r l c e r n i n ~ the p o t e n c y r e q u i r e m e r ~ t s for diphtheria t o x a i d n c ~ . d e d t o e n s u r e a d e q u s t c p t u t e c t i o n a g a i n s t t h e d i s e a s e . A c c o r d l n g l y i t h a s been possible t o i n c l u d e a r e q u i r e m e n t t h a ~ ~ n v o l v c s t h e i m u n i z a t i o n o f g u i n e a - p i ~ s a n d s u b s e q u e n t c h a l l e n g e . e l t h e r hy a l e t h a l c h a l l e n g e d o s e of t o x i n o r by aa i n t r a d c r m l c h a l l e n g e u s i a y

g r a d e d d o s e s o r t h e c h a l l e n g e toxln. I t i s i a ~ e r e s t i n g t u n o t e tha t Lhe l e t h a l c h a l l c n g r t e s t g i v e s a n L n d i c a t i n n t h a t t h e i m b n l z e d a n i m l s a r e p r o t e c t e d a ~ a l n s c a s i n g l e challenge d o s e 0 1 t o x i r l whereas t h c i n t r a d e r m l c h a l l e n g e t e s t , by v i r t u e 01 i t s g r a d e d d o s e s nf c h o l - l e n g e , givcs a m e a s u r e o f how much p r o t e c L i o n t h c I m n i z c d a n i m a l s h a v e a g a i n s t a p a r t i c u l a r d o s e o i t o x i n . C l e n r l y , t h e l a t t e r teqt g i v e s more i n f o r r u a l i o n t h a n t h e f o r m e r . Such a t e s t 1 s r e c n m e n d e d b u t t h e d e c i s i o n c o n c e r r l l n g t h c d e t a i l s o f t h e p a r t i c b l a t t e s t t o adopL i s lrEt t o t h e n a t i o n a l c o n t r o l a u t h o r i t y . IL i s i m p o r t a n t t o include a reference p r e p a r a t i o n i n e a c h t e s t which h a s been c a l i b r a , L e d i n I n t e r n a t i o n a l U n i t s . I n t h i s way t h e p o t e n c y n f the v a c c i n e may a l s o he q ~ i ~ l t e l i i11 I l J ,

Wlltl l e ~ h n i c a l V O ~ ~ I - L series, N o . 2 9 3 , 1964, A n i ~ r x l .

B L G I U N D P ~ ~ ~ . ~ Rev. 1

page 120

Appendix D . 2 6

The s t r a i n oi Corynebacteriurn d i p h c h e r i a e (Parke Wi l l iams 8) from which the c o x ~ i d is nvrde has been shown t o be s a t i s f a c t o r y f u r Lhe purpose of prnducing poten t d i p h t h e r i a v a c c i n c s . There seems t o bc no

purpose In suggeaLL~rp, a change i n s t r a i n . The main aim of good vacc ine

producr ion is to n b t a l n Lhe g r e a t e s t q u a n t i t y of t o x i n dur ing t h e growth phase oE the organisms a n d , t h e r e a f t e r , t o c o n v e r t t h e t o x i n i n t o toxuid by tne most ef f i c i e n r e c h o d .

The UHU Exper t C a m i t t e e on BiulagScal S t a n d a r d i z a t i o n , rccog- n l z l n g t h e l i m i t a t l o a s o f the v a r i o u s methods i n cmlrnon u s e , e s t a b l i n h a d , i n 1951, t h e I n t e r n a t i o n a l Standard f a t D i p h t h e r i a Zoxoid P l a i n i n o r d e r t h a t t axoida c o u l d be assayed by c m p s t a t i v e measurement w i t h t h e a e t o ~ o i d s t a n d a r d s . F u r t h e r n o t e , t h e WHO Exper t Committee on BLological

S t a n d a r d i z a t i o n c s t a b l i a h e d Ln 1955 t h e I t r t e r n e t i o n a l S tandard f o r D i p h t h e r i a Tuxuid Adsorbed becauee t h c l o g doee / response l i n e s of p l a i n and a d s v r b d ~ a x o i d s a r e not p a r a l l e l . Thua a s t a t i b t l c a l l y v a l i d tom-

p a r i s o n of an adsorbed t o x o i d w i t h a p l o i n s t a n d a r d uaa no t p o a s i b l e . S i n c e 1951 t h e L n c e r n a t i n n e l Scendard f o r D i p h t h e r i a Toxoid, P l a i n , has been ceplaced i n 1974.

Although Lhere a r c f c w data a v a i l a b l e tu t h e c o r r e l a t i o n

of a po tency l e v e l i n d b i u l a g i c a l a s s a y with p r o t e c t i o n Ln man, end even ier.cr f o r t h e c o r r e l a t i o n betwaen the p o t e f l ~ y 1 e v ~ . I and t h e d u c a t i o n of imrmnity, a u f f i c i e n t ev idence 1 8 a v a i l a b l e Lo a s s i g n c l m L t l i m m potency l e v e l ebove u h l c h U vacc ine can he c o n s i d e r e d t o be of an acc.epteble potency. Such l e v e l s have been i n c o r p o r a t e d i n t u thc p r e s e n t

Requiremente.

I n t h e preeent I n t e r n n t i u m l R e q u i r e e n t s tor D i p h t h e r i a Toxuid, p b t l f i c a t i o n or r h e p r m d u c ~ Ls r e q u i r e d . D i p h t h e r i a toxoid i n t h e

u n p u r i f i e d farm i s l i a b l e t o g i v e s e v e r e v a c c i n a t i o n r e a c t i r m s in man and much work has been done i n develop in^ p u r i f i e d m a t e r i a l t o a v o i d euch r e a c t i o n s . Even wlrh p u r i f i e d p r o d u c t s , houevcr , untoward r e a c t i o n s

W y occur i n a d u l t * . I n view of a r i s k o f r e v e r s a l t o L r ~ x i c i t y when a

tox in is d e t nxi f i c d a f t e r p u r i l i c a t i u n , che r e q u i r e n ~ e n t s have been f o r m l a t c d t o exc lude this r i s k . There i s e v i d c ~ > c e thaL yur i f i c a t i o n ,

a l t h w g h cnebling, more c o n c e n t r a t e d p r e p e r a t i o n r Lo be used , m y snme-

t 5 W 3 reduce Lhe i w ~ . n $ ~ i n g a c t i v i r y of diphthex.ia t o x u i d , probably 011 accoun t ol: the removal af s u b s t a n c e s h a v l ~ l ~ an a d j u v a n t c i f e c t . Such

Appendix D. 2 6 Appendix D.26

p u r i f i e d prnbucts, i f In tended f o r pr imary iom~unlza t ion , ~ h o u l d be combined e i c h e r w i t h p e r t u s s i s v a c c i n e c o n t a i n i n g whole urgonisms or

with a m i n e r a l a d j u v a n t (or b o t h ) a l t h o u g h they may be used unco~nbined f o r r e i n f o r c l n g i t ~ n ~ ~ u r ~ i t ~ . ~

E a c h o t t h e f o l l o w i n g s e c t i o n s c o n s t i t u t e s a r c c o m e l l d a t i o n . The p o r t s of e a c h s e c t i o n t h a t a r e p r i n t e d Ln largv t y p e havc h e s n uri tLer1 i n c h e form uf requ i rements s o t h a t , i f a h e a l t h admin i s t ra t iu rn su d e s i r e s , rhese p a r t s ay t h e y appear may be used a s d e f i n i t i v e n a t i o n a l r equ i remenrs . The p a r t s or t a c h :sect ion that . a r e p r i n t e d i n s m a l l LyPe a r e comments and recanmendet imts f n r guidance.

Should Lndlvidual couf i r r i e s wish t o a d o p t rhese reguiremetlrs a s t h e bagis of t h c l r n a t i o n a l r e g u l a t i o n 8 c o n c e r n i n g d i p h t h e r i a v a c c i n e , i t i s reconmended t h a t a c l a u s e be i n c l u d e d whlch would permi t modi f i - c a t i o n s of m e n u f a c ~ u r i n g requirernenlb on the c o n d i t i o n t h a t i t be d e m o n s t r a ~ e d , t o t h e s a t i s f a c c ~ o r b of t h e na tLuna l c o n t r o l a u t h o r i i y , t h a t surh modi f ied requirements e n s u r e that t h e d e g r e e of s a f e t y and the potency of t h e t c ~ x r ~ i d s a r e a t lesst e q u a l t o t h o s e p r o v ~ d t d by

tae r e q u i r e m e n t s fo rmula ted bclnw. L L is d e s i r a b l e t h a t t h e World H e a l t h O r g a n i z a t i o n shou ld t lwn be iafatmed of t h e action t aken .

The terms " n a t i o n a l c o n t r o l a u t h o r i t y " pnd :he "nec lonn l c o n t r o l l aburacory" a s ueed i n t h e s c r e q u i r e m e n t s , always r e f e r t o the cot lntry 111 which the t n x u i d i s manuiaccured,

Primary immunizat ion rcfcrs to t h e i n f t i d l c o u r s e o f i n j e c t i o n s which rldrlally c o n s i s t s of two ( o r t h r e e ) i n j e c t i o n s e l all i n t e r v a l of four to s i x weeks fo l lowed by a f d r t h e r i n j e c t i m seven to cwclve months l a t e r .

R e i n f a r c i n g i m u n i t y , which i s blknetimes r e f e r r e d t o as "boos t ing" immuniry, r e f e r s t o subsequen t s i n g l e i n j e c t l u n s , u s u a l l y g i v c n a number c £ years l a t e r .

1 1 E n r e r n a t i o a a l rime and p r o ~ e r name

T ~ F i n c e r n u t i p n a l name shall be "Vaccinum diphtheriae" . 7%-

p r o p e r name shall be the e q u i v a l e n t of t he i r t e r n a t i o n a l name i n the Language of t h e c m n t r y of or igl11.

The u s e o f the i n t e r l u t i o n a l name s h o u l d be l i m i t e d t o coxuids t h a t s a t i ~ f y t h e r rgu i rementa fo rmula ted belrn.

D.1 .2 D e s c r i p t i v e definition

V a E C i ~ u ~ dilhtherlee is a p r e p n r n t l o n u 1 d i p h t h e r i a t ~ x ~ i d p repared by t r e a t l d g diphtheria t l u i n by chemical w a n 6 t o render i t nun-roxic w i t h o u t d e s t r o y i n g i t a imwnbgenic po tency . The p r e p a r a t i o n sheLl

s a t i s f y t h e r e q u i r e m e n t s E o m u l a t e d baluu.

The most comcn mechad t o r t o x o i d i n g L B by the u s e of formaldehyde.

D.L.3 I n t e r n a t i o n a l S t ~ n d a r d a , I n t e r n a t i o n a l Beference P r e p a r a t i o n s and l n t e r ~ a t i o n a l uni te1

The I n t e r r a t i o n a l Standard f o r D i p h t h e r i a Anticoxin ( e e t a b l i s h e d i P

The e x p r e s s i o n of the I n t e r n a t i o n a l Unit as a g i v e n weight of a d r i e d s u b s t a n c e c v n t a i n c d i n an ampoule i s f o r rhe purposes of J c f i n i t i o n only. I n p r a c t i c e t h e I n t e r n a t i o n a l Refe rence m a t e r i a l s shou ld be used by r e c o n s t i t u t i n g t h e whoLe c m l e n t g of Lbe ampoule^ i n a giver. volume and using t he r e c o n s t i t u t e d ma:erial w i t h a c a l r u l s t e d u n i t a g e per u n i t ~ o ~ l r m e depending on t h e volume o f f l u i d used nnd ~ l ~ c declclrrc c o n t e n t of each ampoule.

B L G / U N D P / ~ ~ . ~ Rev. 1

page 132

Appendix D . 2 6

BLG/UNDY/~~.L Rev. l

1934) 1 s s t o r e d i n ampouIes c o n t a i n i n g d r i e d h y p e r i m n e h o r s e serum, and i s

d i spensed as a s o l u t i o n of the aerum i n aa l i r l e c o n t a i n i n g 662 v/* glycerol i n a c o n c e n t r a t i o n of 10 I n t e r n a t i o n a l C n i t s p e t ml. The I n t e r n a t i v n e l Unit (1111 Is d e f i n e d as the a c t i v i t y con ta ined i n 0.0628 mg of Lhe I n t e r n a t i o n a l S t a n d a r d .

The F i f t h I n t e r n n t Lonsl Refe rence P r e y a r n t i o n of D i p h t h e r i a A n t i t o x i n fu r F l o c c u l a t ~ u n T e s t ( e s t ab l i shed i n 1971) i s hyperimmunc: h o r s e s e r u m and i s d i spensed i n ampoules s u n t a i n f n e 1800 Lf e q u l ~ a l r n t s p e r ampoule and f r e e z e d r i ed .

'Cl~e Second I n t e r n a t i o n a l S tandard f o r D i p h t h e r i a Toxoid, P l a l n , Ce6tob l i shcd i n 1974) i s d i spensed in ampoules c o n t a i n - i n g 200 IU pcr ampoule and freeee d r i e d , 7"ne I n t e r n a r i o n a l L'nir i s d e f i n e d as the a c t i v i t y c o n t a i n e d i n 0,10515 ng of t h e I n t e r n a t i o n a l S t a n d a r d .

The I n t e r n a t i o n a l S tandard Fur Diphtheria Tvxuid, Adsdrbcd [ e s t a b l i s h e d Fn L Y 5 5 ! is d i spensed i n arnpadlc. curltdin- i n g 107 L11 and f r e e z e J r l c d . The I n t e r n a t l o n e l ' Jn l t i s d e r i n r d as r h u a c t i v i ~ y c ~ n t a i n c d i n 0 . 7 5 mg 01 t h e 1 r t lu r r . a t ldnn l SLandard.

Thc l n t e r n a c l u r ~ a l S t a n d a r d f o r Sc!iick T e s t Tun in ( D i p h t h e r i a ) ( e s t o t l i s h c d i n IY54) i s dispcnticd i n ampoules c o n t a i n i n g 900 ILJ a n d l r v e z e d r i e d , The l n t c t n a t i o n a l I J n i t i s d e f i n e d a s t h e activily c u n ~ u i n a d i n D.C(%i! tt~g ui Lhe I n r e r n a t i o n o l 5 tnnderd .

A p p e n d i x 0.26

The atiove S t a n d a r d s and R e f e r e n c e P r e p a r a t i o n a r e i n t h e cus tody o f t h e I n t e r n a t i o n a l Labora to ry f o r B i o l o g i c a l S t a n d a r d s , S t a t e n s S e r u m i n s t i t u t , Copenhagen. Samples a r e d i s t r i b u t e d free of c h a r g e on r e q u e s t tu n a t i o n a L c o n t r o l l a b o r a t o r i e s . The I n t e r n a t i o n a l S t a n d a r d s and I n t c r n s t i o ~ a l Refe rence P r e p a r a t i o n s are i n t e n d e d for t h e c a l i b - r a t t o n o f n a t i o n a l s t a n d a r d s and r e f e r e n c e p r e p a r d t i u n s f o r u s e i n rhe manuEecture and l a b o r a t o r y c o n t r o l o f d i p h t l ~ e r l a a n t i t o x i n , v a c c i n v s and Sch lck t e s t t o x i n .

Single h e r v e s t . The t o x i c f i l t r c r l e o b t a i n e d Erm onc ba tch u f

c u l t u r e s inoculated, h a r v e s t e d and proccssed Loge ther .

Bulk p u r l l i e d t o x o i d . Thc p r o c e s s e d puritied ma~ertal prepared . '~ from e i t h e r A s i n g l e h a r v e s t Qr a yvol o f a nulphev ef s l n ~ l e I lu rves t s

It i s t he parenL m a t e r i a l from vhich t h e l i n a l bulk is p r ~ p a r r d .

