Art Met-Analiti Feum Usp Ich

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A Step-by .. Step Approach

to Esta blishing a

Method Validation Prograrn

BY CINDY GREEN, RAC

,!,

he infonnation in this ar tic1e is in tendedto pr ovide asystematic a pproach f or esta blishing amethod vali-

dation progr am. There are a var iety of product types

that r equire some level of evaluation and testing either at theraw mater ial, intennediate, or f inal pr oduct level. Ther e isno question that critical decisions may be made based onthese test r esults and ther efor e, it is logical that one muststrive to ensure the accur acy and re pr oducibility of these re-sults. ecogniing that a11 of us desire to achieve a high

level of conf idence in the reported results, we must also bemindf ul that there are many ty pes of assays used for a br oad

range of r easons. "ll assays used for a11 purposes do not r e-quire the same level of validation. #t is im portant to be ableto pr oceed logically through the rationale that selects notonly which assay should be val$dated, but how that assayshould be validated. This ar tic1e attempts to pr ovide theguidance for ma%ing these two primar y decisions.

&hat's (ew

#n )ecem ber 1**+, the "mer ican "ssociation of har -maceutical Scientists, ood and )rug "dministration, Fed-

er ation I nt ernat ional e P harmaceut ique , anadian /ealthrotection 0r anch, and the "ssociation of fficial "nalyti-

cal hemists s ponsored a conference on "nalytical 2eth-ods 3alidation4 0ioavaila bility, 0io-equ$valence and

har maco%inetic Studies. "s a result, the 56uidance for #n-dustr y4 0ioanalytieal 2ethods 3alidation for /uman Stud-

ies5 was published in )ecember 1**7. "s a follow up to the

1**+ meeting, a second conference was held in 8anuar y9+++. The pur pose of the seeond meeting was to review the

pr ogr ess, im pact, and advances made dur ing the last tenyear s. Scientif ic $ssues related to bioanalytical val$dationwere diseussed and r esolved. The 6uidanee document from

1**7 was discussed and r evisions wer e proposed. "mongthe proposals are the follow$ng4

1. u11 validation is requir ed for a dr ug entity.9. #f metabolites ar e added f or quantification, f ull val-

idation is neeessary.:. artial validation may be acce ptable f or modif iea-

tions of validated assays. E;amples inc1ude4 plat-form changes, change in range, change in sample processing, ete.

<. r oss validation may be used when an assaychange is neeessary during a study. These studiesmust use the same or similar matr i;, and inc1ude=> ?uality ontrol @?A samples or a total of si;? sam ples, whichever is gr eater.

=. or standar d eurves, the sim plest algorithm, ormodel, whieh best describes the res ponse coneen-

tr ation relationshi p should be used with a ppr opriateweighting. nly one lot of matri; should be usedfor pre paration of the standar ds once s pecificity has been deter mined. B=> per cent of the standar ds @or= standards, inc1uding the u pper limit of quantita-

This article first appear ed in JVT May 2000, Vl!"e #, N!"$er %

A!&!st 200' ( Vl!"e )%, N!"$er *

T



Cindy Green, RAC

+

tionA should f all within C1=> for standards and9+> f or the lower limit of quantitation.D. Sample acce pta bility for repeat study samples

should be evaluated using a deeision tr ee. or e;ample, if the original and two repeats ar e within :+>

of the original value, a median ean be used. #f therear e insuffieient samples and only one re peat is r un,a value on the r e peat is aeeepta ble to eonfmn the

or iginal results if the result is within 1=> of theoriginal value. #f the retest value is within :+>, a

mean of the original and retest should be used. #f the

retest is within =+> of the or iginal, the retest valueeannot be used @i.e., no resultA,

B. 3alidation eriter ia f or pr eeision and aeeuraey should

be 1=1=> @eoef fieient of variation for pr eeisionequal to or less than 1=> and aeeuraey equal to or

less than 1=>A with a limit of quantitation at 9+>.or produetion eriteria, DD> of all ? samples

@with a minimum of half of the samples at eaeh ?level being aeee pta bleA must be valid and the mid-

level and high-level ? samples must be C9+> ofthe eriteria. The low-level ? must be C:+>. This

is a modified <-D-9+ rule.7. Fiquid hr omatogr a phy @FA-4rv1ass Spectr opho-

tometr y @2SA-2S has the same. r equir ements asother methods in terms of preeision, aceuraey, and

stability.

*. 2atri; effeets must be evaluated to deter mine pos-

sible effects on assay validation.1+.or ligand assays, the calibrationcurve is non-lin-

ear and should be fitted to an aeeeptable model withappropriate weighting and algorithm. "t least si;

non-ero ealibrator s should be used and standards

should be run in duplieate. Tar get aeeeptanee limitsar e 9+> at the lower limit of quantitation and 1=>at the r emainder of the eur ve. " <-D-9= rule should

be eonsider ed.

Ther e ar e many other reeommendations f r om the wor% -shop that ar e under eonsideration f or adoption into the ne;t

r evision of the guidanee doeument entitled 56uidance for#ndustr y4 0ioanalytieal 2ethods 3alidation for /uman

Studies.5 (umer ous artic1es have been written on validation pr o-

grams, in gener al, and on s peeif ie e;amples detailing howvalidation should be per formed on eomple; assays and

assay r elated equipment. These ar tic1es serve as e;eellentresour ees for eompanies and individuals attempting valida-

tion. 2any of these artieles are cited in this article's bibliog-

r a phy and should be used as r ef erenees and e;am ples ofhow others approaeh method validation.

Initial Questions

The very fir st ste p in the develo pment of a methGd vali-dation protoeol is to deter mine the o bHeetive of the method.

/ow will the method be usedG &hat is the method intendedto demonstr ateG 0ased on the res ponse to these questions,

there will be at least two main ehoiees. or e;ample, if themethod is in tended to monitor patients, r elease final prod-

uet, or determine poteney, level of im pur ity, or contaminantsin ahuman dr ug produet, the method is eonsidered signifi-

eant. &ill the method ' be used to su p por t pr oduet elaimsGSuppor t the seleetion of refer enee r naterialsG Substantiate

the r esults fr om another methodG These ar e only some ofthe %ey questions that must be addr essed prior to determin-

ing the level of signifieance of a method.#f the method is intended to ser ve as a qualitative evalu-

ation for identity, the method is considered a Fevel ## @lesssignif ieantA. #s the method to be used for establishing a limit

of impurity @less than or greater than a standardAG "re the r e-

sults visualG#n all eases, r egardless of the signifieanee, the f ollowing

additional questions will also need to be answer ed4

This is a requir ement for DD> of the ? samples to be

val id with a minimum of half at each level and 9=> of all? samples to be valid f or nm aeee ptanee. re-validationwor% should inc1ude antibody speeifieity, matri; ef feets, and

ealibration range @in the matri;A. r e-study validation shouldevaluate linearity @using dilutions of ? samplesA, preeision

@more than si; runs over sever al days with two or more runseaeh dayA and at least thr ee sets of low, medium, and highsamples in duplieate for eaeh run.