F i r m 1 h t ~ l k . The homogrneuuc f i n a l t a x o i d p t e k e c ~ t i n a J l n g l e n t a i n e r f r m which t h e f i n a l c n n t n i n e r s are f i l l e d eLt11er d l r c c t l y

a r th rough one o r more I n t e r m e d i a r e ~ o n t u i n c r s .

F i n a l l o t . A c o l l e c t i o n of s e a l e d F l n a l c o n t a i n e r s t h s l orc ogeneous w1Lh r e s p e c t Lo thc r i sk of c o n t a m i n a t i u n d u r i n g filling. i n a l l o t must, t h e r e f o r ? , havc been f i l l e d from o s l n g l e contaLnrr anc work ing sessiun.

C E W U L nsNvFAs'runmc; RFQUIRENEnrS

The g e n e r a l r c q u i r e m e r ~ t s f o r m a n u i a c t u r i n g e s t a b l l s h m e n t s curl ta inud r e v i s e d Requirements f o r E i n l n g i ~ e l Sub. tances No. I (General

Hcqui remur l t~ f o r M a n u f ~ c l u z i ~ . ~ L s t a b l i s h m c n r s nod Conrrul L a b o r a t o r i e s ) 1 s l ~ ; l l l a p p l y r o ~ s t a b l i h h m e n t s m d n u l a r L u r ~ ~ ~ y d i p h t h e r i a t o x o i d , w i t h the ~ d d i t i m ~ i)i the f o l l o v i n p :

WrIcten d e s c r l ~ ~ l i o n s o f p r o c e d a r c s t n r t h e p r e p a r a t i o n o f diphtheria t o x o l d adop ted ~y a manufac tu re r sha lL b e submi t t ed f a r a p p r o v a l t o t h e n a t l o n a l c u r t r o l a ~ ~ t h o r l t y . P roposa l s f o r m o d i f i c a t ~ u n s

l l

s h a l l a l s o be subrnl t ted f u r a p p r o v s l t o the n a t l o n a l c o n t r o l a u t h o r l c y

a : b e f o r e t h e i r i m p l e m e n t a t ~ o n .

D. 3 PRODUCTION CONTROL

0 . 3 . 1 C o n t r o l oE s o u r c e m a t e r i a l s

D 3 J l S t r a i n s o f Corynebacterium d t p h t h e r i a e

S t r a i n s of C o r y n c b a c ~ e r i u m d i p h t h c r i a e used i n p r c p a r r n g d i p h t h e r i a t o x o i d s h a l l h e i d e n c i f l e d hy a r r c o r d ol r h c i r hlktory and of a l l t c s r s madc p e r i o d i c a l l y fur v c r i f i c a t l o n u L s t r a l r l charactr!rs .

Thc strain shall bc main ia incd a% a frceke d r i e d c u l l u r e .

A hlghly t a x i n o g e o i c s t r a i n 01 Coryncbacter ium d i p h t h e r i a c s h o u l d bc

used . A s t r a i ~ t t h a t has proved s a r i s - factory i n many L a b e r a ~ o r i e s ia t h e Parke WLlliams 8 s t r a i n .

0 . 3 . 1 . 2 Seed Lot system

The p r o d u c t i o n o f d i p h c h e r l a t o x i n s h a l l be based on a seed lo t system. C l ~ l t u r e s o f t h e working seed sha l l have the same c h a r a c t e r i s t i c s a s c u l t u r e s OF t h e s t r a i n s from whlch t h e p a r e n t s e e d Lot was d e r i v e d . The p r e p a r a t i o n o f s e e d l o t s s h a l l be i n compliance w i t h the requ i rements of P a r t A , s e c t i o n D . 3 . 2 .

l WO T e c h n i c a l R e p n r ~ S e r i e s , KC. 3 2 9 , 1965, Annex I .

B L G ~ U ~ P ! ~ ~ . I R e v . 1

page 1 3 5

Appendix 0.26

0.3.1 . ? C u l l u r e m c d l v r n for p r o d r ~ c t l o n v l lqx!n

The medium shell I;e f r e e from i n g r e d i e n L s t h a t w i l l be p r e s e n t i n the final produc t am1 c h a t a r e known t o cause t o x i c o r allergcc r e a c t l o u s i n man.

S i n c e some medium componan t s m y b e

p r e s e n t i n t h e f i n i s h e d p r o d ~ c t , i t i s p a r t i c u l a r l y impor tan t t o e n s u r e t h a t the Cinal p r o d u c t i s f r e e from s u b s t a n c e s which a r e l i k e l y t o c a u s e t o x i c o r aLlergFc r e a c t l o n s L n m a n . I f t h e medium i s prepared

from p r o t e i n d i g e s t , as f a r example c a s e l n h y d r o l y s a r e o r d i g e s t e d muscle . precautions should be t a k e n t o e n s u r e t h a t the d i g e s t i o n ha. proceeded s u f f i c i e n t l y to f r e e t h e medium from such s u b s t a n c e s . Ne i ther m ~ l i a n p r o t e i n n o t human blond group s u b s t a n c e s s h o u l d b e p r e s e n t i n the f i n a l vacc ine; t h e rnect~nd f o r d e i e c t i n g Lhese s u ' ~ s t a n c c s shllold be ~ p p r o v e d by the

n a t i o n a l c o n t r o l authority.

The g e n e r a l p roduc t ion p r e c a u t i o n s , as formulated i n the requirements P a r t A , s e c t i a ~ i 3 , oi r e v i s e d RequLrements f o r B i ~ l o g i c a l Substances . 1 ( ~ e n e r a l Requirements f u r Manufac tu r ing E s t e b l i s h w n t 5 and C o n t r o l b o r a t o r i e s l l s h a l l a p p l y tc t h e manLfacture of d i p h t h e r i a t o x o i d .

S u i t a b l e methods f o r t h e p r o d u c t i o n of d i p h t h e r i a t o x o i d are given i n t h e Manual £or t h e Produc t ion and C o n t r o l of Vaccines : D i p h t h e r i a ~ o x o i d . ~

WHO T t c h n l c a l Report Series , No. 3 7 ? , 1965, p. 1 5 .

Manual f o r the PrurJuct ion and C o n t r o l of Vecclnrt i : D i p h t b c r l a u i d , w n BLG/LIKDP/)~ . l R ~ V . L , Appeilclix b . 7 .

l.

' , B U / U ? ~ P / ~ ~ . 1 Rev. l

.! page 136

Appendix D , Z 6

0 . 3 . 3 C n n l r u l o i s i n g l e h a r v e s t s

The p r o d u c t l a n s h a l l h e shown t o be c o n s i s t e n t by nhserv inC the growth , p13 and raLe o f cnxin p r o d u c t i o n .

Any c u l t u r e showiny, anomalous growth c h a r a r t e r l s t i c s s h u u l d b e i n v e s - t i g a t e d and s h i o w i ~ C V be s a t i s E a c t o r y b e f o r e being accep ted a s a s i n g l e h a r v e s t .

D.3.3.1 C o n t r o l of b a c t e r i a l p u r i t y

Semplcs 0:: t h e c u l t u r c s used f o r p r r p a r l n g a s i t g l e harvcbl ol toxr>irl s h a l l bc tested Ivr b a c t e r i a l p u r i t y by microscop ic examlnat ivr l of s t n i n c d smears and by i ~ ~ u u u l a t i o ~ ~ i n t o a p p r o p r i a t e culture media. S i n g l e h a r v e s t s s h a l l nu t be ubrd f o r p r e p a r i n g bu lk m a t e r i a l i f contseination has o c c u r r e d a t any s t a g e In t h e i r p r o d u c t i o n .

0.7.3.2 F i l t r a t i o n

Af ter sampling Lur t h e c o n t r o l o f p u r l t y , t l r r c u l t u r e s h a l l be f i l t e r e d u s i n s a f i l t e r c a p a b l e of producing a c l c a r f i l t r a t e .

The c u l t r ~ r e s stluuld br. f i l t e r e d as soon a s p o s s i b l e a f t e r Ihu und n i

the11 i n c u b a t i o n p e r i o d . Fn i a r i l i t a t e f i l t r a t i o n , t h e c u l t u r e s m y bc c e n c r i l u g e d a n d j o r a f i l t e r a i d added becorchand .

I n some c o u n t r i e s any f i l t e r shedding f ibres m ~ y n o t h e v y e d .

The c u l l v r l s may be t r e a t e d w i t h a p r e s e r v a t i r c bcfnrr € i l t r a t i o n . On no account s h o u l d phenol l,c used .

B I C / ~ I N ~ P / ~ ~ . I Rev. 1

page 137

Appendix D. 2 6

D.3.3.3 D e t e r m i n a t i o n a € the c o n c e n t r e t i o n oi t o x i n (LfI

The t o x i c f i l t r a t e shall be t e s t e d by LLoccu la t ion i n cumpar i san with a reference m a t e r i a l c a l i b r a t e d a g n i a n t t h e I n t e r n a t i o n a l R e f e r e n c e P r e p a r a t i o n of D i p h t h e r i a A n t i t o x i n f o r F l o c c u l e t i o n Test o r a n e q u i v a l e n t r e f e r e n c e p r e p a r a t i o n approved by t h e nationai colrLral a u t h o r i t y .

A s u i t a b l e method La d e s c r i b e d i n the Hanual .1

I t i s p r e f e r a b l e t h u t t o x i c f i l - ciates used i n p r e p a r i n g p u r i f i e d t o x o i d shou ld c o n t a i n a t least 50 L+.

This t e s t i s not an a b s o l u t e measure o f pa tency but i t Fs a good g u i d e to the c m n i a t e n c y of p r o d u c t i o n .

The p u r i f l t a t i a n process may e i t h e r p r e c e d e or f o l l o w d e t o x i f l c e t i m . mud Lhe method used shall bc approved by the national c o n t r o l a u t h o r i t y .

L , The a g e n t used ( fo tm@ldshyde or g l u t a r a l d e h y d l ) s h a l l f u l f i l t h e ;,< requ i rements of t h e I n L e r n a t i o n a l ~ h a n r d c o p o e i e ~ o r o t h e r s p e c i f i c a t i o n s . n p p r w c d by t h e r l a t i o n a l c o n t r o l a u ~ h o r i t y .

11 br removed or n e u t r a l i z e d a f t e r The method used s h a l l be approved

l h n u a l f a r the P r o d u c t i o n and C o n t r o l of Vaccirres: Diphtheria old, W O BLG/Um~/77 .1 R e v . I . Appendix D. 7 .

The recand cdiLLon o f t h e I n t e r n a t i o n a l Pharmacopoeia i s n t r o l o f Pharmace l ; t i ca l Preparations",

B L G / u M ) P / ~ ~ . ~ Rev. 1

page 138

Appendix P.26

~ u : / v t m ~ / 7 7 . 1 Rev. 1

page 139

Appendix D. 26

Care s h o u l d be t a k e n t o e n s u r e c h a t r e v e r s a l t s t u n l n does n o t t a k e place on s t a r a g e . Methods for t h e d e t e c t i o n o f r e v e r s a l are s u g g e s l c d i n t h e Manua1.l

I n some c o u n t r i e s the Lol luwing t c s c i s a p p l i e d t u check t h e p o s s i b i l i t y of t o x i c i L y r e v e r s a l . One ~arnpie i s d i l u t e d t o 200 ~ f / m t ( u s i n g 0 . 0 1 6 7 M phosphate b u f f e r e d s a l i n e ) , and a n o t h e r sample t o e c o n c e n t r a t i o n e q u l v e l e n t t o t h a t u f t h e f i n a l bulk. T h e l a t t e r sample i s k e p t s t a n d i n g a t 37°C f o r 20 days. Of e a c h sample 5 ml a r e L r l j ~ c t e d s u b e u t a n e a u 3 l y i n t o et Least I i v e guLr~ca- p i g s , which a r e t h e n observed f o r s i x weeks (as described i n P a r t A , Section 0.3.&.3),

The mcthod of p u r l f i c a t i u n s h a l l be such t h a t no s u b s t a n c e s a r e incorpnraLcd inCv the f i n a l producL t h a t are l i k e l y t o cnuae untoward r r a c t l o n s In w n ,

D . 3 . 6 C o n t r o l of bu lk p u r i f i e d i ~ ~ o i d

D . 3 . 4 . 1 PreparnLion

The b u l k p u r i f i e d t o x o i d s h a l l h e p r e p a r e d e L t h e r frum e s i n g l e h a r v e s t o r a pool of a r~umber o f s i ~ l g l e h a r v e s t s and s h a l l be s t e r i l e .

I t 1% advisable t o sterilize t h e bulk p u r i f i c d t v x o i d hy f i l t r a l i o n . A p r e s c ~ v a t i v e s u c h a s thl~mersal rnay be added t o t h e b u l k t u x a i d .

Manual for Lhe Yruduct ion and ConLrol of Varclncs: D i p h t h e r i a Toxo id , UliU R I & / U N D P / ~ ~ . 1 Rev. l Appendix D . 4 .

U.3.4.2 S t e r i l i t y t e s t

Each bulk p u r i f i e d t o x o i d s h a l l be t e s t e d f o r b a c t e r i a l s t e r i l i r y , a c c v r d i n g to the requ i rement8 g i v e n i n P a r t A , s e c t i o n 5 , o f r e v i e e d Requirements for B i o l o g i c a l S u b s t a n c e s No. 6 (Genera l ~ e q u l r e m e n t s f o r t h e S t e c i l i t y of Biological S u b s t a n c e s ) . l

0 . 3 . 4 . 3 S p e c i f i c t o x i c i t y t e s t

Ench bulk p u r i f i e d t o x o i d s h a l l be t e e t e d for t h e p r e s e n c e of d i p h t h e r i a t o x i n by the i n j e c t i o n of a t leasc f i v e gu inea-p ige , each weighing b e t v e e n 250 m d 350 g. Each g u i n e a - p i g a h a l l be i n j e c t e d

8 u b e u t a n e w e l y w i t h 1 m1 of a d i l u t i o n af p r l f i e d t o x o i d c ~ n t e l n i n g a t least 5 0 0 LP of &onoid . Animals t h a t d i c shall be examined by

gutppsy for s y m p t a of d i p h t h e r i a i n t o x i c a t i o n (red a d r c n a l s ) . !t'h~

b u l k p u r i f i e d toxoid s h a l l p a s s t h e t e s t if no g u i d e a - p i g show symptomS of s p e c l f l c i n t o x i c a t i o n w i t h i n s i x weeks of i n j e c t i o n and i f a t l e a s t 80% of the a n i m a l s survive the tes t p e r i o d .

The gu inea-p igs

a h a l l noL have b t + a u ~ e d p r e u l n u s l y f u r e x p e r i m e n t a l purposcs .

S-e m a n u f a c t u r e r s car ry ouL i n a d d i t i o n a t e a t f a r d e t e c t i n g t h e p readncc of d i p h t h e r i a t o x i n by i n j e c t i n g r a b b i t s o r g u i n e a - p i g s i n t r a d e r a l l y w i t h a t l e a s t 20 L f of p u r i f i e d toxo id ~ n d o b s e r v i n g t h e a n i u 4 l e f o r a p e c L f i c erythemu.

m,3.4.4 T e a t f o r a n t i a e n i c p u r i t y

Each b u l k purifled t o x o i d s h e l l be t e s t e d f o r a n t i g e n i c p u r i t y by &rermin ing t h e I.€ vnlue and the c o n c e n t r a t i o n of ~rutein (non- d i m l y s a b l e ) n i t r o g e n , The Lf d e t e r m i n a t i o n s h a l l be mede ubiag a

reference material c a l i b r a t e d a g a i n s t t h e I n t s r r ~ n t i o n a l R e f e r c n c c P r e p a r a t i o n of D L p h t h e r L ~ A n t i t o x i n fo r F l a c c u l r t i o n T e s t or a n

WHO T e c h n i c a l Repor t S e r i c s , No. 530. 1971, p . 48

R L G / u M ) P ~ ~ ~ . ~ Rev. I

page 1 4 0

Appendix D. 2 6

e q u i v a l e n t r e f e r e n c e p r e p a r a t i o n approved by ~ h c n a t l u n a l c o n t r o l a u r h o r i t y . The method o f L e s t i n g s h a l l be appruved by t h e n a t i o n a l c n o t r o l a u t h u r i t y . The bulk p u r i f i e d toxo id s h a l l p a s s t h e t e a t if it c o n t a i n s n o t l e s s t h a n 1500 Lf per mg of p r o t e i n ( n o n - d i a l y s a h l p ) n i t ragen . I1.3.5 C o n t r o l of f i n a l bu lk

D.3.5.1 P r e p a r a t i o n

.!ia'i 1 The f i n a l b u l k m a t e r i a l s h a l l be p r e p a r e d from bulk p u r i f i e d ,, . . . t o x o i d . The number of L f i n a s i n g l e human dose s h a l l be approved by t h e n a t i o n a l c o n t r o l a u t h o r i t y . P r ~ d u c t s i n t e n d e d f o r immunizat ion of c h i l d r e n s h a l l c o n t a i n between 10 and 50 Lf i n a s i n g l e human duse . YlaLn d i p h t h e r i a t o r n i d must not be used for pr imary i m u n i z a t i o n f o r c h l l d r c n .