Journal of Validation Technology



1. &hat sample-ty pes will be tested using themethodG &ill the samples be whole blood, serum,

plasma, ur ine, stool, pur if ied pr otein, unpurif ied protein, chemical agents, ete.G

9. 0ased on the sample-type, what inter f erenees aree; peetedG #s it li%ely that those interfering sub-

stanees will impact the r esultsG

:. #s the method cell-based, chemical-based, or en-

yme-basedG

<. &hat level of aeeuraey is requiredG



Cind y Gr een, R AC

Figure 1

Data Elements Required for Assay ValidationD.

"ccur acy 3es 3esrecision 3es 3es

Specificity 3es Yes

)etection Fimit (o (o

uantitation Fimit (o 3es

Finearity 3es 3esange 3es 3es

* M ay be required , depending on the nature of the spec ific t est .

LimitTests

(o

3es

(o

(o

AssayCategory

11# .

3es

(o

(o

3es

(o

(o

(o





=. &hat level of sensitivity, selectivity, limit of detec-tion is r equir edG

D. &hat leve! of precision is requiredG #n what rangeis this precision level requiredG

The goal of the questions and the preliminary evaluationis to determine how best to meet the obHective of the method

validation so that it can be documented as suita ble for its in-tended use. 3alidation must be a ble to demonstrate that the

method is f r ee of systematic error s and actually detects thesubstance it pur por ts to detect. The variables that may poten-

tially affect the method should be % nown and investigated ..

Types of Method Validation

There are at least four main ty pes of validation4 "nalyt-ical, 0ioanalytical, 0ioassay, and 2ethod ?ualification.

Each of these ty pes of validation will require slightly differ -ent appr oaches. 0ased on the answer s to the questions dis-

cussed a bove, the type of validation can be determined.

"n Anal ytical Method Validation is the pr ocess used todemonstrate that an analytical method is suit-a ble for its in-tended use. "n analytical method refers to the approach

ta%en when the analysis is performed. Each step of themethod must be described in detail and include instr uctions

for preparing the sam ple, the reference standard, and thecali bration curve @if a p plica bleA. #t must describe the use ofthe instr ument or a pparatus, how the data is to be collected

and analyed, and how the results should be repor ted." Bioanal yt ical Met hod Val idat ion is the process used to

demonstrate that an analytical method is suitable f or use in

AssayType

I d ent ífí c at ion test s @intended toensur e the identity of an analyte

. in a sample. This is nor mallyachieved by com parison of a

pr operty of the sample to that ofa r eference standardA.linpurí t í es Q uant í tat íon@intendedto accurately r eflect the puritycharacteristics of the sample.)ifferent validation characteristicsare required for a quantitative testthan for a limit testA.

I mpurí t íes Límí t @intended toreflect the purity char acteristicsof the sampleA.C ontent / P ot enc y ; Díssol ut íon@intended to measur e the analyte

present in a given sample. "quantitative measurement of themaHor component@sA in the drugsubstanceA.

- --- -Speeif icity

"ccuracyrecisionSpecificity

)etection Fimituantitation FimitFinearityangeSpecificity)etection Fimit

"ccuracyrecisionSpecif icityFinearityange

A!&!st 200' ( Vl!"e )%, N!"$er *

Figure 2

International Committee for

Harmoniation Recommendations

the evaluation of human samples. Suita bility includes the proof that the method can determine the concentration of a

particular analyte in a given biological matri; in a r eliableand repr oducible manner . The results of bioanalytical meth-ods are often used to su pport registr ation of new dr ugs or

changes to e;isting ones. The bioanalytical methods must





Pre-qualification Requirements

#n all types of method validation, at least some pr e-qual-ification must be perf ormed. #n all cases the following items

must be

evaluate

dand

mor

e

e;tensive evaluation is neces-

sary f or those that may have a higher potential to af fect theassay4

1. quipment. Equipment must be calibr ated or cer-

tified prior to use.

9. Personnel. #ndividuals assigned to the validation

Cind y Green, RAC

Figu r e 3

Validation Characteristics ersu s Type of Analytical !rocedur es

. , / Test for t"p!rltles-

Type "# !rocedure Identification $uantitation Limit Dissolution, ./ . %easur ement

. . , , - ,/ /./ , / 1Cntentl3tency4

"ccuracy (o Ies (o Ies

recision4epeatability (o Ies (o Ies

#ntermediate recision (o Iesa (o Iesa

Specifiity Ies Ies Ies Ies

)etection Fimit (o (o$ Ies (o

uantitation Fimit (o Ies (o (o

Finearity (o Ies (o Ies ange (o Ies (

o

Ies

a, When reproducibility is performed , intermediate pr ecision is not needed.b. May be needed in some cases.

E

demonstr ate the claimed perf ormance and reliability inorder to place a high level of confidence on the test results.

Some e;am ples of bioanalytical methods include4 chernicalmethods @6as hr omatogr a phy J6K and /igh ressur e

Fiquid hr omatogr aphy J/FKA, 2ass Spectr ophotome-tr y @2SA and com binations of 6-2S or 6-2S-2S, etc.

0iological methods, such as enyme-lin%ed immunosor bent

assay @EF#S"A and rnicrobiological methods may also beused. 2any of the princip#es and requirements are similar tothose used in analytical methods.

Bioassa y M et hod V alid ation r ef er s to thevalidation of biologically based material s, either used as test samples or

as reagents for the assay. ell binding assays and antibody-antigen-based assays ar e e;am ples of bioassays. The use of

cells or biological r eagents pr esents challenges that arecompletely different from those f aced with enyme or

chernical reactions. E;tensive pre-qualif ication is r equir edof the biological r eagents used.

Method Qualif ication is the pr ocess of determiningwhether the method meets basic criteria for perfor manceand accuracy. This is generally accomplished using pr elim-

inary spi%ing and r ecovery data, perforr ning a basic assess-ment on sta bility and selectivity, and per forming precisionand accuracy with higher lir nits than e; pected with valida-

tion @f or e;ample, C9+>A. The num ber of e; per iments completed for qualification is limited in number and a

r equirement for additional o ptimiation is .li% ely, 2ethodqualif ication is used during the dr ug discovery stage of de-


Figure !

Comparison Ta&le Representing 'DA(

)*!( and ICH Requirements

C+r iter ia

"ccuracy 5 5 5 5

eproduci bility 5 5

Sensitivity 5

Specificity 5 5 5 5

Finearity 5 5 5

recision 5 5 5

)etection Fimit 5 5

uantitation Fimit 5 5

ange 5 5

ecovery 5

uggedness 5 5

,- 4

velo pment. 0y the preclinical phase of development, the pa-

r ameter s for the method should be more e;tensively evalu-ated. or e;ample, the precision and accur acy lir nits should

be tightened @for e;ample, C1>A, selectivity should beevaluated, recovery calculated, sta bility investigated, and

the limit of quantitation, r ange, and linearity defined. 0y theclinical phase, an even more e;tensive evaluation of the

method should be per f ormed ta% ing each par ameter and per forming a higher level of testing.



should be adequately tr ained with res pect to safetywhen handling chemicals, biological agents, etc.

They should also be trained on the use of the equip-ment. Tr aining recor ds should be maintained and

competency should be assessed.:. Supplies. The supplies necessary for per forming

the validation must be identified with r egard to thesup plier and descr i ption. Special requir ements may

be necessary, such as ster ile pipet tips, chemically

c1eaned container s, etc. These s pecial requir ementsmust be documented and the su pplies assigned for

use compar ed to these r equir ements.<. Materials. The material s must be evaluated to de-

ter mine whether the quality and composition meets

their intended use. or e;am ple, the gr ade of mate-rial should be identif ied and documented. The ma-

ter ials should be sta ble, stor ed pr o perly, and handledin a manner to minimie potential contamination.

=. Ref erence Standards. Ther e ar e three ty pes of ref-erence standar ds4 Lnited States harmacopeia

@LSA certified r eference standard, commer ciallysupplied mater ial f rom r elia ble supplier s, and ma-

ter ials of % nown pur ity and performance as deter -mined intemally or by a certif ied la bor atory.

D. Quality !ontrol @?A Materials. 6enerally, fourlevels of ? samples ar e used. The Fimit of ?uan-

titation @F?A sample is the same concentr ation asthe lowest non-er o standar d. The low ? sample

is less than or equal to thr ee times the F?. Themedium ? sample is appr o;imately midway be-

tween the high and low ? samples, and the high? sample is B=-*+> of the highest calibr ation

standar d.B. Sta"ility. Stability of both the test samples and the

r eagents must be detennined prior to initiating themethod validation. Thesample -stability may re- .

quir e an evaluation of freee-thaw cycles, storage

Cind y Green, RAC

at 9-7M @or f r oen conditionsAN or stability on theinstrumentation used for the analysis. eagent sta-

bility may r equire evaluating both reconstitutedstoc% solutions and diluted concentr ations stored at

both 9-7M and froen conditions.

Validation of Assays

Ther e is no shor tage of guidances, publicationsN and r ef -

erences for method validation. There are a var iety of a p- pr oaches, many being ver y similar in principle. See Fiur e l !

"ccording to the LS, ther e are f our categories of vali-dation for compendium methods. They are as follows4

1. "nalytical methods for quantitation of maHorcomponents of bu#% drug substances or active ingr e-

dients @inc1uding pr eservativesA in finished pharma-ceutical pr oducts.

9. "nalytical methods f or determination of impuritiesin bul% dr ug substances or degr adation com-pounds

in finished pharmaceutical products, inc1udingquantitative assays and limit tests.

:. "nalytical methods for determination of perfor-mance char acteristics @e.g., dissolution,drug r e-

leaseA.<. #dentif ication tests.

"ccording to the #nter national ommittee for /ar mo-niation @#/A, recommendations ar e made in Fiure "!

#n the #/-9?"4 Te;t on 3alidation "nalytical r oce-dur es, validation characteristics #ersus type of analytical

pr ocedur es ar e shown in Fi ure :.The #/ ?904 3alidation of "nalytical r ocedures, de-

scribes the main o bHective of validation as the a bility of ananalytical procedure to demonstrate that the pr ocedur e is

suita ble for its intended purpose. The document stresses thatwell-characteried refer ence materials, with documented

.. purity, should be used throughout the validation study.

0iological based assays must be validated for precision,accur acy, sensitivity, and specif icity, at a minimum. Onow-

ing the error in pr ecision is very important. The higher theintr a or inter assay pr ecision, the higher the number of repli-

cates that must be r un and the higher the number of runs thatmust be per f ormed. "ccuracy can be deter mined by s pi% ing

pure pr otein into the a ppropr iate sam ple matr i; or using

amino acid analysis to esta blish pr otein concentr ation.&hen cell-based assays ar e run ther e must be a consistentsupply of cells. This requir es esta blishment of a cell ban%

A ! 6 ! s t 2 7 7 ' ( Vl ! " e r s. N ! " $e r *



Cind y Green, RAC

f or the cells used in the assay. Testing must be done as part

of the pr e-qualification todeter mine possible adver se ef -fects f r om mito gens, ser um, preser vatives, p/ var iations,

and ionic str ength varia bles. These ar e % nown to inhi bit orinterfere with cell gr owth and mas% bioactivity.

"nimal-based tests ar e %nown to be the least . preciseE -with cell-based assays having more precision @B-:+>A and

binding assays @antibody-antigen assaysA with the highest

precision @:-1=>A. otency r e por ted in hase 1 is gener allya value or result only. #n hase #l, the r esults ar e gener ally

com par ed to refer ence material and in hase ### an acce pt-a ble r ange should be esta blished.

P aramet ers

"lthough there is not com plete harmoniation as to the parameter s that should be evaluated, the par ameter s listed in

Fi ure < should be considered."n e; panded table entitled 5"nalytical 2ethod 3alida-

tion - erf or mance riteria5 is shown as an "ppendi; pull-

out.

"ccur acy is used for gener al assays, as well as quantita-

tion of impurities. The recommended data set includes ninedeter minations over a minimum of thr ee concentration lev-

els covering the s pecified range. e producibility @precisionA is detennined using a mini-

mum of nine determinations covering the s pecif ied r ange.or e;ample, three concentr ations with thr ee r eplicates of

each concentr ation. "n intermediate precision determina-tion can be made using ty pical variables, such as multiple

days, analysts, equi pment, etc. The data should be r e por tedusing standard statistical par ameters, such as standar d devi-

ation, r elative standard deviation @coef ficient of var iationA,and confidence inter vals for each set of pr ecision data.

S pecificity deter minations requir e the initial evaluation

of blan% s using the a ppr o priate sample matri;. #t is recom-mended that si; lots of blan% matri; be used and tested ac-

cor ding to the established procedure with r es pect to sample pr e paration. S pecificity is the a bility of the method to distin-

guish and quantitate the analyte of inter est in the presenceof other constituents found in the sample. "n evaluation asto the potential eff ects f r om degr adation pr oducts, im pur i-

ties, etc., must be determined.

Finearity is evaluated using a minimum of five concen-tr ations and the simplest wor% a ble regr ession equation. Fin-

earity is used to determine the concentra.tiP'f lQN.Rf un%nownsam ples by comparing the measur ements against a standar d

cur ve.


recision is determined as repeata bility with a minimumof nine detenninations cover ing the s pecif ied r ange, e.g.,

thr ee concentr ationsthree re plica te s each. " minimum ofsi; determinations should be used at 1++> of the test con-

centr ation. Standard deviations should be r epor ted using a

*=> onfidence #nterval.The detection limit is gener ally based on visual evalua-

tion. #t can also be based on signal-to-noise @a ratio between

thr ee- or two-to-one is gener ally accepta ble for estimatingthe detection limitA or using a third method of utiliing the

cali br ation cur ve to estimate the detection limit.

The quantitation limit can be based on visual evaluation,signal-to-noise r atios, or calibration curves. #t is the lowest

amount of analyte in a sample that can be quantitated witha r elative standard deviation of 1+> or less.

ange is deter mined by the pr ecision and accuracy at the

lowest and highest e;tr emes of the intended assay r ange. ecover y is the amount of analyte detected when it has

been spi%ed into a matr i; and compared with the theoreticalstandar d. The determination should be made using e;tr acted

sam ples at thr ee levels @low, medium, and highA. o bustness @r uggednessA .should be evaluated for all

methods. obustness ta%es into consideration such varia blesas operators, la boratories, envir on-r nental conditions, time,

etc. )eterminations should be done using a homogenoussample @or com posite sampleA tested on sever al days, in dif -

ferent laborator ies, using differ ent instruments, etc.