P roduc t$ i n t e n d e d f o r Lhe i m u n L n a t l u n b f a d l r l t s o n l y may cotlCaLl> l e s s than 10 LE i n a s i n f i l e hurmn dose. I n some c u u n t r i e s 2 - 5 Lf 1s utled f o r t h i s purpuae.

When i n t e n d e d For primary irumunizatlon t h e p l a i n toxo id s h o u l d be combined v i t h p c r t u s s i s vacc ine a n d j o r a m i n e r a l a d j u v a n t IalumLnLum o r ca lc ium c a r r i e r ) .

0.3.5.2 P r e s e r v a t i v e

I ' B U / U N D P / ~ ~ . ~ Rev . 1

I i . i

- :

.,. . :+-* . , X : ... W b; . ~- ,-. . -

l.... -<' .. . I '

' 1 ::

page 141

Appendix D.26

A s u i t a b l e p r e s e r v a t i v e s h a l l be added t o t h e f i n a l bulk. The ' ; ; : l

The a d t u v a n t u s e d , i t s p u r i t y and i t s C o n c e n t r a t i o n s h a l l be approved by the n a t i o n a l c o n t r o l a u t h o r i t y .

p r e s e r v a t i v e used s h a l l have been shown i n the amount p r e s e n t i n the f i n a l b u l k t o have no d e l e t e r i o u s e f f e c t on t h e t o x o i d or on o t h e r v a c c i n e components v i t h which t h e t o x a i d may be combined, and t o

: I

c a u s e no untoward r e a c t i o n s i n man. The p r e s e r v a t i v e and i t s con- , . I

c e n t t a t i o n s h a l l be approved by the n a t i o n a l c o n t r o l a u t h o r ~ t y .

: i '

AlurninLum o r ca lc ium compounds a r e used a s m i n e r a l c a r r i e r s . The c o n c e n t r a t i o n of aluminium s h a l l n o t exceed 1 . 2 5 mg and that ol crlciurn s h a l l n o t exceed 1.3 mg per s i n g l e human dose .

I n s m e c o u n t r i e s therte upper lFmFts f o r the c o n c e n t r a t i o n of m i n e r a l c a r r i e r s are considered t o be t o o h i g h and about half these c o n c e n t r a t i o n s a r e used .

I n some c o u n t r i e s t h e adsorben t i s p r e c i p i t a t e d in p r e s e n c e of t h e r o x ~ i d i n order t o get b e t t e r a d s o r p t i o n and h i g h e r s t a l r i l i t y .

D.3.5.4 S t e r i l i t y Lest

Bach f i t i a l bulk s l l a l l be t e s t e d f o r b a c t e r i a l s t c r i l i t y a c c o r d i n g t o t h e requ i remeniu g i v e n in P a r t A , ~ e c t i v n 5 , of revised Requirements fo r Bio logLca l S r ~ b s t a n c e s No. b ( C e n e r a l Requirements f o r the L c e r i l i r l o f a i ~ l o ~ i c r l Substanoer ) . i

D.3.5.5 S p e c i f i c t o x i c i t y t e s t

Each f i n a l bulk s h a l l be t e s t e d for t h e p r e s e n c e of d i p h t h e r i a t o x i n by t h e L n J e c t i o n o f a t least f i v e g u i n e a - p i g s , e a c h weigh ing between 250 and 350 g. Each g u i n e a - p i g s h a l l be i n j e c t e d s u b c u t a n e o u s l y

th r q u a n t i t y e q u i v a l e n t to a t l e a s t f i v e s i n g l e human d o s e s . h l s that d i e s h a l l be examlned by a u t o p s y f o r symptoms of d i p h t h e r i a

. i a t o x i c a t i o n ( r e d a d r e n a l s ) . The f i n a l b u l k s h a l l pass t h e t e s t i f

g u i n e a - p i g shows symptom of s p e c i f i c i n t o x i c a t i o n w i t h i n s i x weeks i n j e c t i u n and i f a t l e a s t 80% of the rnimls survive t h e t e s t p e r i o d .

YHO T e c h n i c n l ReporL S e r i e s , No. i 30 , 1973, P. 48.

B L G ~ u N D P / ~ ~ . ~ Rev. 1

page 142

Appendix 0 . 2 6

U . 3 . 5 . 6 Potency t e s t

E a c h final bulk * h a l l b c t c s t e d f o r iuununizing p o t h c y by comparison with a n a t i o n a l r e f e r e n c e m a t e r i a l c a l i b r a t e d a g a i n s t t h e a p p r o p r i a t e i n t e r n a t i o n a l s t a n d a r d . Thc t e s t s h a l l i n v o l v e t h e i n o c u l a t i o n o f Eroups of gu inea-p igq (weigh ing 250-330 21 and t h r e e d i l u c l u n s O F both thr : f l n a l bulk and r e t e r e n c e m a t e r i a l s h a l l b e used. A l t e r i n m n i z a t l o n t h e an imals s t l e l l bc c h a l l e n g e d e i the r b y a l e t h a l c h n l l c n g e dose o t t o x i n g i v c n b y t h e subcucancous route o r by graded d o s e s of tnxir l give11 by t h e i n t r a d e r m a l r o u t e . S t a n d a r d R t a c i ~ t l c a l methods s h a l l he used 111 c a l c u l a t e t h e p o t e n c y

of t h e f l n a l b u l k . Thp Inethod allupted sad its t n l e r p r e t a t l c > n s h a l l be approved by t h e n n l l u n a l c o r ~ t r u l s u t h o r i t y .

I n somr c o u n t r i e s por fncy t c s c i n ~ is n o t c a r r i e d ou t on e a c h f'irral b u l k

but uo each f i t ra l l n ~ . I n s u c h c a s e s t h e p r o v l s l o n s o f Part A, s e c t i o n D.S.3 @ r e o p p l i c a h l e .

~ u f f i r l e n t a n i m a l s should be u s e d

t o a c h l c v e a 45% c o n f i d e n c e i n t c r v a l amal l r r Lhan 5CflV t o toUtflo,

T h e d e t a i l s o f s u i t a b l e methods f o r po tency t e s t l n g a r e g iven i n Lhe h n u a l . l More i n f o r m a t i ~ n and hence g r e e t e r p r e c i s i o n i s o b t a i n e d u s i n g t t l r same numbcr of an imals by the m u l t i p l e i ~ ~ t r a d e r m l c h a l l e n g e method.

I n some c o u n t r i e s potency i s t c s t e d by ~ ~ i i x i n g s e r a o f i m n i z e d g u i n e a - p i g s with graded doses of t o x i n , fo l lowed by a t i c r a t i o n of t h e r e s u l t i n g mix tures i n c e l l c u l t u r e s .

M n u a l f o r the P r o d u c t i o n and C o n t r o l of Vaccine.: D i p h t h e r i a Toxoid, WHO BLG/~~NDP/J?.~ Rcv.1, Appendix 0.11.

BLC/UNDP/~~ .I Rev. 1

page 1L3

The po tency of t h e Einal bulk s h a l l be approved by the n a ~ l o n a l c o n t r o l a u t h o r i t y . The potency of d i p h t h e r i a v&ctines c o n t a i n i n g

an a d j v v s n t and used f o r primry immunization of c h i l d r e n s h a l l be not l ees t h a n 30 1.U. per s i n g l e human dose . F o r t h ~ s purpose Lhe

r e f e r e n c e m a t e r i a l ,,Red s h a l l be one c a l i b r a t e d against t h e I n t c r n a c i o n a l S tandard for D i p h t h e r l e Toxoid, Adsnrbed.

P r o d u c t s In tended on ly f o r buoster i m n i a s t i n n of a d u l r s m y c o n t a i n Leqs than 70 I.U. per s i n g l e human dose.

p l a i n d i p h t h e r i a coxold shalL not bc recommended for primrry i m n i x a t Lon .

D . 3 . 5 . 7 Terr f o r r r ~ l d u a l f r e t d c t o x l f y l n g auet)lr

Each f i n a l bulk s h a l l he t e s t e d f o r r e s i d u a l f r e t f o r m l d c h y d e , If t h i s h m s been usecl for d e t o x i f i c r ~ L o n , by a method approved by t h e n a t l o f i a l c o n t r o l . au thor i ty and s h a l l con ta i l1 n o t m r e than 0,02X of r e s i d u a l f r e e formaldehyde.

A s u i t a b t e t r 3 r I s a cv lo r imet rLc

d e t e r m i n a t i o n o f che r e a c t i m product o f fnrmsldehyde end f u c h s i n - s u l f u r w s a ~ l d ,

S i m i l a r t e s t s for t h e p resence uf o t h e r d e t o x i f y i n g a g e n t s L~lutaraldoh~de) s h a l l bc i n c l u d e d . The t e s t s used and the l imi t s

0f c o n c e n t r a t i o n of svcll ck~emica l s s h a l l be approved b y t h e n s t i u n a l

The s t a b i l i t y of t h e v a c c i n e s h a l l be shown t o t h e s a t i s f a c ~ l u n of the n a t i o n a l c o n t r o l a u t h o r i t y ; a t l e a s t t h r e e c o n s e c u t i v e b a t c h e s af f i n a l bulk s h a l l be t e s t e d t o prove s t a b i l i t y d u r i n g t ime end c o n d i t i o n s o f s t o r a g e . When any changes i n the p r o d u c t i o n procedure

! I '

I ~ ~ , G / U N D P / ~ ~ -1 Rev. 1

p a t e 1b&

Appendix D. 26

are made, t!~e vac,:itie produced h y t he new mvthod shall be showr~ to be stable.

i Thc vaccine shall meet Lhe requirements f o r potency (see Part A , S . . section D. 3.5.6) chrcrughout the d a t i n g period ar t h e recommended $ . \ P i u r a g e Lemperarrlr*. 1 i: I.

Mznlltacturcr; shuuld Lc encouredet! to c s r & l ~ s h t h e t irne/( nmperaiure Y P 1 B t l c n h h i p ~ of The s L a h i l L t y wit!] rcspec l to pa lency .

W.3.5.9 Tesr f a r pH

:. ; I The pH of the final b u l k shall be recorded

I :

1 ' The pk s h o u l d be b e t w r e n 6 . 0 a n d I

I i 6.7.

D . 4 PTLLZXG AND CONTAINbKs

The requirements cnncerr~ing r l rc f i l l ine a l ~ d cuntainer. g i v r ~ i Ln ParL A, sec t io r . G , of Requirrmcnts for Hiolugical Subs~ances K,?. L

;. ~

(~eneral Rrquirements f u r M n n r i f a c t u ~ i n ~ EsLablishments and Cunlral

1 : ' ; ~aboratoriesll shall apply.

Sicglt- and mulliple-dose containers may 5 , used. Attentian Is drawn to the r i s k assccialed w i t h the use uf multlpie-

d u s e conLarners, and also Ln the fact that it is inadvisable for the p ~ r l o d @ t Lime brcwnen t l ~ c ciLhdrawal nl . the first atld the rL11a1 dosew t o be ~jndt~lj? (~rulon~ud. The tilling of an excessive number of doses inLv ~ u u l t i p l e -dnsp

c o n t a i l ~ e r s should, therefore, b e ~voided.

j : WHO 7 e c h n ~ r a L Report Series, La. 323, 1'365, p. 11

B W / U N D F ~ ~ ~ , I . Kcv. 1 page 145

Appendix u.26

It is rec~nmne~~ded chat a vial once

opened should he used an t h e same day and not reused on subsequent days. I f

srsra~e and r e u s e are absolutely unavoidable, the vibl m i l s t be held in e refrlgesator at 5-C 3'C and never

for ruore than onc working x e u k .

D.5 CONTROL TESTS OP; Y l X A I . PRODUCT

D. 5.1 ldel~tity test

An Lclen~ity tesr shall be pertormed on at l ea s t une labelled container from each final lot.

Flocculation o f tte t o x u i d w i t h diphtheria antitoxin may serve as an identity test. Tests on t v x o l d con-

c t l i o i u g an alurnj nium or c a l c i c ~ m carrier may b e done a f t e r t h e cnrrier has been

dissolved with e solurlon u l sr>dFrlnl c l t r a t e g r ~ f t e r t h e toxo ld has hcen cluteri by n iultable ; n c t l l O d . I f thr: carrier cannot be removed, tcsL.s can ba madc by specific n n r i r o x i n ncctraiiration or by antitoxin production in animals.

.2 Sterility test

Bach final L ~ L s h a l l be tested for sterility according Lo the quirements give11 in Part A , section 5, o f he revised Kequirements

Rioloplcnl Substancgs No. 6 (~equiremet~ln for the S t e r ~ l i ~ y of

logical Subscal icrs) . '

YHO Technical Repart Series, No. 530, 1973, p . 4 8 .

BLG/uM)P/~~.~ Rev. 1

p a g e 146

Appendix D. 26

D . 5 . 3 Potency rest

A pntancy t e s t s l r a l l be made a c c o r d i n g t o P a r t A , sec t ion 0 .3 .5 .6 (page 11) on e a c h f i n a l l o t if s u c h e t e s t 118s n o t been performed on Lhe f i n a l b u l k .

D . 5 . 4 I n n o c u i t y t e s t

Each f i n a l l o t s h a l l be t e s t e d f o r abnormal t o x i c i t y hy t h e i n J e c t i o n o f one human dose b u t no t more than l m l i n t u e a c h of f i v e mice ( w e i g h i n g 1 7 - 2 2 g) and a t Least one human dose b u t nut more chen 5 m1 inLo e a c h of twu g u i n e a - p i g s (weighing 250-35i1 g) hy the i n t r a p e r i t a n e a l ruule. Tbc reuts s h e l l he approved by the n a t i o n a l c o n t r u l n i j t h o r i t y .

fie anlmals shnll s u r v i v e f a r e t l e a a r seven days r i ihot l t s i $ - n i f i c a n l s i g n s uf t u x i c i c y of Lhe p r o d w c ~ .

D . 5 . 5 T e s r Cur adluvanl c u n t e n c

Each f i n a l l a t s h a l l be t e s t e d f u r ad juvanc conLent by a e l h o d approved by the n a t i o n a l c o n t r u l authorFLy ( s e e P a r t A , s c c t i o n 11.7.5.7), if this tcsc has not been done on t h e f i n a l bulk.

I n s a n e c n u n c r i e s t h i s t e a t Lci

spplied L o the final bulk o n l y .

D.5.6 Tcst for preservative contcnc

E a c h f i n e l l o t O E adaurbcd t o x o i d s h a l l bc rested t o r t h e c o n t e n t uf the p r e s e r v a t i v e ( s e e P a r c A , 3.5.21 i f r h i s t e s t h a s noc been d o n e on the f i n a l bulk. The t e s t method s h a l l be approved by t h e na t i ona l conlrol a u t h o r i t y .