Acce $t ance % r it eria

The following acce ptance criteria ar e r ecommended f ordetermining the validation status of a test method. " method

is considered 5valid5 if it meets these criteria. Accur acy! The mean value between batches is within

C1=> of the nominal value at the low, medium, and highconcentr ations of the ? contr ol. The accuracy should not

deviate mor e than C9+> at the Fimit of )etection.

&e $r oduci'il ity ($r ecision)! The coefficient of variation

between batches for the low, medium, and high concentr a-tions of the ? contr ol should be 4d=> and 4=9+> of theF? ?contr ol when thr ee separ ate batches ar e tested.

Sensitivity. The lowest standard must be acce pted as the

F? of the method, when thecoeff icient of variation at theF? ? is 4=9+> when thr ee separ ate batches are tested.

*$eci ficit y! #nterf erence f rom other components, such asimpurities, degradation pr oducts, or place bo ingr edients,

must not be pr esent. or automated equi pment, the re-. s ponses of inter fering pea%s at the r etention time of the an-

alyte must be 9+> of the res ponse of a F? standard.



es ponses of interfering pea% s at the retention time of theinter nal standar d should be 4==> of the res ponse of the con-

centration of the internal standard used.

+inear it y! The acce ptance cr iteria using five or morestandards ofvarying %nown concentrations with r esults f it-

ted using a straight line equation, must be 9+.*++ withthe slo pe remaining within 1+> of the original, acce ptable

value. The I-intercept must be .7. with C9 times precisionof the method.

Pr ec ision. The coefficient variation between thr ee ormor e batches for the low, medium, and high ? controlconcentr ations must be 4=1=> and 4=9+> for the F? ?

sample.

et ect ion +imit ! " signal-to-noise ratio as deter-rnined

by com parison of measur ed signals from samples at the low-est concentr ations, the blan%s must be :.:4 1, pr oviding that

the standard deviation of the blan% s and samples ar e r elia ble.

Quant it ation +imit ! The quantitation limit must have ar elative standard deviation of C1+>.

&an e! or an assay of a drug substance or finished drug pr oduct, the range must be 7+-19+> of the test concentr a-tion. or content uniformity, a r ange of B+-1:+> of the test

concentration must be included. or dissolution testing, ther ange must be C9+> of the specified range. or determina-

tion of an impurity, the results must be 19+> from the re- porting level to the specification. or impurities, the level of

acce ptance must be based on the required level the impuri-ties ar e to be controlled.

or assay and purity tests when perforrned together as asingle test, the linearity must cover a range from r e por ting

level of impurities to 19+> of the assay s pecifications.

&eco#er y! " recover y of 1++> is desirable, but not al-

ways achievable. Fower r ates of r ecover y are acce pta ble, pr oviding the recovery is pr ecise, accurate, and r e pr oducible.

&u edness! The method must meet the criteria for pr eci-sion and accuracy when used by multiple technicians, on

multiple instruments, in dif f er ent laboratories, on multipledays, etc. when the same sample or composite sample is used.

Cindy Green, RAC

Re#alidation

"s part of the method validation progr am, cr iteria must

be esta blished as to when r evalidation will be r equired f orall assays. Each company must Hustif y whatever criteria are

chosen. rossover or equivalencystudies may suffice f orminor changes that have a minimr Um potential impact on the

method validation. Some e;amples that should be consid-er ed for full or part$al r evalidation include4

• epeated failure to meet validity criteria• ailure to meet calibration curve acceptance criteria

• hange in matri; or sample preparation

hange in r eagent forrnulation, solvent, column

matri;, etc.

rossover studies may be considered f or 4• hange in technician

• (ew model of the same equipment

• hange in % ey r eagents or supplier s

#n all cases the e;tent of the studies r equir ed will be de- pendent on the potential impact of the change and the pos-

sible impact on the in tended use or obHective of the method.

$ocumentation R equirements

rior to initiating a pr e-qualification, qualification, or val-

idation, formal protocols should be written, reviewed, andapproved. " suggested format for a protocol is as follows4

• over age• Table of ontents

• urposeStudy bHective

• es ponsibilities

• )efinitions

• re-?ualif ication equirements@surnmar y of completed studiesA

• Study )escr i ption

• Sample )escription

• 2aterials and Supplies

• Equipment #nforrnation

• Test 2ethods• /ealth and Safety #nforrnation• Standard perating rocedures

• Test rotocol @Test functions describing each pa-rameter , how it will be tested, and the acceptance

A!&!st 200' ( Vl!"e )%, N!"$er *



Cind y Green, R AC

criteriaA• )ata /andling and "nalysis

• eport equirements

The r esults of both pre-qualification and validationshould be documented in for mal validation reports. 1N... f inaltest r eport @or validation re por tA should include4

• over age

• Ta ble of ontents @with list of f igur es and tablesA

•

Summary of the Studies• esults

• )iscussion

• onc1usion

• " p pendices

The standar d o perating procedur es and test methodsshould be wr itten, reviewed, and approved by the Study )i-r ector or the res ponsi ble la borator y super visor or manager.

E; periments should be r ecor ded in bound, laboratory noteboo% s or on pre-num bered and issued assay f orms. The note-

boo%s should be r eviewed and signed by a second individual%nowledgeable in the wor% being r eviewed. The noteboo% s-rnust be signed and witnessed in a timely manner . "ll noteboo% s and assay for ms ar e subHect to inspection and must be

r eadily availa ble. " r elia ble and organied system must be in place f or handling and storing raw data gener ated in su pport

of method validation studies. aw data must be reviewedand signed by the individual assigned to the wor% . "t a m$n-

imum, the following infor mation should be recorded in thelabor ator y note boo%s or la bor atory recor ds4

• )escr iption of the method being validated and its

intended use

• Surnmary of stability studies @with refer ence as to

where the data may be f ound for samples and %eyr eagentsA

• )escri ption of the e;per iments or test f unctions performed to evaluate the method @e.g., sensitivity,selectivity, pr ecision, etc.A

• )ocumentation of r eagents @e.g., sup plier , catalognumber , lot number , e; piration date, concentration,etc.A

• Standar ds and calibr ation data @e.g., standards usedwith lot numbers, e;pir ation dates, su p plier , cer tifi-cate of analysis, how standards wer e prepared, etc.A

• ormulations and ca1culations @with refer ences, if

not o bviousA


• rocedure used for re-assay or discar ding data @orrefer ence to in-house procedureA

• )eviations from or iginal pr otocol or cont4rolledmethods @with investigation or follow u p require-mentsA

• "cce ptance criter ia @with Hustification for accep-tance levels assignedA

)ocumentation is a critical element of method validationand must adher e to the most str ingent standar ds. The results

from the validation studies will be used to su pport the choiceof methods, perfor mance of those methods, and the accep-tance criteria used. "ll Hustif ication for the use of selected

methods r esides with the validation documentation.