I n some c o u n t r i e s t h i s t e s t i s a p p l i e d KO t h e f i n a l b u l h o n l y .

BLG/UNDP/~~ . I Rev. 1

page 147

Appendix D . 2 6

D.5.7 T e s t f o t pH

The pH o i e a c h i i n a l l o t s h a l l be recorded, i f t h i s t e s t has not been done on t h e f i n a l bulk.

The pH shou ld bc between 6.0 and 6 . 7 , Zn some c o u a t r i e s t h i s t e s t L E a p p l i e d to t h c f i n a l b u l k o n l y .

D.S,8 Insyec tLun of f i n e l c o n t n f n e r s

Each c o n t a i n e r i n each f l n e l 1 ~ t shall be i n s p e c t e d and ~ I I O S ~ showing a b n o m u r l i t i e s , such ss c luawlnp o r p r c s e n c e of y a r t i c l e s , s h a l l bc d i a c e r d e d .

The reGuircments $ i v c n i n P a r t A , ~ c c t l o n b . u f r c v ~ s e d R e q u i r e m c n t ~ f u r B i a l o g i c u l S ~ ~ h s c a n c c s Nu. l (Lerrera L Re u i rehfen t s l o r l l a n u t a c t u r l ~ l ~ S s t a b l l r h o e n r r and c o n t r o l 1.aborxroric.J ahall e r p l y .

W r i t t e n r e c o r d s s h a l l bc k e p t u f a l l t e s t s i r r e c p s c r i v t . r r E t h e i r r e s u l t . The r c c o r d s s h a l l be of a t y p c npproucd by t h e n a t i o n a l c o n t r o l authority.

A sample of a s u i t e b l e s u m r y p r o t u c ~ l to be uacd f u r d ~ p h t h e r i a toxoid i s i i ~ c l u d e d i n Appendix D + 1 ~ ' t these r e q u i r e m e n t s .

The requirement$ g i v e n l n P a r t A , h e c t i o n 7 , of r ev i sedRequi remencs for B i o l o g i c a l Subs tances N n i ( ~ c n e r a l Requirements f o r Manutac- tltring t a r o b l i s h w n t s and C m t r o i ~ s b ~ r a t o r i e s ) 2 ~ 1 1 ~ 1 l a p p l y .

W O Technica l Repurc S e r i e s , No. 323 , 1965, p. 17.

WHO Technica l Repor t S e r r e n , No. 3 2 3 , 1965, p . 18.

BLG/uNDP~?~. l Rev. 1

page 148

Appendix D . 2 b

Thc l a h e l p r i n t e d on o r a f f i x e d t o each c o n t a i n e r and t h e l a b e l on che c a r t o n e n c l o s i n g one o r more c o n t a i n e r s s h e l l s h a v a t l e a s t :

t h e words "Vaccinum d i p h t h e r i a e " and/or t h e p r o p e r name of t h e p r o d u c t ;

the word "Adsorbed" i f a p p l i c a b l e ;

t h e name and a d d r e s s o f t h e m a n u f a c t u r e r ;

the number o f Lhe f l n a l l o t ;

t h e t e m p e r a t u r e o f s t o r a g e and t h e e x p i r y d a t e i f k e p t a t t h a t t e m p e r a t u r e ;

t h e recowlended s i n g l e human dose and r o u t e o t a d m i n i s t r a t i o n

I n a d d i t i o n , t h e labeL p r i n t e d on o r a f f i x e d t o t h e c o n t a i n e r o r t h e l a b e l on t h e c a r t o n o r t h e l e a f l e t accompanying t h e c o n t a i n e r s h a l l c o n t a i n t h e f o l l o w i n g i n f o r m a t i o n :

t h e f a c t t h a t t h e t u x o i d f u l f i l s t h e requ i rements of t h i s document ;

t h e n a t u r e and amount of p r e s e r v a t i v e p r e s e n t i n t h e t o x o i d ;

t h e n a t u r e and amount of a d s o r b i n g a g e n t (iE applicable);

t h e c o n d i t i o n s recommended d u r i n g s t o r a g e and s h i p p i n g ;

a n i n s t r u c t i o n t h a t t h e adsorbed v a c c i n e s h o u l d n o t be f r o z e n ;

817 i n s t r u c t i o n t h a t t h e adsorbed vacc ine shou ld be shakcn b e f o r e use :

i n s t r u c t i o n s f o r the u s e of t h e toxo id and i n f o r m a t i o n about c o n t r a i n d i c a t i o n s and t h e r e a c t i o n s t h a r may f o l l o w v a c c i n a t i o n .

D . 9 DISTRI BITTION AW S H I P P I N G

The r e q u i r e m e n t s g i v e n i n P a r t A , s e c t i o n 9 , of r e v i s e d Requirements f o r B i o l o g i c a l S u b s t a n c e s No. l (Genera l Requirements

B U / ' ~ I N ~ P / ~ ~ . I Rev. 1

page 149

Appendix 0.26 . f o r Msnofac tu r ing E s t a b l i s h m e n t s and C o n t r o l L a b o r a t o r i e s l L s h a l l app ly .

D . 10 STORUGE AND EXPIRY DATE

The s t a t e m e n t s c o n c e r n i n g s t o r a g e t e m p e r a t u r e and e x p i r y d a t e s a p p e a r i n g on t h e l a b e l as r e q u i r e d i n P a r t A, s e c t i u n 8, s h a l l be based or, exper imenta l evidence and s h a l l be s u b m i t t e d f o r a p p r o v a l t o t h e n a t i o n a l c o n t r o l a u t h o r i t y .

D. 1 0 . 1 S t o r a g e c a n d i t ioc-S

The manufac tu re r s h a l l r e c a m e n d such c o n d i t i o n s o f s t o r a g e . and s h i p p i n g a s will ensure t h a t t h e t o ~ o i d conforms t o t h e

r e q u i r e m e n t s for potency u n t i l t h e e x p i r y d a t e s t a t e d on t h e l abeL.

Storage a t a t e m p e r a t u r e of i 5 ° C f 3-C h a s been found t o be s a t i s -

f a c t o r y .

D.10.2 E x p l r y d a t e

The e x p i r y da t e shell be f i x e d w i t h t h e a p p r o v a l o f t h e n a t i o n a l c a n t r u l a u t h o r i t y end s h a l l r e l a t e t o t h e d a t e of t h e l a s t satisfactory potency t e s t , the d a t e of a p o t e n c y t e s t be ing that on which t h e test sn imals were first i n o c u l a t e d with t h e t o x c i d . When

toxo id i s i s s u e d by t h e m a n u f a c t u r e r , t h e e x p i r y d a t e s h a l l n o t b e more t h a n t h r e e y e a r s Irorc the b e g i n n i n g of t h e po tency t e s t .

PART B. NATIONAL CONTROL W 1 ; I E m N T S

. D. 1 GENERAL

The g e n e r a l requirements fo r c o n t r o l l a b o r a t o r i e s c o n t a i n e d i n

, P a r t E o f r ev i sed Requirements f o r B i o l o g i c a l Subs tances Xo. 1

WHO T e c h n i c a l Report S e r i e s , No. 323, 1965, p . LB.

BCG/WDP/?~. l R e v . 1

page L50

Appendix D.26

(General Requirements f o r Uanufacturing Establishments and Control LaburatoriesIL shall apply .

The natiohal centra l authoricv s h a l l :

Specify t h e potency requitements;

Apprvva the method of manuiaclure;

Approve the method of deroxi f icat lon and p u r i I l c a c t o n .

Approve the coucentr~.tIon of preservative nnd ad juvant ,

Approve the ecsc For s t a b L L i ~ y ;

Provide Lhe eatia~>al r e l c r c n c e materjals.

i D.2 RETEASE AM) CERTLPICATIOh c

4 toxoxd nhal l bc c~lrased only if it f u i f l l x Part A nf the present requiremehis.

A staterner~L signed by the appropriate u t t i r i a l of thc natrohal cuntral labnrstocy a l ~ a l l be piovided a t the reqircst uf the manufac:- tur lhg cstabliutlnrent and s h a l l cerllly whether the l o t pf toxoid i n q b s s t i o n *pets a 1 1 ~ l a ~ i u r ~ a l rcqui~ements a6 w e 1 1 a s Part A of Cbe present rrquircmrnts, Thc c r r l i t l c a t e shall stare he date of Ch? 1 . 4 ~ ~ s a t i s f a c t o r y potency t e s t , t h e l o t number, the ~lrrfilber under which he l o t was released, and the nusber u p p c a r i n ~ on the labels or he containers. I n addi t ion , r copy of the o f f i c i a l I I R ~ inna l release document s h a l l be attached.

'Phe purpose of the c e r t i f i c a t e i s t a f a c i l i t a t e t h c exchange oL d i p h t h e r i a toxoid betweea countr ies .

Seri No.

B L G / U F ~ D P / ~ ~ . 1 Rev. 1

Appendix D.27

OKprint from bf>,)d flEN)l/, ~ ~ ~ , , , , j : ( l , t , , , c Ttrbroig iil Rrporr SPrit'.!. 1 v 7 3 , YD. !lo

GENERAL REQULREMENTS FOR THE STERILlTY OF BIOLOGICAL SUBSTANCES

(Requirements ior Biological Sub&oce NO- 6) l Revised 1973)

REQUIREMENTS FOR BIOLOGlC AL SUBSTANCES AND OTHER SETS

OF RECOMMENDATlONS

WHO tXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATLON

Twenty-fifth Report

Anrlcxcr 4 and 5

WORLD HEALTH ORGANIZATION GENE\'A

13'3

B L G / U N U P , / ~ ~ . 1 Rev. I

Appendix D.27

\VHO E I P E R C CO\ l \ l ITTEE O N B IOLOGICAL S T A N D . ~ R D T Z ~ T I O K

Geneva. 3J-30 Apri l 1971

D r H. Cohm. Direclor, Nal ionr l Inr t~ to le o f Publmc Hcalrh. Rilrhoirn. Wrlher- land%

Pruic*.l?r S. G. Ilzdguror. Dlrrctor. T a r r w r i t 51rlr lnniluhc h,, tile Sc.mdJrdudllvn Jnd Control ol'Blolog:csl Preparrlicms. Morcor. USSR

P:oresor D. G. Elanr. Dircitor, N s l i u n ~ l lllrrltuts ror tliologlcal S ~ i u ~ l a r d - and Contml. I-ondan. England ((lnirrnnn)

D r 5 ~ 1 % ~ G u p l a i ~ k . Chicl. Biological Oi\i,ion. The Govcrnmenr Pharnl;irru!iial O~gmiznion. Bangkok, Thailand

nc p. K m e . oimctor. Drpaitmcnt of B~ologicdl Slandardlzarbr. SraMnr Serum- inrli lul, C o p h a p n . Denmark

nr H. klirihdmry, Asrociatc Director, Rdri S tas lnrrhutc lor %rum and Vac~ inc r r v l ~ x r i ~ n . Tehrr~n, Iran

Or R . M ~ i r d h . Direclor. The Second Department oi Hac!rriohe!, National Lnrl!tulc or Hcallh. T d y o , Japan

DC I.. B . sellgm40n. ~ r . ~ i r r r t o r . Dlvirvon o i r o n t r o l Att iv i t i rr , I lurclu ol 'B io lo~?s. iood and Drug Adrnlnirtralion, Ruckvillc. h l d . 1154

UTA. b ~ u ~ % l t o a r n . C h d Mlds~s ! URloer. B8010gii;rl Slandzrd~r~~ciun, WHO. GC,,*"&. S,L~~ZC'IJ"~ (s<<r<r,?ro

BLC/WP/~ 7 . 1 Hev . l Appendix D.27

Annex 4

GENERAL REQUIREMENTS FOR THE STERalTY OF BIOLOGICAL SUBSTANCES

Pa ec In1 roduclioo . . , . , . . . , , . . , . . . 41 Grnrml cvnstderariunr . . . , , . . . . . 4 I Pall A . Manufacruring rcyuiremonts

l . Teminclopy . . . . . , , , . . 41 I. Central precnulions ayainst microbi~l c~nt r r r l~~ l lon in rnanl~hcturc . . . , +1

3. G c n e h l precautions againsr microbial conlamjnation from materials used Tor

manufacrure . . , . . , , . . , . . 47 4 . Cisncral prccauloos again91 microbial

cnnlamindtion in slrrilily 1cslin8 . , . 47 5. Slerdily tcrts . . . , . . . . . . .

48 5 . 1 Samplin~ , . . . , . , . +. , . . 48

5 2 Stcriliry tcSa for baclsria sod fungi . . 49 5 3 Sterilily 1-t for mycoplasn~s. . , , , 5: 5.4 Stc'llr~y test f o r virus- , . . . , , 5: 5 . 5 Tcst3 fur S ~ F C ~ ~ C micr~-or~nisnls

. . 52 6 . Records . . , . . . , . . . , . . 53 7 . Addjt~owi wmpirs. . . . . . , , . , 5 7

h r l R. Ndtlnnal conlrol rqulrcments

I . General. . . . . . , , , . . . . - 5.3 2. Kcrcdsc and ctrtiticatinn . , . . . . , . 5.1

-4ppcndl~ I Media for lhr: d~Itcti~,n of aerobic and ~nacrubic bactctia and Fungi . . . . , 54

appendix 2 . R~?crduh fur sierilily Its1 usinp rnemhranr filtration . . . , . , . ,

5 5 Appendix 3. Tcsls for mycop1asn:a . , , .

. . . . 6 Apprndix 4. Acknowkdgemcnts . . , , . , , . , . 7 -

BLG/UNDP/I~. L Rev, l

Appendix D.27

IntroducCiw

General requirements for the sterility of biological substances (Require- ments for Biological Substances No. 6) were formulated by a W H O Study Group in 1859.' These general requirements were applicable to any hiolog- ical product from which the exclusion of microbial contamination is imperative, and they have been quoted in all the sets of requirements for indi~idual biological substances that have since been formulated. The twenty-fourth Expert Committee an Biological Standardization agreed thar i l ~ view of recent dcvelopmcnts In methods of sterility testing of bidog- iwl products and the improvements in control measures that were now fcasibie, the general requirclnents for sterility sbould be revised. This tusk was rherciore undertaken. Sinr~ the preparation of a set of amendments or alterations would be an unsarisfactory way of revising the reqil~rements, it was decided to provide a complcte document embodying all the rqu i re - men15 concerned. Where provisions famhdated by the earlier Study G r o u ~ were retained unchanged they have been emhadied as such in the p e n t revied rquiremtnts.

In p repr ing these revixd requirements account has been taken of the opinions of connultants, thc rebvanr regulations and requirements that have km formulated in a n u m b a d counrries, as well as information from both published and urkpubli~bed reports. In addition, opinions and data relevant to these rtquiremcnts have bctn received from a number of t x ~ r t s to whom gratcful acknowledgement is made (SK Appendis 4,

The rcquircmcnts in this document apply to all immunological biolog- ical substaner, i.e., vaccines and =fa, that must be sterile and arc u r d for edrninisuation to man. Many of the ptovisions, howcvcr. my be uscd for t h t control or sterility uf uthcr biological p m t i o n s , or of blood and blood products, with suitabIc modifications, where ntmsary .

wrd Hfth Org. rerho. Rrp, dv.. 19M),No, 200,Tl1e mcmbtnOflhcStudy Group were: Dr M, wrn Bcntron, Statms Scrurinrlilu~, cop hag^^^. Dmmmrk ; n r P. h. Bonnd. Ccntrt dc T r a ~ f ~ s i o n - Rt/nimation dcIiArmk, Slat. Frm- (Ropportrurl ; P . dc

G&r, tnstitutc of Microbiology, Rio d t Juciro. Brazil ; D r M. Pitlrnan, Dinslon df

Biologies Stsadudn. N ~ t ~ o n s l Imtitutm of Hrallh, BLthtsda, Md., USA (Chairman); Pr G Pcnno, Labomrory of Microbzology, Isritutn Su~t ia r r di Sanirl, Rome, Italy ; Dr R . H . Regarncy. Ind i lu t A'tlygitnt d t I'UnivenilC, Gcncvd. SwRz.crland; Di Surniatnu. Par t r i~r Inttitutc, Bandpn~. Indonesia (Vicc-Chairman) ; Dr 1. O'H. 'I'obin, P i ~ l ~ g c a l Slandards Control Labwatdry. Medical Rcacarch Council, Humpstead. Lmdo?. Endand (hpF1cur): Dr G. V. Yymdchikov, N. F. Gamalcja lnstltuts of Ep~dcm~olagy and Microbiology, MOSCOW. l.ISSR. Secvetwiar: DC B, K . Bhatlachqa, Mcdial OWlzf, Biol~@crl b~undardiratlan. WHO; Dr N. K. Jcmc. Chid Medical mcer , ~io lof id Srahdnrdization. WHO. m d U S"1.r~. - - p~

Wld Hlrk Urg. rcrk#. Rep. Ssr., 1972. No. 166, p* 19.