A%&'T T( A'T(&R

Cindy Green, RAC , has oer %0 years of combined

e ! perience in Regulatory Affairs, "uality Control , and

"ualit y Assur ance in the areas of in itro diagnostics,

traditional drug products, biological products, and cel#

therapies. $he has been consulting for o er )0 years.

Past products inc%ude r egu&atory submissions, com'

pany and supplier auditing ( compliance under con'

sent degree, generat i on of $"Ps, alidation pro)ects,facil*ty design pro)ects, and o many others. Cind y + as

responsi ble for the submission of the first urine based

- test , and the first detection test for anthra ! .

$he also filed the first -/" for a combination cel#

therapy and instrument product. Cindy receied her

Regulatory Affairs Certification in )66% and is a

current member of RAP$, -$P0 , A$M , AA M- , "RCA,

and W11A. $he is an "RCA board member and

has ser ed on the 0ditorial 1oard for 23 since its

inc eption. Cind y can be reac hed by phone at &45 6 7'

485'9:58, by fa ! at 1*2843*)%9:*02 or by ema*i at

c%ndyn+rs;seanet .com.

A!)*&+,$M*T

The author e; pr esses her sincer e appreciation to her dear

friend and colleague, )onna 2atuie% . &ithout her manyhour s of r esear ch, this article would not have been possible.

mailto:c/[email protected].













0#0F#6"/I

Anicetti, V-R- Bihar" Cnference, p-2%%9**-

Bianalytical Methds Validatin fr ;!"an <t!dies- De9

ce"$er )66:-

Br=n, >-, >ernande?, @- 1eds4- )66'- Deelp"ent f

<pecificatins fr Bitechnl&y har"ace!tical rd!cts

6) '69::- De Bil <tand- Basel, ar&er-

B!ic, A-R, et al- )660- .Methd Validatin in the Bianalyt9

ical a$ratry-. @!rnal f har"ace!tical E Bi"edical

Analysis :,n- :9)2 #269%'-

B!nch, E - -)66%- A<FC+>DA a$ratry CGM II <e"inar -

1)6 March-4

C!rrent 7pinin in Bitechnl&y 6%)6928- )66:-

De<ain, C- )66'- .Test Methd Deelp"ent and Valida9

tin-. Bihar" 1April4 #09%-

Giand, T-E-, et al- )6:2- .<!$"ittin& ;C Methds t the

C"pendia and Re&!latry A&encies-. har"ace!tical

Technl&y 1<epte"$er4-• The Gld <heet 2:, n- #- @!ne )66*-

G!ideline n Validatin f the i"!l!s A"e$cyte ysate

Test as an End9rd!ct Endtin Test fr ;!"an and Ani9

"al ar enteral Dr!&s, Bil&ical rd!cts, and Medical De9

ices- Dece"$er )6:'-

.;ar"ni?in& the Validatin f Analytical rced!res-. Bi9

har"- A!&!st )666-

• ;!$er, -, )66:- .<yste"s F!alificatin fr ;i&h9erfr9

"ance iH!id Chrrnat&raphy<yste"s-. Bihar" 1N9

e"$er4 #89#-

IC;9F2A Tet n Validatin f Analytical rced!res,

March )668-

IC;9F2B Validatin f Analytical rced!res Methdl&y,

Ne"$er )66#-

McEntire, @-, )66*- .Bitechnl&y rd!ct Validatin, art

8 <electin and Validatin f Analytical TechniH!es-. Bi9

har" 1@!ne4 #:9'6-

a!l, - - )66)- .J< erspecties n Analytical Methds

Validatin-. har"ace!tical Technl&y 1March4-

Re&!latry G!idelines fr Validatin f Bianalytical rce9

d!res, AA< rshp n Bianalytical Methd Validatin-

@an!ary 2000-

<hah, V--, et al- )662- har"ace!tical Research 6, n- *

8::962-

<"ith, A- and Ritter, N- )66*- .Cnsideratins in the Valida9

tin f Bianalytical Methds fr rtein Characteri?atin-.

Jnited <tates har"cpeia and Natinal >r"!lary 1J<

2*+N> )64 K)228L Validatin f C"pendial Methds p-

2)*69)82-

CDER 1CMC %4- )66*- Validatin f Chr"at&raphic Meth9

ds 1Ne"$er4-

Validatin est 11. rshp - )692) May )66'-

Cindy Green, RAC

6FSS"I TE2S

"ccur acy4 loseness of determined value to thetrue value. 6enerally, recovery of added analyte

"nalyte4 " specific, unique chemical moiety in theformesA in which it would be foundin5a biologic matr i;.

"nalytica# rocedure4 The analytical procedurerefers to the way of performing the analysis. #t shoulddescribe in detail the steps necessary to perform each

analytical test4 Thismay include, butis not limited to,

the sample, the reference standard, and preparation ofthe reagents, use of the apparatus, generation of the

calibration cVrve, use ofthe formulae for the calcula-tion, etc.

"ssay @content orpotencyA4 To providean e;act

result that allows an accurate statement on the contentor potency of the analyte in a sample.

0ias4"n individual error inherent in a method, whichmanifests itself as a positive or negative deviation of themethod aver age from the accepted reference value.

0iologica# matri;4 " unique material of biological

origin that can be pr e pared in a reproducible manner.E;amples are blood, ser um, plasma, urine, feces,

saliva, sputum, and various discrete tissues.

alibration urve4 alibr ation standards are usedto construct calibration curves from which the concen-

tration of analyte@sA in control and un%nown samples isdetermined.

)etection Fimit4 The detection limit of an individ-ual analytical procedure is the lowest amount of ana-

lyte in a sample, which can be detected but notnecessarily quantitated as an e;act value.

6as hromatogr a phy @6A4 hromatography

based on the volatilied sample transported by the car-.rier gas as the moving phase through the stationary

phase of the column where separation ta%es place bythe sorptionldesorption process.

/igh er f ormance Fiquid hr omatography@/FA4 Separation based on interaction and differen-

tial partition of the sample between the mobile liquid

phase and the stationary phase.

A!&!st 200' ( Vl!"e )%, N!"$er *R-



Cind y Gr een, RAC

6FSS"I TE2S

Fimit of )etection4 The lowest concentration of ananalyte that the analytical pr ocess can r elia bly dif f erenti-

ate f r om bac%ground levels. The lowest concentration ofanalyte that can be re pr oduci bly detected under the stated

conditions. " par ameter of limit and qualitative tests.

Fimit of ?uantification4 The #owest and highestconcentrations of analyte in the calibr ation curve that

can be measured with esta blished accepta ble accur acyand precision.

Fimit of ?uantitation @F?A4 The lowest concen-tr ation of analyte that can be deterr nined with acce ptable

pr ecision and accuracy under the stated conditions. " pa-r ameter of quantitative assays. The lowest concentr ation

of an analyte that can be measur ed with a stated level ofconfidence.

Finear ange4 6enerally ta%en as the r ange overwhich the pr ocedure has been demonstr ated to give a lin-

ear detector r es ponse. " r e pr oducible nonlinear res ponsecurve,. however , can also be accepta ble. (onlinear ity is

cer tainly the case with irnmunological pr ocedures ..

Finearity4 The a bility to elicit test r esults that ar e di-

rectly,or by a well-def ined mathematical transf or mation, pr o por tional to the concentr ation of the analyte of inter est

within a given r ange. 6ener ally e; pr essed as variancearound the slo pe of the r egression line drawn through thetest results. The linearity of an analytical pr ocedure is its

a bility @within a given rangeA to o btain test results whichar e dir ectly pr o portional to the concentr ation @amountA of

analyte in the sam ple.

2atr i;4 The biological f luid or tissue in which the an-alyte Rs to be deterr nined.

2ethod4 " set of all of the pr ocedures invol ved in the

collection, proeessing, storage, and analysis of a biologi-cal matri; for an analyte. The com plete analytical proce-dur e by which samples ar e analyed.

2odel4 The selected r egr ession and weighting used toconstruct the calibr ation cur ve.

@!r nal f Validatin Technl&y

recision4 The degr ee of agr eement among individual.test results when the procedur e is applied re peatedly to

multiple samples of a homogeneous sample @gener allye; pr essed as elative Standard )eviation J S)KA. The

E -closeness of re plieate deterrninRtions of an analyte by anassay. r eeision can be f ur ther subdivided into withinday

precision or intr a-assay precisiWn and between- day pr e-cision or inter assay pr ecision. The pr ecision of an analyt-

ical pr ocedur e e; presses the closeness of agr eement@degree of scatterA between a series of measurements o b-

tained f r om multi ple sampling of the same homogeneoussample under prescribed conditions. r ecision may be

consider ed at thr ee levels4 r epeata bility, inter mediate pr e-cision, and r e pr oducibility. epeata bility e; presses the

precision under the same o perating conditions over ashort interval of time. "lso ter med intraassay precision.

#nter mediate pr ecision e; presses within labor ator y var ia-tions @e.g., differ ent days, different analysts, dif f er ent

equipment, ete.A e pr oducibility e; pr esses the pr ecision between la bor ator ies @collaborative studies usually a p-

plied to standardiation of methodologyA.

?uantitation Fimit4 The quantitation limit of an indi-vidual analytical pr ocedure is the lowest amount of ana-

lyte in a sample that can be quantitatively determined withsuita ble pr ecision and accur acy. The quantitation limit is a

par ameter of quantitative assays for low levels of com- pounds in sam ple matr ices, and is used par ticular ly for the

deter rnination of im pur ities anclor degr adation of pr od-ucts.

ange4 The interval between the u pper and lower lev-

els of analyte that have been deter rnined with precision,accuracy, and linearity under the stated conditions, gener-

al#y e;pressed as the usable portion of the standard cur ve.The r ange of an analytical pr ocedur e is the interval be-

tween the u pper and lower concentration @amountsA of an-

alyte in the sam ple @including eoncentrationsA for whichit has been demonstrated that the analytical pr ocedure hasa suitable level of pr ecision, aceuracy, and linearity.

ecovery @or e;tr action eff iciencyA4 ecover y is r e- ported as a per centage of original dr ug carr ied thr ough

the sam ple purif ication steps of the method.



Cindy Green, RAC

,&SSAR &/ TRMS

B.eplicates 0independent replicates12 #ndependent

replicates are separate aliquots ta%en from a sample orstandard @or dilution thereofA that are processed indepen-

dently.Replicates 0dilution replicates12 Lsed in immunoas-

says, dilution replicates are separate aliquots ta%en fromserial dilutions @e.g., 11+, 111++A of the same standardthat are used to ensure that the e;tent of binding is withinthe range of the calibration curve.

Ro"ustness2 The robustness of an analytical proce-dure is a measure of its capacity to remain unaffected by

. small, but deliberatevariations in method parameters and provides an indication of its reliability during normal

usage.

Ruggedness2 The degree of reproducibility of test re-sults obtained by the analysis of the same samples in dif-ferent labs andor on different instruments, generallye;pressed as the lac% of influence on test results due tooperational and environmental variables.

Sample2 Sample is a generic term encompassingcontrol samples, blan% samples, un%nown samples, and

processed samples. ?uality ontrol Samples are used tomonitor the performance of a method and to assess the in-

tegrity and validity of the results of the un%nown samplesanalyed in an individual assay. 3alidation ontrols areused specifically in the validation of bioanalytical meth-ods to define accuracy and precision. 0lan% Samples of a

biological matri; are samples to which no analyte has been added. They are used to assess the specificity of themethod. or internally standardied methods this wouldcontain the internal standard. " )ouble 0lan% is a blan%sample that does .not contain the internal standard. "nLn%nown Sample is a biological sample that is the sub-

Hect of the analysis. " rocessed Sample is the final e;-

tract of a sample that has been subHected to variousmanipulations @e.g., e;traction, concentration, etc.A

Selecti#ity2 The ability to measure accurately andspecifica11y the analyte of interest in the presence of com-

ponents that may be e;pected to be present in thesamplematri;, genera11y e;pressed as bias, enhancement, or in-hibition of sample signal.

Specificity2 "bility of a method to measure only whatit is intended tomeasure.Specificity is the abiUityto uni

equivocally assess the analyte in the presence of compo-nents that may be e;pected to be present, Fac% ofspecificity of an individual analytical procedure may be

compensated by other supporting ar$alytical procedure@sA.

This definition has the following implications4 #denti-fication, to ensure the identity of an analyte. urity Tests,

to ensure that a11 the analytical procedures performedallow an accurate statement of the'content of impurities of

.ananalyte, i.e., related substances test, heavy metals,

residual solvents content,etc .

Stability4'ThR 'chemical stability of an analyte in agiven solvent or matri; under specific conditions forgiven time intervals.

Standard cur#e2 The relationship between the e;per-imental response value and the analytical concentration

@also ca11ed a calibration curveA.

Standards2 "n "nalytical Standard is an analyte of%nown purity and molecular composition. "n "nalytical

Stoc%Standard Solution is dissolved in a solvent which isfurther diluted to generate wor%ing or secondary standard

solution@sA used to prepare calibration standards andorcontrol samples. " alibration Standard isa biological

matri; to which a %nown amount of analyte, as an analyt-ical stoc% solution, has been added or spi%ed. alibration

standards are used to construct calibration curves fromwhich the concentrations of analyte@sA in control and un-

%nown samples are determined. "n #nternal Standard is atest compound added to both calibration standards and

samples at %nown and constant concentration to facilitatequantification of the target analyte@sA. " eference Stan- .dard is a calibration standard used to chec% instrumental

performance, e.g., sensitivity and chromatograph, prior torunnmg an assay.

Thin-,ayer !hromatography 0T,!12 Separation is

based on rnigration of the sample spotted on a coated @sta-tionary phaseA plate with one edge dipped in a mi;ture ofsolvents @mobile phaseA.

A!&!st 200' ( Vl!"e )%, N!"$er *



7.

Inter"etiate presicin

.

t Appendi

<f2=> '

i-it, . / f"-!

Rd

3,,M0/

tl t='<=?