BLG/UNDP/'~~.~ Rev. 1

Appendix D.27

The Study Group on General Requirements for the Sterility of Biolog- ical Substances I (in 19591 pointed out that confidence in the sterility of bidngical preparations depe~lded on two important considerations ; first, adequate control tests for the sterility or biological preparations in their final containcrr, using a suficimt numhrr of random rwnpln, and secondly the use of suitable precautions with rcspcct to source materials and manufacturing prwedurcs. T h e Study Group considered available infor- matiuri on sampling proccdutes adoptcd in several couniries and also discussed a suggested schedule for sampling of fiml containers to cnrurp an acceptably low probability of the rrtease of contaminated final conminers.

Theoretically, sterility may he defined as the absence of all micro- organisms capable of rn~ltiplying.~ Sterility tests employing a reasonable numbrr of samples are capable of detecting contamination only in a lot with a high prcmtage of contlminated units. Accordingly, it is not possible to delecl, on the basis of acceptable confidew limits, a low prr- centage of contaminated uaits in a homogeneous lot of final product. If the finished product is a single final container of bulk material, tben the matrol measures are appliable on the basis of an assumed homogeneity of material in such a single final container. Sterility, therefore, cannot be assured in the control laboratory, but must te built into the produd during processing. Experience in many countdes over the years has confinned that greatrr reliance must b. placed on appropriate tcchniqus and p r w - dulrs throughout the manufacture of the product (including in-proass Ymlity testing at various stages) rather than simply depending on sterility tests made on a number or samples of the final batch as the sole basis for criteria of sterility. Notwihstauding the limitations of sterility tests. every eRon should bc h d e to use the t tmt effective praadure and to strengthen and validate the tests in the light of Ule most recent knowledge and cx- per ience .

The tesu for nerilily mentioned in the previous &"ern1 yu i r cn rn t s have k n reviYJ in thc prevnt document. both in naturi and in smp . They now include recommendations for tea p r o d u n r for mycoplarmr trnd for tests rrsing membrane filtration. Tcsts for viral contarnillants are not dealt with in detail because requirements mmra ing such contaminants of biological products and appropriate tcsts for viral sterility have heen included in thc sets of Requirements for Biologicat Substanms a that have k e n Comulated For individual products. Similarly, information on whcd

. & In I he case c l a producl ronsisting of Itving micro-organisms, =.g,, ccrmir. vaccines, sterility mosisls of the ahsm* of concamination by oibtr nicro-organisms. Pratifalfy, hmerrr, osscrtions q r d ~ o g stcrilily retalc to the probability that all units in the lot are slcrile. For ec*mple, in mmc counfncs, a 101 of a product i s acccprcd ar r t c r ~ l i il. there bc n o m m than one living micro-organism in one million units.

a * U.'J'~ ffjfh 078. rcchfl. Rrp . Srr.. 1973, No. 530. p. 62. for a compleie J f r f .

T, B L G / ~ ~ W P /7 7.1 Rev. l

and sterility tests are to be made will h found in tb. aqu i rnzn t r

for individual products and this question is not discvsvd in detail in the present document.

h c h of the foliowing sections constitutes a recommendation. The parts ul each ectian that are printed in large type have bsn written in t h e form of requirements m that, if a health administration so desirrs, these p r l r as they appur m y bc included in dclnitive nationd requirements. The m r t s of each section tbat arc print& in mall type are commenrr and recomincnbtian~ for gu ibncc

Sanvld individual cmnt"es wisb to adopt tkrc ~ q u i ~ e m n t s a5 [he basis for thcir national regulrtionn concerning serillly of Mologid pmd- ucts, it is recommended tbat a clauu be ittdudrd that would pdt modih- cations of mnanufacturing requirements on the condition that it be demon- strated, to the satisfac(ion of the national mnlral authority, that such modified requirements ensure a drgrrc of sterility o l the produn at least equal to that provided by the rquiremnts formulated below. It is desirable that the Wodd Health O t g n i a t i o n should tben b. i n f o m d of the action taken.

The terms national control authority " and " natiaoal cmtrol labor- a toq '' as used in these requirements, always refer to the country in which the product is manufactured.

l. Termin-

Contamination : The prevnce of l~ve extoneous rmcrodrgnirms. The ,cm ex,raneous applies not only to micraorpnism that m y enter the pruduO during proccrsing, hut alto to those that my bc p r ~ n t in tbc materials used for preparing the product, such 3 s SV40 virus in tissues used to propgate certain viruses for vaccine poduction. Micro-orp.nim$, as tefrmd to in this document, include bacteria. fund. uirurss. nckcttsia~, mycoplasmas, and chlamydia.

Brlk mowrid is n qusntity of partly or wholly proursed material, orcxnt in a r i n g container at any stage ptior to distribution intn final containers.

Finvl bulk mtairriol is the homoyncous finished pfcparation yrewnl i u a dndr container from which the And containem are filled, r i tb~r dircctly or indirectly, through onc or morr intermdiaie containers.

I n some cases a final bulk may conslitutc the manufacturer's -~~ -~

ftnal product.

Plodur( lot : ,411 finished material, in rcaltd final containeS. &n derived from the u m e final b~&, all Of which, n the last s t a v of

BLG/UNDP/I 7 . l Rev. 1

Appendix 0 . 2 7 processing capable of alteang its composition. has k e n processed together and theretore has a uniform con~position,

In ths case. fur cxalnp[e+ a vacrirlc swh a lot may he termed a vaccine 101. The ! e m s pruduct hatch end vnccinr. batch may be used as appropnatc.

Final lot : A collection of scaled ha1 containers dcrivcd from a sinslr filvl bulk, which are homogeneous with rerpst to the risk of contamination during fitling, sealing and, if applicablc, during drying. A final lot must therefom have been filled from a single container, and T

, t applicable, dried in One continuous opnation, e.g., in one working rcsrion, the procrsing bring so arranged that each such operation yields a single homogeneous batch of the product.

A flnal lot may cu~lsist of the whale or part of a product lot. In appmpriatc cases Ihc terms Lml batch, filling lot, filling

batch o r drying lot may d.

E V ~ V manufactutifig ~rabliuhm~nt should ia addition hnve a separatc and independcat quality audit to w k h every batch of ~ W d u c t would h $ubjsc~od a t the cnd of procesbing; the purpose of such an audit would bt to ensurn that the measurer adupttd for control of mimbial conlamination of thc product have b a n carried aut gatisfarto~ly.

The general manufacturing requirements given in Part A of thc revised Rcquiremtnlr for Biologkd Substances No. L (Gcnenl Requirements for Manufacturing ~ h l i s ~ n t s and Control Leboratorie~)~ shall apply which the addition of the following :

The requirements conccrnine building and equipment given in Part A, section 2 , o f the above q u i r m c n t p arc applicable. Thc most desirable siluarion might be the provision of scparate buildings for each product. This, however. i s h s i b l c o r aeccs. saw only in special circumsl*rrccs or for very large-scale manu- facture. The sitantion uwaliy tncwntcrsd is one in which more than ono product is mmufacturd in on= building or Iabomtory.

If the-manufscturiog pr-s rcquim~ that micro-organisme be cultured a d pmanaed to yield Mmentralcd rubpnsiuos thew may b a o m ~ the SOurcw of scrawls o r may conuminote qulpmcn!, clothing, and personnel. The potential for cram- mntamtnation is high and bacrisrs should b cstahllshed to Phvcnt conlaminalion o r Iht tnvjronmcnl and cross-conta- minalion.

B L G / U M ~ Y / ~ ~ .l Rev 0 1

Appendix D. 2 /

The virrious mawrcs to avoid contamination arc c5scnlirlly b8rricrS that may be spatial, remporal, or opcnlionol. Thc nature of these barners dcpmds on thc risk of contamination and on the mshanirms by which conlarninatinn nnd croS4- coptarnination may occur, and i c wiLl valy with tht nature of the potential hazards inmlucd T h e barden mry include (n) mcas- urcs 10 rcjfrict ccrtaio orpnisms and/or uperations to separate buildin@ or separalc arm5 within the one building. (b) restric- tion of the movement of personnel and ma~erisls, (c) the trca1- ment of air to remove or dnlroy aerosols con~aining micro- organisms and to conlral relarive humidity, (&l the es~ahlish- mcnt of pressure gradients to conlrol the direction of air move- mcni, (P) the provision ol. sp&al work slations such as laminar flow stations o r biological safety cabinets, and ( f ) wearing of suitable clothing. including head-covering and footwear, by

I€ the same area is used for the successive manufacture of different products, the area as well as all equipment it contains shall be adequately cleaned and disinfected aftcr the proces5ing of one product has beer1 com- pleted and btfore the proassing of another product is co~nmenad-

Manufacturing procedures involving thc handling of microbial sports shall bt confined to a separate building or to a sepalate area within s building. Thosc involving livc virurcs shall be arrangdd so that ar any time each distinct virus is confined to an area that i5 physically iwlated by ap-iatc barriers. Virus product testing l h a ~ requires thc excluuon of adventitious agents shall also be subjected to this rcquircment.

The preuutions against contarmnation given i n Pan A, e t i o n 3.4, ai the revised Requirements for I3:obgical Substances No. 1 (General Require- ments for Manufacturing Establishments and Control Laboratories) ' shall apply. Thcre should be careful control of thc air filtration and

routine microbial counts of the air in the manufacturing areas should be c a r r i d o u t during manufacturing operations or period- ically using an appropriate method of air sampling. The results of such counts should be checked against criteria, establishtd for the particular rnanurac!uring area, and adequate records of the counm should be maintained. Where air filters are uscd in manufacturing areas. fillem that remo%'e 99.90% + 0.010'0 of particks ~ c a t e r than 0 . 3 pm in diameter are suitable. 'I'hc effi. ciency of the filters should be checked routinely.

The re~otnrnendrtiut~s conwrning filling and containers given it1 Par1 A. bcction 4, of the revisecl Requirenients fur Biological Substances No 1 (General Reqvircmntr for Manuhcluiing E~tablishmc~tts and Control Laboratories) "hall apply.

' Wid Hlth U r g . terhn. Rep Str., 1966, No. 313, p. 13. ' Wlrl Hlth Org. tecbrr. Re,,. Gr., 1466. No. 923, p . 16.

B L G / U N D P / ~ ~ . ~ Rev. 1

Appendix D.27

Filling o l w r a i i o n s shall be c o n d u c t e d ill such a way as to avoid a n y contamination or a l t e r a t i o n af the p r o d u c t .

T h w up-rslions should w k e place in contrullcd arcas specially Jestgnrd Tmr he purpoar, in whrch steps arc taken to r t d i t r ~ 10 a minimum thc number ur micro-organismr 10 which the product k i n g fillcrl is espnscd. The use of Ian~inar Anw work stoltons, disinrecfing ngenls, air liltcn, and steriliad clulhing, i9cluding hwd-coucring and Footwear for perrunnel

conducting fllina operdtb~nx, is rccnmmentled. Germicidal lamp-; and dihinfwting acrasl?ls should not used during filling opcrstions.

The operirtions wherc liquid preparations ore filled should be checked. This may be done, c.g.. at least twicc each year at Ihc clrr3c of 3 working day, by filling not less than IOOO con- tdincn w ~ l h J nutrient medium containing no antibiotics or bactcricidal s c b s ~ ~ n c e s , and incubating the cornplere batch of filled cunl;rinrrs a[ 30'- ?6'C fur at Icast 14 dnys. If containers filled 2nd incuholed i n this manner $how a contamination rate above Q. 39b, some coentricr do n n t consider the procedure w ~ c p l a b l c (or the tilting of otcrile prepur?lions with or without an anlinticrobial i g r n t .

The p-nce o f micro-orgunkms in the air nt rllc filling point ihuuld be munitnrcd, for example. by mcms 01' wltle plates or s!i\-sanrp!ing ~ L T ~ C C L

A l l m a t t r i a l from an area in which virus is heing processed sha l l be a u t o c l a v e d he fo re be in3 sent to t h e witshin? and sterilizing d r p r t m e n t s , irrespective o f w h c t h e r each area has i t s o w n facili t ies or common facilities are employed.

A common washing and steriliution 3 r d ic1r glassware and culture medi;r may be nsed where l u o or murc virus vaccines are being produced, provided lhat only heat-bterilieed n~arerials are issued (or use in [he production areas and that each production area has ils own equipmcnl suitably marked. All media that ore s1crilk.d by filtration alone should be prepared in a sepsr- ate arc3 with ils oun cquipmwl for Ihc prcparntiorr of such media.

In controlled areas, apart from specific organisms or cells beinp. ptocessed, all material intruduccd from thc main washing and stcriti i l ion area should pfcfcrsbly bc sterilized by heat. I t E. preiernblc that auloslnrrs or c h ~ m k r s used fur thc slerilization c~f materials rajsing in nr gut of an srra Irc of tlle " dauble-cndcd " lypc. built into the wall in ;r suitnhlc niantirr and ~ohjeclcd ro control pr~:cdrrrcs such thdl ~ h c only passap way for malcrbls bcfwecn areas cont;ll~ing ~unslcrilirsd ;~nd e~crilized mnreriab b throuph the autocl:~ve or srcrilizn~inn ch,lmber itsclt'.

Cell cu l tu rc s for vacc-lne p r o d u c t i o n shall be prepared 111 a special a r e a separated from ntiy area in which virus is be ing h a n d l r d . \Vhere different

46

Appendix D.27

cell liacs a r e h a n d l e d in an arca measures shal l be taken t o p r e b e n t cross- c o n t a m i n a t i o n of one cell l i ne with another.

The room and e q u i p m e n t used for hlling a lire v i rus vaccinc into con- tairiers s h a l l be used solely ior this particular vaccine , unlcss it is a d e q u a t e l y disinfected when it my be used later for the filling of other products.

Whcn animals srp uscd fo r a b ~ a i n i n ~ tissues for cell culture. appropriate recommendation$ should apply.'

3. Gcwd precauutloas agninst microbial c ~ ~ l i o a from mmteriak nstd ibc nuMf.m

Precautions shall be taken to avo~d c a n t a m l n a t ~ o n f r o m source or o t h e r ma te r i a l s uscd for m a n u f a c t u r e that m y affeci tk sterility d the final product.

All s t r a i n s of micro-organisms used as seed n~ateriak for biological

preparations sha l l be maintained in a manner that wil l assu re freedom from c o n t e m i n a t i n g micro-organisms.

h cr~ntamination v i a sced lot iormduca lltc pus~ihilily of 6uWqucnl contaminPtinn or a n u m k r or production lots, smal prccnulion(i are ncmsmry urhsn prcparrng or rnunipu- l a b 8 seed 101s.

All blood and blood-derived source materials shall be obtained u n d e r

conditiclns that e n s u r e a safe and effective final p r d u c t . I n the case of human bbod m a t e r i a l I h c r e q u i r e m e n t s f o r ~ U I I ~ C C material given in Part A, section 3.1, of the R e q u i r e m e n t s for Human l m r n u n o g l o b u l i n (Requ i r e - ments for Biofogical Substances No. 14) ' sha l l a p p l y .