*.

Cind y Green, R AC

Q

Linearity Range Ruggedness Ro&ustness

NT f i e cncentratins o er @rug $ubst ance of ini shed Nt def ined- <h!ld $e cnsidered d!r in&E

r an&e, n standard dil!tins r Produc t :09)20 f specifica9 deelp"ent f analytical prce9

synthetic "it!r esO linear r e&res9 tins- d!re t sh= relia$ility =ith r e&ardsin analysis 1cr relatin ceffi9 C ont ent Bni f ormit y = '09)%0 f t "ethd par a"eter ariatin-

cient, y9intercept, slpe, r esid!al specificatin 1r =ider4 rides a $asis fr syste" s!it9s!" f sH!ares4 f si&nals er s!s @issolution 3esting = 0 1 -"23 a$ility tests and criter ia-cncentratinO plt f act!al data -mpurities 3ests= fr" reprtin& Ea"ples f ariatins sta$ility f

# ers!s r e&ressin line- leel t )20 f specificatin analytical tests, etr actin ti"eO fr1Ref er t 0% A 0%:4 iH!id Chr "at&raphy 1C4

"$ile phase p; and c"psitin,cl!"ns, te"per at!r e, fl= rateOf r Gas Chr"at&r aphy 1GC4

IC; G!idelines

F2A .Tet n Validatin

f Analytical

rced!res,. 1%+684

F2: .Validatin f

Analytical rced!res

Methdl&y,. 1))+6#4

J< 9< @9+++AMn&raph K)228L

.Validatin f

C"pendial Methds.

- -

- -, - - Acc!racy - / - .

prug $ubstance= assay +1 analyte +1 n=n

+ p!rityO c"parisn ers!s a secnd =ell9char9

acteri?ed prced!reO r inlerence frrn preci9

sin, acc!racy, and specilicity res!lts-

@rug Product= assay +1 synthetic "it!res ,+1the dr!& prd!ct c"pnents t =hich n=n

H!antities +1 the dr!& s!$stance hae $eenaddedO assay +1 spied dr!& prd!ct r crn9

parisn ers!s a secnd =ell9characteri?ed

prced!reO r inlerence fr" precisin, acc!ra9

cy, and specilicity res!lts-

-mpurities= assay +1 dr!& s!$stance+prd!ct

spied =ith n=n a"!nts +1 i"p!ritiesO il

i"p!rities r de&radants are nt aaila$le,

accepta$le t c"pare ers!s an independent

prced!re, and !se dr!& s!$stance respnse

factrO "!st $e clear as t =+=, area, etc-

A "ini"!" +1 nine deter"inatins er a "ini9

"!" +1 three cncentratin leels, cerin& the

specilied ran&e 1i-e-, three replicates each at three

cncentratins4- Reprt acc!racy as recery

+1 spie r as difference $et=een "ean and

accepted tr!e al!e =ith cnlidence interals- /

@rug $ubstance= assayed ers!s Reference

<tandard- r c"pare ers!s secnd "ethd-@rug Product= recery f standard additins

t "atri r f spies t sa"ples--mpurities= recery f i"p!rity additins t

sa"ples, r f i"p!rity t dr!& s!$stance

!sin& respnse factrs-

As per -C= a "ini"!" +1 nine deter"inatins

er a "ini"!" f three cncentratin leels,

cerin& the specified ran&e 1i-e-, three repli9

cates each at three cncentratPns4-

repeatibility

Nt less than 1NT4 nine deter"i9

natins cerin& the specilied

ran&e 1i-e-, three replicates at three

cncentratinsO r NT si deter"i9

natins at )00 +1 the test cn9

centratins4 -

Assay +1 a s!fficient n!"$er f

aliH!ts f a h"&ene!s sa"ple t

$e a$le t calc!late statistically alid

esti"ates f the <tandard Deiatinr Relatie <tandard Deiatin

1R<D4 1cefficient f ariatin4-

As per -C = NT nine deter"inatins

cerin& the specified ran&e 1i-e-,

three replicates at three cncentra9

tins, r NT si deter"inatins at

)00 +1 the test cncentratin4-

ReH!ire"ent depends n circ!i

stances +1 intended assay !se-

Typical ariatins 1days, ana9

Iysts, eH!ip"ent4 need nt $e

st!died indiid!ally, "ay $e <t

ied !sin& eperi"ental desi&n

1"atri4-

7ptinal, "ay $e cnsidered

d!rin& standardi?atin f a pr

cedure $efre it is s!$"itted t

J<-

>DA CDER Reie=er

G!idance

.Validatin f

Chr"at&raphic

Methds,. 1))+6*4

>DA <!$"issin

G!ideline

.G!idance fr <!$"ittin&

<a"ples and Analytical

Data fr Methds

Validatin,. 12):'4P

-,

Acc!racy st!dies fr dr!& s!$stance and prd9

!ct are rec""ended t $e perfr"ed at :0,

)00, and )20 leels +1 la$el clai"-

Replicates at least in triplicate at each leel,Recery st!dies +1 analyte spied in the fr"a9

tin "atri in the linear ran&e f the anayte-

<!$"issin "!st incl!de data de"nstratin&

@rug $ubstance= acc!racy er ran&e r

interest 1ca :09)20 f thery4-

@rug Product= acc!racy er ran&e f interest

1ea :09)20 +1 la$el clai"4O recery fr"

sa"ple "atri-

-n)ection Repeatability= NT ten

inQectins, =ith R<D NT )

Analysis Repeatability( R<D +1

"!ltiple analysis $y sa"e analyst

1cnd!ct =ith acc!racy st!dies4-

<!$"issin "!st incl!de data

de"nstratin& fr @rug $ubstance=precisin er ran&e +1 interest 1ea:09)20 +1 thery4O tr @rugProduct= precisin er ran&e f

interest 1ea :09)20 +1 la$el clai"4-

As a "ini"!", analysis n N9

t= separate ccasins,

7ptinal

fr @rug Product precisin a

t9day, analyst9t9analyst, anc

as a l!rther indicatin +1 reprc

>DA cGMs

2) C>R 2))-)#81e4

2) C>R 2))-)6*1a4124

2))-)#8 Testin& and release fr distri$!tin-

1e4 The acc!racy, sensitiity, specilicity, and reprd!ci$ility f test "ethds e"plyed $y the fir" shall $e esta$lished /

2))-)6* a$ratry recrds-

1a4 a$ratry recrds shall incl!de c"plete data deried fr" all tests necessary t ass!re c"pliance =ith esta$li

124 A state"ent f each "ethd !sed in the testin& +1 the sa"ple- The state"ent shall indicate the lcatin +1 data th

tested- 1)) the "ethd e"plyed is in the c!rrent reisin +1 the Jnited <tates har"acpeia, Natinal >r"!lary, As3

dr!& applicatin and the relerenced "ethd is nt "dilied, a state"ent indicatin& the "ethd reference =ill s!ffice4-