Particular attention should be pad to any regulations that are appticable to the manulacturtr and:or organistlon that collccts such human material.

The a p p r o p r i a t e r e q u i r e m e n t s for m n u l a c t u r e given in Part A. seclion 2, sha l l apply to those areas in which, and those p r ~ e d u r e s by which, sterility teaing is done.

Thc r ~ s k o l accidental con~amination irom #he environmcnl should he kept to a minimum by l l ~ r usc ordlsinrcctinf, agcnls, gcmicidal lamps, and air fllicrs. Gcrmicidal lamp5 end disin- itcling acrosnls rhoulrl not be urcd during actual testing rrpcra-

.

1 et, for c.iilmple, Hru/fk t ~ ~ p ~ ~ i ~ of the s ~ ~ p r l y und u . 7 ~ *if rrn~~-hrrrwfl l ~ r i n l ~ f e ~ for bl~medlc.nl pur(rarr,, rz,ior, OJ tr l r h ~ ScipnrGr Groiip I I I L ! H I I ~ O r s - ~*.~.llfl. Rc81). Scf.. 1971. No. 470).

HlrA Org. rr',hn. Rtp. Sd'r.. 1967, Xcl. 161 , P. 49.

B LG/UNDP/~ 7 . 1 Rev . l

Appendix D. 27

tlons. The lest manipular~ons should bc carried out in a filtcred air environment ur under a brnlnar flaw hood. and thr uperatom 5110uld be dressed in sleriliad, i'llcclricslly nAlrrsl clothing, including had-covering and footn.ear. The zir pres- sure in the lesling room shuuld be grearer than the exterior area. The perftlrmnncc of rht Iarninar Raw hood should k munitorcd by scttlc ploles or slit-barnpl~ng devices and thc car-iency of rhe 61tcrs and germicidal lamp chccked mutincl,.

Sterility testill; shall not be car r i rd o u t in produc t ion areas.

S. Sterility tests

Sterility tests shrill bc p e r f o r n ~ c d u h c ~ ~ e r e r specified in the reyulrenlcnls fu r indibidual p r o d r ~ c t s .

Tests for sleriliry are often perfurnled adranta$causly a t various s u t g ~ s o f rnanuhcture. In addition ro how given in hquiremcnt~. The t s t s described In ihese rcquimcnis may L* u d for this purposc.

Detailed n r l s and ptecautLDns for the stcnlity t n t pra- cedur6 should be writI.cn and carclully fallowd.

me staff performing the sterility tars should haw cxpri- cncc in thc duties assignd. Sopewhion n l iht work and Inter- pretalim of sterrlity krls should bc undertaken by an individual with training 111 scicntlfic subj~~is. who should alsa have triijncd in m i c r u b i o l o ~ or k ~ i v e n trsining in that subject.

Where sterility lests lisr bactcria and fungi a r e required: such tcsis shall be made 4s described in Part A, section 5 . 2 . Appropriate rests for viral a n d

r~ckc t t s ia l sterility. hen r c q u i ~ d , shall bc m a d e as speciftrd in the require-

mcnts for t h c i ~ k i i \ i d ~ ~ o l products . Tcsis for r n p p l a s m a s , whrn required. shall be made as descrihcd in Part A, secti011 5 . 3 .

The specified n u m b e r uf suitable samples shall bc taken r rom [he producr. S u c h samplcs shi~ll be t aken 31 least from1 ewh final bulk, as well as f rom each final 101.

A sample shall be takcn from each h ~ ~ l k t r l be rested i ~ t such a m a n n e r a5

t o bc reprcsentat i i fc of t h e rnaterial tu be ~ c s t e d . T h c smuunr 1akerr shi~ll be sutlicient to perform t h c rests a n d ally repenr tests tha t map 1~ retlr~iretl. S I I C ~ s a n ~ p l e s sl:oIl be takcn so as to n ~ a i n t a i n intact the level uf s ter i l~r) of the material , o r , if this is not possible, ~ h c samples shall be raken : ~ t the s t a y of further prcc-ersiy.

BLG/UNDP/ 7 7 .l Rev. 1

Appendix D. 27

Sinct any microbial contaminants in a liquid may settle out, thorough mixing i$ q u i n d befort the sample is takcn.

5 .1 . 2 Sampling from final lot

Samples of find containers from each final lot to be tested shall Dc t a k e n in such a manner as to be represcnut ive of thc lot to be tested. Appropr ia te p r i o d i c samples shall betaken, i n c l u d i n g s a m p k s a t ~ h t b g i n - ning a n d t h e e n d of t h e filling o p r a t i o n . -

I f a product lot h bllcd through several ourlcts from a ~irr#le bulk, surnpler should be taken from each oi~tlet (filling [ut) so as to be rrprvscntativc of the filling as.~mhly.

The n u m b e r of samples taken shall be at least that approved by' the nat ional con t ro l authori ty, providcd thnt far final lots cnntainirlg 500 or more containers , at least 20 snmplcs shall be t akcn , including samples at the keginning and the e n d of t h e filling optration. -

Fur Anal 101s c~ntaining fc#tr than 500 wntaincfi. not W than 10 conlclinep from the lot should bs l h o for a sample, exccDt that for lots of lejs that 10(, containers. only 10% uf the Iot need bc taled.

A rrrg~mtcd rule 1 for culculaiing the number of samples

is 10 iakc 0 . 4 \, N ssrmples, where N in the nutlthcr of Anal

The ru l to rc media used for sterility tests fo r b a m r i a and fungi shall be those approved by ihe natio11aI control aulhori ty. S u c h mcdia shal l have btcn shown to be capable of suppurlirrg the growth of a widt variety of m i c r o - u i ~ i s m s , with haih aerob ic and anaerobic growth charactsistics, including thc t y p found in the environlnent of the manufacluring opet-

Morc than unt culture medium, gcnemlly, will LW nsedcd tn fulfil I h e ~ criteria. The media used in many countlies for rtarilicy tests atc Ruid thioglycoldtt medium and r o y k a l ~ a s c i n d i g d medium.' Any other mcdia that are used, howcvcr, shruld hnvc bcen dcmunsiratcd to hevcat kas t equivalent growlh supporting propertim.

For tcstin8 the grvwth supparting qualities of each cullure medium. streins of micro-organisms should be wed with exact- ine nutritivr and aesobic-annerobic teqtirernenrs. in an inac- ulum of only ;L small nu~~lber of the arganisn~s ( las rhrfl 1001

- - ' Wld H1111 Urg. ~eckw. R F ~ . S r r . , 1960. No. ?{m, F. 31. a The formulae of these culture media arc give* in A p ~ n d i x 1 .

B L G ~ U N D P / ~ ~ .l Rev. 1

Appendix D. 2 7

T h r media should be incubated at the temperatures a t which they will be used in the stecilitv tpsl.

, . - - Each hat of dehydrated medium o r each lot of medium

prepared from basic ingredients should be tesred for its growfh supporting propenies, since every lot may not support the %rowtb micro-orpnkms as well as desirable. 11 is desir;lblc that 101s so tcsrcd should be kcpt separate and reserved for use i n slcrili~y tcsling. This ir J safeyord against occasional unsalishctnry cornpnnenis in a particular lot, ar destruction o f certain cnrnponenls by over-heating w over-rrerilizbtion, which m a y ciluu differences in growth raponsc.

Irr somc countries an additional test is made to verify ihc srowth rupponingprvpcnies o f the mcdium, arter thc completion 0r.l s[efili(g 1cs1 ill whith no grorvrh has occurred in !he medium by inocllla!iort with a 5nr:lIl nnmbcr of ru i t~h lc u rga~r i~ms (I& Ihsn 100).

B L C / / U N D P / ~ ~ 1 Rev . l

Appendix D.27

The q u a n t i t y of medium pui into each v ~ c l for conducting the sterility

test shall b. suficiently Iarge 10 ensure that the volume of ma te r i a l inoc- dated does not impair the growth s u p p o r t i n g propxties of t h e medlum l

by dilution. * I1 vcavlr of i nocu la t ed media rhal l k clearly identified by label l ing

th.! is aciquate t o ident i fy tbe p r o b u c t k ing tested. e a c h m e d i u m used. and each temperatux vf incub6on.

5.2 .2 .3 I ~ c u k t i n r r

AU vessels shal l he incubared at the appropriatt ternpewturn fur the m e d i a . Tht tcmpcntllm selected rhall be t h a l c a p p r o v e d by the na t iona l cvatrol authority and sha l l include 20"-25:C and 30-36°C'.

Prior to conducting A s t ~ t i i i t y test on al ly p r o d u c t , it shall have h e n 1 d e i e r m ~ n e d r h e t t r or not t h e nmter ia l to h Tested itwlf has t h e proprly of killing or inliibiting the p i w ~ b of m i c r o - n ~ a n i s m r . or c o ~ l t r i n r prcwrv- ac i r c r or other subs l ancv$ that h s ~ i t h h effect If s u c h m c f i c i i s shown, the Ycrility NISI shall be nude using a su i t ab l c ~ o c r d u r e to counterrcl the effect.

~f only on. tmpcrall;rc is stltcrcd for n p:lr~icular ~ c d i u m , gemrP)Iy fluid thioglyc&rc nudlurn is incubalrd At '0"- 3G"C and cuein d j p t m&um At 10'-21''C.

In so.mc countries a d d i l i a ~ l tempemluW5 Ure used for psyrhrophilic and thrrmophjlic organhms,

lr is &inus that the incubatnon Pppamlu5 should haye a

Ihr lfih'hi["fy t.Ikc1 It1 ;I prtp.lration to for i'y bcovcrcome by incrtusing the volume ofc",lure nr2dium u"~'. 'i' lhqt lhc ~nhibitrrrr r e render& intpctive, or if Frr~"r 'r 'oncnn filtcrcd. the inhibiton removed by mt.mhrunc '''mlll'll.' The volunlc or medium rrquin;d to overcornc rhc

e R ~ l shtluld bc determined. Oncc criabljshcd. rksc 'lAdniitics mfiY hC u x d b r subseqbcn( sfehjicy terts unless rhan@c is m;ldi. ill the ctrrnyc~sition o f the prt,ducl,

*,, veslal. ilxubted for a p c r t o d of *art 14 day' and *'l1

b. Mmined it and (m the !a* day (lf i*cubiltiOn 'Or

=.,iknce of mraobial g r o w t h .

. . If prcprPlion imcu[ared into the ~ ~ t ~ e s ~ ' ~ has .- . l. an Ertsnt that it is dfifulc Io rccngnirr -.> ." . '. . ... wherhe, no, growth

has taken place, subc"laurrr nu' less ., . . :', .d ,. than .O ,,,l $hwld h made from the cloudy bcCwcen

5 2.2. 1 i / ru~ t t~ l l l l f 'C.. ,~.; I,: lhlrd and scurnth days the start of Ihc test- me

.,+,g llwl and trsnrlcr vmse15 M lncubaleJ alld observed For of hulk nlalrri;ll. ;if least 5 mlrst be used for for a af nut Icss rhan 14 days. C 7.; .

i r lncu la t ion intoeachcullure m e d i u m altd foreach temperatureof incubation. For s t e r i i i l ~ testing a final 101 from each of the final containers 5 . 2 . 3 MPmbrme jiitrnriun of sanr~ier

20 ml. an a m o u n t of'at least 1 .O shall h inoculated pcrlormance t s ~ s \rith t k aid of membrane jnto c'hute m e d i u m and f o r each tempemture incubat ion, the filtrstio,, rhc tat aampln is

a valuable mclhod d improving in each f inal c o n b i n e r is less t h a n 1.0 ml, the

snlount to inocu- tfi(5tlnz in ctrtatn situations' lated be tbc ent i re

cantent 01. t h e conrainer for each culture nledium tlrm manipulalions invo[ved in lhm prdures

,ional thos for the pcriarmance of the fc" dtrcribcd in each of incubation. If the volume i n each final con-

sE,ion 5.1.2 may. however, be a murce Q' cxtrancous

greater than 20 ml, b u t not more than 100 ml, the amount to be mn-inarioe H~~~~ routine use of p ~ i t i v e and negative

shall be least 5 mt for each c u l t u e medium and for each coamls ad,.isablc for validating the results A

1emperatu1-e of incubat ion. control k the occasional use of known 'On- - -

~

ke Ap&--37d1x I for a description of rhc procedure liltration, I Sc *I*lx I a *er"pla a procedure ror 5tcrllitY tests using membrane

50 filtration.

5 1

BLG/UNDP/~~.L Rev, I

Appendix D . Z_L

laminated solutions containing a k w micro-organisms of dif. kring types ; if tht ptoduc~ does not contain a pmenative, approximately la mble microbial cells may bc uwd in the total volume employed. If the product contatns a prcscrvative, the positive control may be added ro the wash fluid

II no evidence of growth is found in any of the vessels inoculated for the test for srerility in Parr A, seutiun 5 .2 , the bulk material or final lot, which- tver is applicable, meets the requirements for r h ~ s test. If evidence of growth is found, the preprdtion tested fails to meet the q u i r e m e n t s for thc t en for steriliry. unless it can be demonstra~ed to the wtisfaction of the national contrcll authoriry, by retests or by other means, (hat the test was invalid.

The decision belwtcn failure o f l l l c product ro pass the t o t 31rd invalidily of the lcsl prurrdure requires compcrenl judgc. men1 vr a tnincd individual.

For viral vacr-ines made by growing the virus in animal tissues or cell cultures, sterility tests Tor mycoplasmas of virus culture fluid and control fluid specified fur the panicular product shall be made by a method approved by the national control authority.'

T h e ~arnplcr takrn may includc muterial prior t o clarifi- catiun or tlllrrtion in ~ h l : rase br live v a ~ i n e s pmduccd from i l l vltro living cell culturcs or animal tissun. In thr casc or inacfiviltcd v i m vaccines producrd from such cell crllt~lrrs nr tissutr. tt~c sample may bt taken prior lu inactivation, from circh virus hawer1 pvul and from cvntrul puvlcd fluid.

Control ~ultures should be included in each rcsl

Thc sterility tes ts for the detection of exlrnneolls viruscs in producls where their prcserlre is unaweptable shall be prformcd as spciticd in the rrquirements fot the individual prnducts.

The tcsts to cnsurc thc non-surcival of ~nicro-organisnls in inactilatcd iacuines and additional tests for ~ h c abstncc of specific extraneous micro- orpanislns in such products shall be perrorl~lcd as spcified in rhc reqll1r.e- menls for the indi~iduc3l products.

BE/UNDP/~ 7.1 Rev 1

A p p e n d i x D . 2 7

7 -

Records shall be permanent and clearly indicate all steps in the sterility testing of the product, including temperatures and incubation times, as well as the relationship of the samples under test to the ~nanufacturing operations followed in the production of thc lot under test. The records shall be of a type approved by the national control authority. Written records shall be kept of all tcsts performed, irresptctive of their results. They shall be retained throughout the dating period af the produn, and be available for ~nsprction by the national control authority. Records shall be mainlained of all culturcs kept in the establishment. Such records shall include labelling for the ~dcntificarion of the culturrs and the purposes for which the cuitures are uuscd.

Fnr each sterility test performed a record sholl be kept of the name ;md identifyills numbers of the product under test, the quantities inmulaled, the batch. type and tests of culture media used. the tcmpecaturn v l incuba- tion, the dates of inoculation ar~d thc results.

7. Additiotul snmpl~s

For each final lot for which sterility tcsts are made, additional samples shall be retained 3s reference material throughout the dating period of the product, in P manner that ensures thr identity of the lot or batch of tbc product.

11 i a dc>irablc that, wlrere p655ibIt, manuhcturrfl dhnuld rrtilin tufficitdt ndditionvl ssmplfi to pcrmil npetitiub of the srerility trst4.