Analytical %ethod Validation , !erfor mance Cr iteria

Reprd!ci$ility *pecificity Dete3tin Limit -uantitation Limit

-rr n9 Nt r eH!ir ed f r "ar etin& a!thr i9 I D "est s# discri"inatin fr " c"9 By ByL ?atin dssier- p!nds f clsely r elated str!ctr e , A. Vis!al eGa" 1f relia$le A. Vis!al eGa" 1f r elia$le H!anti9

liely t $e pr esentO sh= psitie detectin4O tatin =ith accepta$le acc!r acy

If standardi?atin f analytical pr 9 respnse f sa"ples =ith analyte /. <i&nal9t9nise rati and pr ecisin4O!d9 ced!re is needed 1as fr c"pen9 and ne&atie respnse f sa"ples 12) t %)4O r /. <i&nal9t9nise r ati 1)0)4O rI dial s!$"issin4, then dne as an =ith!t analyte- !. <tandard deiatin f the !2 <tandar d deiatin f the

inter la$r atr y st!dy- $ssay and Im pu rit y f r chr 9 respnse and the slpe- r espnse and the slpe-

"at&r aphic prced!res, r esl!9 >r A and B, incl!de chr 9 Validate !sin& sa"ples at r $eltin f ad Qacent peasO f r nnspe9 "at&ra"O f r B and C, alidate the li"it-cif ic assays, er all specif icity =ith - !sin& sa"ples at r $el= the

s!pprt fr " ther prced!r esO f r li"it-assays sh= discri"inatin f ana9

Iyte in presence f i"p!r ities r

eGcipients 1spiin& st!dies4O f ri"p!r ity tests sh= discr i"inatin1and r esl!tin4 f analyte 1spiin&st!dies, r c"par isn f i"p!r ity

pr file fr " ther =ell9char acter 9i?ed "ethd4O pea p!rity tests ar e!sef!l 1dide array, "ass spec4-

7ptinal, "ay $e cnsidered d!r9 ID " est sh= psitie respnse, As per IC;, "ay $e fr " si&nal9t9 As per IC;, "ay $e deter"ined $y

+- in& standardi?atin f a prced!r e selectin fr " c"p!nds f nise r ati f 2 ) r % )- analysis +1 sa"ples =ith n=nS7 $efr e it is s!$"itted t J<- clsely r elated str!ct!r e liely t tr atins +1 analyte and $y esta$lishin&

$e present the "ini"!" leel at =hich the analyte

Impu r íty "ests# sh= apprpriate can $e deter "ined =ith accepta$leacc!r acy and pr ecisin +1 sa"ples r acy and precisin- >r instr !"ent

spied at appr priate leels- ds =ith $ac&r !nd nise, "ay start

$ssays# !naf f ected $y presence +1 Ir " si&nal9t9nise r ati f )0), $!ti"p!rities r eGcipeints- sh!ld incl!de analysis +1 a s!ita$le

n!rn$er +1 sa"ples n=n t $e near r pr epared at the H!antitatin li"iU-

FT Nt nr"ally eGpected if inter"edi9 Representatie chr"at&ra"s lr Cr !de "ethd t eal!ate the leasi$ility Analysis repeata$ility and inQectin

ate pr ecisin is acc"plished- str essed and nn9str essed sa"ples that eGtr ane!s pea detectin is t !se the r epeata$ility data at the H!antitati-incl!de i"p!r ities test "ethd, pr eser a9 ciai"ed lr detectin li"it Ir " he ar ea li"it- Jse f reler ence standard attie1s4, etc- =ith the r elated place$ sar n9 the anaiyte- Rec""endatins Analysis the li"it- Reliance n siHnal9t9

pie- Representatie chr"at&r a"s t $ility and inQectin r epeata$ility data at nise rati nt rec""ended-sh= selectiity $y the additin +1 n=n tin li"i- Jse +1 an additinal relerenceeGtr ane!s c"p!nds- the a!antitatin n li"it leel in the test

<!$"issin "!st incl!de data Nt eGplicitly r eH!ir ed- I"plied Nt eGplicitly reH!ired- I"plied

?ar ta$ility =ith r e&ard t la$9t9Ia$ day9 lr @rug $ubst ance= specilicity +1 the !nder acc!r acy, pr ecisin, linearity, !nder acc!racy, pr ecisin, linearitO

id cl!"n9t9cl!"n "ay $e incl!ded and li"its lr de&r adatin pr d!cts r and specificity reH!ire"ents- and specificity r eH!ir e"ents-

Wd!ci$llity ties, =hich sh!ld $e adeH!ately

char acteri?ed- >r @r ug P r oduc t specificitythe "ethds and li"its lar de&radatin

r i"p!rities, =hich sh!ld $e adeH!ately

tilied and character i?edO de"nstratinther Iresh r de&r aded place$ inter fer es-

I

Q and dc!"ented- <!ch alidatin and dc!"entatin "ay $e acc"plished in accrdance =ith 2))-)6* 1a4124-

ilPshed specif icatins and standards, incl!din& eGa"inatin and assays, as f ll=s

that esta$lish that the "ethds !sed in the testin& f the sa"ple "eet pr per standar ds f acc!r acy and relia$ility as applied t the prd!ct

ssciatin f 7fficial Analytical Che"ists, B f Methds, r in ther recHnl?ed standard r eferences, r is detailed in an appred ne='l. The s!ita$ility f all testin& "ethds !sed shall $e er ified !nder act!al cnditins f !se-



cl!"ns, te"per at!re, fl= rate

As per IC; NT f ie cncentr a9 As per IC; @r ug $ubstance or Assay f aliH!ts f h"&ene!s Nt co er ed .

tins o er assay r an&e- * nished Pr od uct :09)20 t- lts in dif ferent la$s $y dif fer entspecificatin- analysts, !sin& per atinal and

Contact Bniformity '09)%0 f enir n"ental cnditins that "ay8- specificatin 1r =ider 4- dif f er $!t ar e still =ithin the speci9

@issol uti on 3est ing = 0 1 -"23! fied par a"eters f r the assay-

De&r ee f reprd!ci$ility is deter 9"ined as a f!nctin f the assayar ia$les, and c"par ed t assay

pr ecisin !nder nr "al cnditins--3

Assay linearity f r :09)20 f <ee acc!r acy and linear ity reH!ire9 Nt coer ed . Data $tained fr " st!dies f r

n la$el clai"- Assa % l m urit ies combi ' "ents- r $!stness th! h nt !s!all nation method = linearity fr )20 f s!$"itted, ar e rec""ended t

tar &et cncentratin d=n t li"it f $e incl!ded as part f the "ethd

alidatin-i"p!rity- Typically, r e&r essin ceff i9cient 1r4 is NT 0-666-

<!$"issin "!st incl!de data Nt eplicitly reH!ired- I"plied Nt eplicitly r eH!ir ed- I"plied Nt co ered.

45

,

de"nstr atin& the fll=in& @rug !nder acc!racy, pr ecisin, linearity, !nder pr ecisin reH!ire"ents-

$ubst ance= linear ity oer ran&e f

and specificity r eH!ire"ents-inter est 1ea :09)20 f ther y4-@r ug Product linear ity er ran&ef interest 1ea :09)20 f la$el

clai"4-

-

Pr oided by @onna M atui eD X M ar c h 5 E EE

J T7 7EN T;I< C; ART >J <IXE

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