Part B, National Coatrol Requiremots

1. General

The general rquiremcnts for control labratofits even in Part B uf thc revised General Requirements for Manufacturing EstaMishrnefits and Cortlrol Laboratories (Rcquire~nents for Biological Substances NO. l ) shall apply.

2. R e l w and certification

The requirements given in Part B, section 3.3, of the rcviacd General kquirements for Manufacturing Establishments and Control Laboratorits (Rcquiremmts for Biological Snlhstancrs No. l )? as well as the relevant pro\isions in the requirements for the indiridual products shall apply. --

)I'Id Hlrh Org. teclrrr. Rep. 5cr.. 1964, No. 313 . p. 19. ' I ) /d Hlih (h*. tcchn. Rep. &r,, IYldt , No. 321. p . 21.

BLG/UNDP/~~ .l Rev. 1 Appendix D.27

;ip-k r MEDIA FOR THE IIETPCTION OF +GRURIC AKD ANAEROBIC

BACITRIA AND WCI *

~-cyatinc . . ~ . . , . . . . . . . . . , . 0.5 g spdium chloride ~ . . . . . . . . ' . . . - . 2 . 5 g glucose W,tftzO~.H,OI . . . . . . ~ . . . . .

5 . 5 g agmr . . . . , , . . . . .

. * . . . . 0 . 7 5 g yeast extracl, water soluble , . . . . . , . . , J,O g pancreatic dlgcst orcascin . . . . . . . .

. . 15.~1 g distilfed wattr . . . , . . . , . , . . , , . l w . m3 thiogtycolic acid . . . . . . . . . . . . . . .

0.3 m1 (or Saditlm th~oglycolatc . . .

. . - - . resnzllrin sodium (0.10% fresh soluiion) . , , . 1.0 m1 Final p H - 7 . 0 - 7 . 2

Thoroughly grind the first %l* ingrcdicnis, i n the urdsr given aburc. i n nrorlnt. Stir in some heat4 water. Iransfcr 10 a suitabk cotruiner, add the remsinder of the

wslcr and complete the rolution. by hrmlng in a boliing water bath, takinp special film t~ ensure fornplete solution of :he L-cynline. Add #he thioglycolic compound, then I W sodium hydrmidc so lhat the pH of the conplctid and sterilized medium .iill b 7 0-7.2 . Rehul the s o ~ u l i ~ n , but do not boil. filter (if necessary) thrnufi a mallrntd finer papcr and add the r~lazurin solution. Distribute in10 suitable vr*reIs ond stenljrc hy auwclaving for 18-20 minutcs at 121°C. Cool promptly to :ii°C a ~ ~ d %:me a t 10'-30°C avoidingcxcessive light. I f the uppermost portion nE thc mcdium hn, chan- ped IL* a pink colour and this exceeds onc-third of the depth c ~ i the ~ l ~ e d i ~ ~ n l . I I i'l

u"guirable roruse, but may be rcsrorcd o ~ ~ c c by het~ttng in $IC.~!,I. hlcdir~nl mnrr than 3 Wcucrlis old should not be used.

pancreatic digest or carein . . . . . . . . , , . 17.0 g

pilpaic dipx.1 olaoybean meal . . . . . . , . , 3,0 g sodium chloride . , . . . * . . . . , . . , _

dipntassium hydrogen phosph~tc . . , . . , . :." g - .5 g

g l u a u [GHIzO*.H:O) . . . . . . . . . . , 2.5 g di31iited water . , . . I . . . . . . . . . . linwr r n ~

Final pH -7.1-7.5

1)issalve the sfilids i n :he water, Ivilrrn~nF slightly, thcn cool thc s o l r ~ l i ~ ~ ~ > tn ~,~l,rn

lcmPeraturc. Adjuu (he rcacllnn with I N sadium hydroxide ir nmr,,,,, ,hat the orth the compltkd and ~terilited medium will be 7.1 -7 .5 . Filter, i r n ~ e s ~ ~ ) . clJriry. *wrihutc into cu~lubic resscls and sterilize in, an au~oc la \ .~ ror 18-20 rninlllsi J t I 2 I :C.

-

* The quality of' the components should h In accordant,, +rirh ~,,~,..;~,.~,r,,,,,~ /;V

Rcfl~rn's ~ r l m l i ~ ~ J ~ ~ i,? thr InI~trr~ifiorn~rl Phcrrrrrr,roaooir, Genr\d, wor(,j lth ~) rc : l l l l - Z"[lon, 1963. unless o l h e n ~ i x s~accd.

I Reagent 9ua l i l~ sodium th~oglyrol;lte in more 514hlc than thiuglycol~c ~ 1 3 .

54

B L G / U N D F / ~ ~ .l Rev+ 1

Appendix D.27

AppPollq l

PROCEDURE FOR STERlLlTY 'TEST USLNG MEMBRANE FILTRATION

- - A su~rable unit consists of r closed =emir and a r ~ c ~ r a c l c , repararc4 from one

another by a properly supports4 membrane of apprupriatc porosity Mcmbwne ~ntntrally suitable for sterility wting have a nominal porosity 010.21 pm or 0.45 W, a diarnrlor of approaimately 47 mm, and a flbw rate a l rnl ofwater per minurc at a p-urc or 70 t m o l rncrcury, preferably asvemblc m d rtcrilitc the entire unit with tht mcmbrant Id p l m , prior lo uu. Whcrc the sampla 10 bc tested b an oil, i t may bc -y to rtcrilUt h mtmbnns scpatately and, afru lborough dryin$. -bit the unit, usin% q r i c pmrauhlw Kf U& entire rnemhne is to be cultured (a btlow), at J w t IWO 51td units ar t M up. 1 00.11-

FfMid A : 1 g of pcptic diet of anilnal lir~uc l or the equiwltnt in dirtillcd

wsrcr 10 make ooc lit=, dlw or eatrifup to clanCy, adjut to pH 7.1 102, dirpcnse into fisk.5 in tmml quarrliLia, end ~ W d h at 121°C for !&-m nunu-.

f/#id B : I f the tcsl 8arnplc contains bdthin or oil, W Fluid A to uch lit= of which has ben added I m1 of ( p t . r t ~ l p h a a o n y ~ p ~ l ~ ~ h ~ ~ h ~ o l , to PH 7.1 * 0 .2 , dlrbenre into hb. and ntcrik~ at 121- for 18-20 UlYDules. .

r - - -

Nate : Any s k b c diluent h a t dOQ m t madrut rrrtimimhhl lrtivity and does not d s t BC porosity of the rnsmbmmc m a y be suitaklc for ~olvsolvia~ a prepantion under rat for s t n i h y

TM p.arrdw

Liquid¶

The numbtr or mmpk contnintrs or rbc vdums of bulk sunpk lhrt is s ~ i f i e d for conducting the s d b t y t a t U mkm. )ucptially transfer the q u i d volumes from cach container d i m l y to m m m n b m fllGci pnviouly mohttncd vitb stcr ik sntcr or rhc diluting Buid used m to two r t d c vs* C a t pwt iog prior to w f r r to a moist mem- brane. lmmtdiattly draw each sunpk thou& Ihc fllter with the nid ool vacuum.

I f the substance is m visfourj liquid ar wspcnsion w t ndrptabk to rapid Blrration, artptically add a w&ied quantity QC FSuid A to the p w l d m p l e to in- t h e flow nte. Swde enzyme p ~ a n t i o m such 1s pcuiC11ltnn~ Or d l u b be a d a d to the

hililutins tluid to aid in diuokin$ inwluble substarn. If thc rjllbsrsnuc under fni haa i n b c a t antimicrobin4 prapcrrin or conrrids a pr#mtivc, wash the lilter with suf&icnt IOaml purrions d Fluid A. If UIC rubelum u~Mcr imt contains lsithin or oil, rsplace Fluid A by Fluid B- Tkt number of portions or Uuid used should b. suflkient to allow gowih of a small inoculum oforganisms (spproxjmatcly 50) sensitive to thc aolimicrob~al subrtance rn the p r w m of tbc residual inhibitoty material on tbc membrane.

Upon completion of fihmtion, ttanskr each entirr membrane to 100 m1 of the cukure medium (or nppronirnnlcly onehalf of a c h rnanbnnc to 100 rnl of each of two cultum mcdia) selected for the s~crt l~ty test. The rarnplcs ore thcn incubated A I the sdcctcd

-- r ~~~~~l~ : peptone, &red, R , .S dascrihcd in Sperifiroriri~r f i R e a ~ m J r menf i~n- l

fn rhr ~ ~ , ~ , ~ ~ r i ~ ~ ~ / phorptocopmd. Cicncva, Wmld klcal1h Orgalllmtiy. 1963: P. 117. aulor)aved solution [ 2 tn 100) t r clcar and is nculral or m4rly so In It5 reaction-

5 s

BLG/UNDP/~~. 1 Rev. l

Appendix D - 2 7

temperaturesand Tor Ihc appropriate periods and examined as described In Pan A. section

5.2 of lhcse requirements. Thc period of incubation membrane-hltcred test samples used by some workers is sharter than the leday period pmrc r ikd in this $cction. sinc+ growth occurs more rapidly when micro-organismsconcentra~cd on Alttn a r t inoculated.

TESTS FOR MY TOPLASbrQS

All varielies of mycuplohma are I I V ~ capahlc of growth ill thr aame medium. Same slrdins require special culiurr media. Suilabte medin rhould thcrclvre be se lw~ed ha\ing rcplrd to pflnicular sondl t~uns , e .g . , ihc tissue in wl~ieh a virits is grown o r snllrcc of ar~rmal senrm, and the growl11 requlrcments of the polcniial conli~minanrs, <.g.. the need for yeas1 cxlrac.l. Sblid and >crni-solld or liquid mcdta contalnlrlg serum rracrianj rich in certain lipids ( b t c r ~ ~ l a ~ r n d phosphol~pids) iiru used Ir! proude rhc nutrilive rcquiremcnls. Cnritrol cu:tures un solid and sc~n~-s<?lid or hqoid m e d ~ a or knnrc~b fa*tidious slralnr nf nrjcuplasma, bvlh srcrol-rrqhtrln~ and ~lon.rtcrol-requir~ng. are included in each 1-1 to show that the medin are capsblc of s ~ r ~ ~ o n i n g the growth of mycoplzsrnas.

Samples of the nlateriai of nut Iew 111irn 6 rnl for a h g l e lest (i.e., using onc solid and one semi-solid or liquid medium) arc stored ciiher h twccn 2- nnd 8.C for no Iollficr than 24 h o u n o r a t -10°C or Irtwrr ifstnrcd fur lonner t l~nn 24 hozrrr - . .... -.*.

Nnr less than 2-0 m1 d each sample arc invmlaled and evenly djstrilrutul over the surhce of a! l a s t 10 p l a t e o f each solid mcdiun) 11x4, and not lass thnn t .O nll ~ r c a c h sample is inoculated into r ~ c h o r J I b34t 4 I I I ~ C S of each semi-slilid o r liquid n~cdium used. Each r l t k should conrain I n m1 o r medium. h r p e r volunvs o r i nmulu~n , C.&.,

making a total v l 5 0 rnl of rnelcrial i n 4 0 0 m1 of medium. have been uscdudvantagwusly. I l l t racc~~tr i fuga~ion herore teating such samplcs hoa also h n used.

The plates clnd r u k s arc itlcubatcd at 36°C t I .WC: for no1 kss Illan 14 days. C)w half of each set I$ incubated aerubically in an environmcnt o r adequate humidity, .and Ihe other haH is incubalcd anaerobically in ;In environmcnt ~ r f S-IO"C carbon dioxtde in nitrogr-n, alsn of rdequatc humidity. At I ~ C end of 3 day. and = p i n ut 14 Jayn, 0.5 ml Crvm cach of 2 IUI>CX bcine incubated a~ruhically are I-umbinrd ilrld ~nucularcd on tv nor Ims than 4plutcs T h e ? t u b heingincuba~rdanacrnbrc.tlly arctreliled Itkcwiw. Slnce some mycoplanmas may not grow un media In I'rlri dishcr, sui-h suhf111~~1reb un sohd m ~ d i a may also be m8dc in

All rrlaterial seeded originully on solid rlrrd~a is auhculrured on the day 4Clcr the pritllary i~loculslion. *n thr third d i ~ y and on the sixth day, o t ~ orhrr ruiuhlc ro l~d and liquid media. and th&e subcultores src incubalcd si~r~ilerly, half J C ~ O ~ I C B I I Y ,tnd ~ h c other hnlf ~naerobically n s described, and e ram~ncd after 10 days, wh~lf (hc nrrpnst cultures are examined r l lhc cnd o f rhc l 4 d n y periud. All plates (and tub(\l after incubation for not 1-5 than 14 days arc obscrvcd fur the growth o f mycopkian~ar by apprcrpriale procedorm.

If no cvidcnic is found of Ihe prewncc of rnycnplasrnas the matcrisl,e.g.,>iruspi>ol, meets the r equ i r c~~~rn ta U T thesc rests. I f evidence o r pruwth o f niycoplasma-, is found. the myroplasmas should be idenlificd and thr suurcc trdrad.

' Non <trrulreql~iring mycopl.isordr will gror! on m c d i ~ co t i~a~n ing sterols. In orricr to illduce ~n)*vupldsmus tv !row In IrquiJ n ~ e d ~ i l , s~mililr \ u h c r , l t t ~ r ~ ~ ~ ~ i n l l U

he don?, bul tfircc. or imlr p:l%s.~pcs rnuy be needed.

Appendix D.27

For ddrction a n d i d c ~ W i u t i a n o l mycop la smuthe~o l l~wing p d u ~ m w l P t d - -. bui athm rnclhods arc i3vailnHt.

1. Pat W& of I t IY~~~ptumm

M 0 ~ i - 1 co\ooio o r specific mlorntion (blue) by Dicncs's stain or col0niCS $pmd on blxk$ of agar in pttri d k h c ~ . and viewed under ths micro=opt ;

(b ) ~ 0 ~ ~ r a ~ ~ micmlmpy OI mloni~ ( s t r i p o r P ~ ~ U O I for i l o ~ u l a ~ mdia. tilnmenis and grazlnuln :

(C) exnmilu&rn U.&T 11,. rh tmn m i c r ~ ~ c ~ p e u f lhe pellel ~ c l ~ r r j f u w d from yrni50lid cullurc :

{dl .xamip,don under the light microxope ( X iW) of a film s h i n 4 Gemsa-

2, For srpnrnntlar d p - e o p b s t ~ s MO

(0 , e n z ~ m ~ t i c &m ; i e m r a a t i o n of gluwu. bfeakdown or at.inhc and d u d i o n oTrfiphcnylld-le. u s h ~ drn lomt ion of phenol red as indicator for 'he h t two e f f ~ l ~ and red calwr of the t c l r m k dcnvativs for Ihc last :

I r bcrnoIni5 in 4% s w p s i m of washed p i w a p ~ g e l l s . a d d 4 and 10~11htd for .-, IBA4 hours airobically a l 37-C:

(C) D1cma&o'plbn of a 4% supension of guincnpil relh p o u l m the p** $date or thation aT red h l d corpuscles by the mlonim aftcr incubation L 37% Dr

3. Far m* a,, a *is. ,&.ycop~arna svum ( p r c p a d in the ybhi l ) i such

inhibit,, n igh t Ub. &monst*6 by d e p m i h g p ~ r di$e imp l rpa t cd w t h the m 2 4 hour p w t k ~ u l r u r n and observing Lbcrn from the thifd day.

l m r n u n o f l u o ~ e o c ~ tcch&lues tor thi5 purpux an b e ~ n g ~ev t lopcd .

Thc unpublished working dmumcnt W H O / B S / ~ ~ . ~ M Z ; WHO1 PIi.4R\l/73.474 on rlliak thew requirements were byd was prepared by ihc lollowing e r p r i s and mcrnlxrs of the WHO Wrdarial :

D, E. A. Chriatrnsin, ~ o e t r o l b p e n m e n t , Statcns Seruminrlilut, Cop~ l~hMCn. Dcnmark 11. Coben, n i rmmr , National Institute o r Public Health, Billh*ven, N e l k r " lands (Cvrtnritnnr)

Wr 1. IXsborder, Directar. Microbinlogy Seflitm, National Public H t ~ l l h bburrltnr)', Paris. France (Conrul;anO

Or L. F. D~&O", Dj=[or, Nntional Biotogical StandarQ labotatory, Common- wealth Department uf H c ~ l t h , C a n h c m , AusIralia ~ C ~ n Q l l ~ o n f )

BLG/UNDP/~~.~ Rev. 1 Appendix D.27

Pr~fers*r G. Edsafl. Head, Deparrmcn~ a t Mir-robmlogy, London School or Hygiene and Tropical Medisinc, Lundnn. tnglanil (Cnnru~ron,)

D. G . Evanr. Dircctn~, Naljonal Inslit~rlc Tor Biological Srsrtddrd5 J I , ~

C-orllrol. Lo~ldon. England (CDIISU~IO,I~~ - . . , Mr J. W . Lightbown. D~vision o r Biologi~al Standards. National Tn~lilut; Ibr

niol*.Sical Star.dords and Control. London, Enghnd (I :o ,r~don,) Dr 'C. J . u a c r k . 16541 S Werll~nd Drivc, Cjaithenb<lrg, Md., (l$jA Mr A . P Mccl~iovski, Phzrn~accutical 0 6 c e r . Pharmaceuticals, WIIO. C i r j r c i a ,

S u.ltzerIa.d

Dr 4 S . Olitjchnnhr. Chief Medice1 Officer, Bidogii,ll s l ; l ~ a r t i i ~ ~ r i C I ~ l l WJI(I, Cicllc\s, Swlrirriand

Dr M. Grlnlc~-P;rchrcl1, Scientisl. Biological S r ~ n < i ~ r d l r a l ~ ~ r ~ , wllf l , Suitxrl.tntl

Gralcful ac l t lowledge~ncn t is m d e to the e x p r t s ;rnd jnstltr~tiun.; listed helow Tor th r i r comments and adkice it~ld for supplying additional dara :

hjr J . M. Ballcy, Execuii\c I l H i C C r . .T.llc Aawi.loti~>n <>l.r!,r British Pharlll;lL, Illlcal Indurrry, London, England

Prolcscor A. J . Birndoni, Lims 1425, 8ueno.; A ~ W , Arg~lllillil Vr T. bitan. Hcad. Chernis~ty Deparrnlrnf. lnriiiu~c For ~ h c Cut~lrnl oi Drug.

Z~greh, Yugorlavr:r { ;l;" CJrrnl B ~ c l ; , Food and Drug Allministration, W;~>hingro~t. T) ( '~ . ~ S A

A . Brunzcil. Assuci~tc Pnrresrur, Thc Galenical Sc .c~ iu~~ , nivisirln 0 4 phsrnIJc,, : '# ! 3 . Dc~~clln.n1 17 Drugs, Natjanal Board of Health and WeIldre. Slocll>uln> ,.jrvcJej, -. . > -

Dr J . R. Buriarlck, st.11~ Ir).xt~lutc for the Conlrd or Drugs. Prague, C L , . , - ~ . , ~ ) ~ ~ ~ L , ~

Prorcssor A . Ca'd. L.hisi. Labnra~ary of Chcoiisrry. Istituto Supcriure 4, > : r O i l i , Rome, Italy

58

B & / u N D P / ~ ~ . 1 R e v . 1

Appendix D.27

M! K C. Chatterjet', Bombay Culkge ol. Pharmacy, Kalina. Santa Cruz. Bomhuy Indha

Dr E. A. Christenren. Control m p n m s n t , Srnrcns SEruminslitut, Cowhagen , Wninarlr

Dr V. F. Dabty, Deputy Director (Technical). Cumnronvcal~h krrrm Laboratories. Parkvik. Victoria. Australia

MF I. Daridsun, Ckntml Veterinary Lborarory, Wcybridse. Surrey, kngland

Dr Ni$ ~ i d l n g , Apo~keas C t o ~ l l a b o n l o r i u m , Sob*, S w d t e

Pc~ferrur J . Dony, SEYnlific Diretor, &1@hn Pharmaceutical ~slociaiion. Dmg Conml -a, B w k . BCIgiw

Mr 1. Evllns, A t h n ~ Cbicf, Dms Microbidagy Blanch. Division o i Drug woloey. M c t of Phameuc ica l R m h and Tmling. ~ m h i n p o n , D.C.. USA

Mr 1.7. Faragher, Cenml Yctuinary I-rboratoq. Wcybridg, Spmy. Enannd Dr M. Fors, kcmtay C-l, Wordic P h s r m a c ~ i a Council, Slockhulrtl. Svrdcn Dr Ch. B. Gerichkr. D i M o r , M s t o n Q € Lpbra~oncr . MiniPtry of Health, Jeru-

sal-, lrrarI

Dr D. Gho'lh, Dimtor, Gcnval D ~ g r Lnborstory, Calcutta, India

Mr N. Godd~rd, Ssrtorius-Membnnfiltcr GmbH, GlJttingcn. Federal Republic or G m n y

Dr l. J . Gmybon, C-onwalth Scrum b b o n l o & , Parkviilt, viclorie. Australia

Ur f. Hs&y, Dcm, ~ h c &hod of ~ka-y. Univcniry of Londoa, and Chairman. Ehkh Fkannecopods Commiuion, Londmh. h @ d

Dr L. H+lict. Department of Medical Misrobiolla~, Slanford Univcnily School d Medicine. Sunford, CaliToraL, USA

Dr John Hampton. National BioIo@L S~n&v4$ L a h r a ~ p r y . CnnberrP, Australia

P r n f w r R. Hazard, Faculty of Mcdicinc, l ~ t i t u l r of Ph~macnlagy , ~br is . F rancc

Rol-r G. Htplann, Dimtor , Paul Ehrlicb lartituw, Fcdsra) Agency for Sera urd Vaconer. Frankrun-am-Main. F&rd Rtpublk or GCWMY

Dr L. Hig-Made , F h l Contml sod Unlo@~sl ~tandardirstion Scftion, lnuilurc of Imrnuuology. hgrcb, YuWlevlP

Dr N. W . Holm, Head, A ~ ~ : & n l o r Jkpnrneni. ~ a n i s h Atomic Encr~y Cnrnmhirro. Rescarch ErrabEshmeot Rise. Roik~lbc. Denmark

Dr D. W. HO-, Asishat D i m o r , Natio-l Biologi-l ~ u n d a r d r Iabaralori (Viral Product.. SBCljon), Parhv~llt. ViFturiP, Australia

Mr E. C. Hulre, Deputy Dtmtur, Central Vdcrinary Lsboratory, Wcybtidgr, Sumy, Englamd

Dr T. lnauc. a i d , Department oC Drugs. Nationnl ln$titutc or Hrmcnic Science, Tokyo. Japan

Dr S. Iwahara, Chitr. Depanmcnl OF Micmbiolo~(~. N a u w l lnstilule of Hygienic Licncrs. Tokyo. Japan

PTO[TSO~ L. 18nner. Shrc Serum lnstitutc, llclhinki, Finland

Mr C. A. Jullllsun, Scientific Director, British Pharnlaropacia Commission, I ondon, E n ~ L a d

Mr S. C. Jolly. nirrrcnr, DFp.Irlmcnl of pharmaauri t~l Screnccs, The Philmlnceu- llcrl Scicty or Great Britain, London. England

Dr J, Kanrlo, bledic;i\ Omccr, The Y~ccinsllon Rrsrsrch Ccnlcr. T ~ ~ Y v . japafl

59

BLG/UNDP/~ 7.1 Rev. 1

Appendix D . 2 7

L'r 1. C - Kelscy, Deputy Dirwtor. Public Health Laburarory $cr\iw7 C r n ~ ~ n l public Health taboratory. London, EnglnnJ

D r K. Kihara. Nolional lnstitutc of Hcqlth, Tokyo, Japan

Mr E. )Cnoll. Chid. Srerflit)- Trsting Brnnch, National Csntrr for Antihio[ic Analyrjr, m c : or Phar~t~accuticst Krscarch and Tcsling, W.lshingtnn, IIC.. ~ I S A

1. Kramtr. Acting Director. Division of Drug Biology, Office r ~ f Ptiarmaceutical Reararch and 'Iksring. Washi~~grnn. 11.C.. USA

Prrlrcrsor K. G. KrebE, Dircctor, F Mcrck AG. Darmstadl, Federal Rcpublic of Germany

Dr Hanne Krirtenscn. St,ntcns !kru~uins~itut. Coptnha#m, Uen~,lsrk

Dr M. Kuroharia, r h ~ c f . Uepwrln~cnt of GenernL Biolo&o Conrrul, Nntinnol lmtircte or Health. Tokyo. Japan

D r A. hfonjafific, Dirrcror. Jnb~itutc of Hygiene and Epidemiology, un~ss~ls, Belgium

D r E. I ang. Assiqtant hfanager. ClBA-CiEIGY I. irrrilcd. Basle, Switxrland D r H. 1 ansberg. National In r t i t~~rc or Public Hcallh. Bilthovm. Ncthcrlandv D r P, L r o u x , Scientific Director. SPKCIA. Paris, France

Dr F I . !.v. Irradiated Products Ltd.. Elgin Estale. Swindor~, Wilt,. England

Dr I . P Luwenrhal, Chief. Departmznt of Biologi~x Rtrwrch. Division uT Corn- municablc Disease + Immuno lo~ , Walter R e d Arm) Institrttc o f R-rch. Wsltcr Reed Media l Ccntcr, Washiopoon. D.C.. USA

Dr J. Lyng. Srnrens Semminstitut, Copcnhaacn. Dtnrnark

Mr A . G Mathews, Chief of Qualrty Cctr~trol. Con~n~nnwcalrh Scrum Laboratories, P~rkvillc, Victoria, Australia

Mr F . A. Maurina, 3485 Bedford. Dcrroit. Mich.. USA

Dr J . A. M<KirI. Director Gcncral, I ~ b o r ~ r o r ) ' TentR far D I S ~ ~ S E C.nn<rot. Health Protection Branch, Oitawa. Onlario. Csnada

Mr H. F. MelJahl, K3bi Lrd, Technical Department. Stockholnr, Suc&n D r H. Mirchnmsy, Asrocia~e Director, R J L ~ Sutc In>titure for Semlm 2nd Vaccine

Production, Tehcran. Iran o r R . M u m y . Spec]-(1 Assistant to thc Dircclur. National Inrtitutc of Allcrpy and

Infectious Diswser. Natiunal lnsl l l~tcs of Health, Bc lhe~d~ , Md.. IJSA Dr R . Ncftcr, Dirtclor. Virology Scrtiun. Nntiuaal Puhlic IIenlth Lobnralory.

Pans. Fmncc Dr E. Niemincn. Director, I r~harntor) for rhc Fxflrrrination of Phnrm.~ceu!icd

Producu, Helsinki, Finland Miss M. C)mnndik. Baclcriologisl. Ctnlral Inslilutc of Hyyisnc. Ankara. Turkey

Mr B. v. Pdlrl. Dr Vikram Ssrabhui Mar?. Ahrncdrh~d. ("Jia Profe~lOr G. Pens~i. lstltuto Shfprriore di S.rniti, Rome. Itdly Prtlfc3*or hi . Pernamw$hr, Faculty a(. Ph;rrm,rrcu!ic~l Sciences. Thc un i vev i t ~ t7f

British Columbia. Vancourrr. Canada p ' A Pucch. Director, hhntl\<l l i ir Srrrlr,n. I . j ; l ~ i ~ ~ ~ t Lahor<lrory for !h<

Control or Llrucs, hlonlpellier, ir i lnrc~ o r Chaloclll Purunirnarld~. D~ rc~ to r , N~~lalr : , I Blnn~I (>I~trcI TI , ;~~ RCL) I rgl'i\ S~,citl\,

Fa l~~Lok . Thai l~nd

B L G / ~ J N D P / ~ ~ . 1 Rev. 1

Appendix D.27 -

J D. vdn Ramshors[, Chief, Wpr tmen~ oC Bio?o8jcal Standards. National Instjture of Public Hcalth. Bilrhavcn. Netherlands M, K. Rangn&ar, Cmmmiainner, Food and D r u ~ Adrninhtmtion. Maharashtra .

Srsle. Bombay, India

Dr 0. Ringrtz, A-iate Profmsot, Swedish National Bactcrivlogiul LaboralOry. Sweden E. B, Seligmann, J ~ , D imtor , Division of Control Aclivirin. B u e U of Biologic>, F& and Drug Administration, Rochville, Md-. USA

Proffiwr p. L, S"ov, Flat 17. IbTcplyi p ~ ~ u l o k . MostoW, USSR Dr J . Spaander, Dirtclor Gcntral, National Insljtult bf pllblic H d 8 . Bdth~vcn.

~ e t 6 c r l n n d ~ nr 1, SWki, airc D c p n r n ~ n t or Sgmhctic Chemistry, National Instituc*

Hygienic Science, Tokyo. laps* Q, l , ~j T ~ ~ ~ ~ ~ , nesd, Quality Control, r-tltuco Sierotcrapicb e Vaccinogcno

T-no " Sclavo ", SUO~. Italy F, ~ ~ ~ i f e k . pharmaccu&nl and Biochmcal Recarch Iwtit'Jrca Prague.

. .. .

Budapnt. )i&ary MI V-I. Sprtorius-Mcmbranfilter G r n b ~ . G ~ u i n g n , Fcdcral Rcpublis of Gcrmary

Dr l . Volckringr.r, Honorary Innpsror-Gcncrsl of Hal th . 3 me Louis ~o l l nnd . Monzroup, Fmncc

p,,f-, M. WelCch, Dimor, Faculty o f M t d i c i ~ . -sir d M i c m b i ~ l o g ~ . vc@tg of Up, &@urn

Dr W. W . Wri@t, Acting Diratm. O ( B a or p h ~ m l i a r R m h s n d T a ~ . B~~~~ of rug, ~~d and Drug ~dministration, Washmmm, D.C., USA

Valvrblc suggestions wcrc alvl obtnincd from tbr repon of n diw-on 0. recvmmenda~ions for WHO RaJUircmtntS that took piam at the poaium on Sterility and Steribty Testing d Biolu@ Substan- from 9 to 12 April 19'73 in Madrid, Spain, by t h lntema~im%l A*socialion

L I L G / ~ J N D P , / ~ ~ . 1 Rev. L

APPENDIX D. 28

GKWERAI, REQULBEMEXT3 FOR MANUFACTLTRWG ESTABLISHMEKTS AND CONTROL LABORATORES

(REQUWEMENTS FOR BIOLOGICAL SUBSTANISES No. 1)

Pafl A . Gemrnl rsquirtmtnls for manufacturing establi$Lmtnrs . . . . . . . . . . . . . . . . . 1 . Pcmnmt

1. Buildiops and cquipmcnl . . . . . . . . 3. Production control . . . . . . . . . . . . 4. FiUiog and cnntaimrs. . . . . . . . . . . S. Tesis . - . . . . . . - . . . . 6. R s c ~ f d s . . . . . . . . . . . . . . . . 7. Sample . . . . . . . - . . .

. . . . . . . . . . . S. Labclling. . . 9. Distribulioa and dip pin^ . . . . . . . .

. . . . 10. S t o w and =pig date. . . . Par1 9. General ~ i n m c n l s far wntrol Iaboraturk

2. Building and equipment . . . . . . . . 3 3. of adivitia . . . . . . . . . . . . . . 20

The procedures required for controlling biological substances during tlunufacture arc differeat from the control procedures applied to final products by cconuol aurhoritics. Control a t the manufacturing level ia a matter of national coocc:t.rrl, whcrcas control of final products, including