174362793 Bioburden Control

8/18/2019 174362793 Bioburden Control

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Bioburden

:The Burdenon our

Biological Operations


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Objective To understand

Issues related to Bioburden

Sources of Bioburden

To Implement Methods & Systems to Control Bioburden

Ultimate Goal To Prevent Loss of Time !esources and Money


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Bioburden

Bioburden is normally defined as the number of bacteria livin" ona surface that has not been sterili#ed

The term is most often used in the conte$t of bioburden testin"also %non as microbial limit testin" hich is performed on

pharmaceutical products and medical products for 'uality control purposes( Products or components used in the pharmaceutical ormedical field re'uire control of microbial levels durin" processin"

and handlin"( Bioburden or microbial limit testin" on theseproducts proves that these re'uirements have been met(

Bioburden testin" for medical devices made or used in the US) is"overned by Title *+ of the Code of ,ederal !e"ulations and

orldide by IS- ++./.(

http://en.wikipedia.org/wiki/Medicationhttp://en.wikipedia.org/wiki/Quality_controlhttp://en.wikipedia.org/wiki/Microbehttp://en.wikipedia.org/wiki/Title_21_of_the_Code_of_Federal_Regulationshttp://en.wikipedia.org/wiki/International_Organization_for_Standardizationhttp://en.wikipedia.org/wiki/International_Organization_for_Standardizationhttp://en.wikipedia.org/wiki/Title_21_of_the_Code_of_Federal_Regulationshttp://en.wikipedia.org/wiki/Microbehttp://en.wikipedia.org/wiki/Quality_controlhttp://en.wikipedia.org/wiki/Medication


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According to United States Pharmacopeia (USP) ;IS 117!7 PA"#S 1$2

0atural bioburden is defined as1• The amount of livin" microor"anisms on an item 2e("( tissue ormedical device3 prior to and after manufacturin"• Bioburden can be described both in terms of 4U)0TIT5 and T5P6of microor"anisms

7 8hy is %noin" your bioburden important9: Sterili#ation ;ose: )septic processin": Process Capabilities 2manufacturin" of pharma and non


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Bioburden Bioburden1

Population of viable microor"anisms on a ra materialcomponent a finished product and>or pac%a"e(

Measured in CFU (colony forming units) per unit of product

Sterile1

,ree from livin" or"anisms 2Microbes3

)septic

!emoval of Bioburden


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Sources of Bioburden !a Material

Processin" 6'uipment

6nvironment

Personnel

Pac%in" Material

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Why Bioburden Control is

Critical Product ;e"radation >Spoila"e Safety

6fficacy

)ppearance Material ;e"radation & Life Cycle

!eduction

)ffect e$perimental data & !esults

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Solution for Bioburden

Control

Use everythin" sterile

Commercially and Technically not feasibleand not re'uired too(


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Material Sourcin Lo Bioburden Products< 6$pensive

Material inherently lo in bioburden

-r"anic Products

Inor"anic salts

Material inherently hi"h in bioburden

)'ueous li'uids< ith > ithout salts & su"ars

0atural Products : 5east 6$tract Peptone

8ater< ,resh 6very ;ay


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Raw Material !andlin

Minimi#e openin" and

Use clean tools for dispensin"

@eep the lid on:reduce moisture in"ress

!efri"erate hen instructed>possible

)void lo and hi"h bioburden processin" to"ether

-pen Critical Material in Controlled air : L,?Biosafety hood etc(

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"rocess #$uip%ent Immediately clean after use

Use bioburden reduction a"ents here everpossible : 0a-? )cids ?yprochloride

Use )utoclaved "lassare for critical operations If in doubt clean> saniti#e a"ain

Store either in very dry state or ith biostaticli'uid< no not store et> moist

Periodically clean e'uipment thorou"hly forremovin" soil> "rime from hard to clean areas

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Controllin Bioburden in

"rocess ,ilter Buffers and solutions ,ilter Intermediates< for stora"e

,ilter intermediates before loadin" on to the

columns ,ilter all critical ;S> ;P

)void multiple openin" and handlin"

Tubin" are "ood source of bioburden : 8et

difficult to clean Use Secondary Pac%in" : )dditional Shield


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"acin Material Use )utoclaved Glassare and handlin" tools

)lays use sterile tips and pipettes

,alcon tubes are sterile as lon" as you handlethem that ay


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Bioburden 'pplications


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Device Bioburden

'pplications

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Microbioloical (estin of

"roducts 4uality assurance and manufacturin"controls shouldbe such that or"anismscapable of proliferation and contamination

of the product are ithin acceptable limits(


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Microbioloical )i%it (estThe principle of the Microbial Limit Testis that each viable

microbial cell present in a sample ill hen mi$ed ith ana"ar medium and after incubation form a visible separatecolony(

7 The enumeration of these colony


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Microbioloical )i%it (est7 0on sterile pharmaceutical products are alloed certain

microbial bioburden based upon the product specifications

7 8ith the absence of patho"enic microor"anisms that

chan"e chemical composition by spoila"e and mi"ht affectstability and inte"rity of the product and pac%a"e


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Microbioloical )i%it (estThere are * methods used in enumeratin" the microbial content of

a product(

7 *embrane +i,tration uses filtration apparatus(

7 #ota, P,ate-.ount *ethod (#P.) involves direct platin"(

USP di/ided *0# into 2 tpes

3uantitati/e test

;etermines number of bacteria yeast and mold present in a

"iven pharmaceutical samples( 3ua,itati/e test

;etermines the presence of specific patho"en as salmonella sppStaphylococcus aureus Ps( aeru"inosa and 6nterobacteriaceae(


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Microbioloical )i%it (esto Salmonella and 6 coli 2to$in producers3 are "ram ne"ative

rods lactose fermentors indicates fecal contamination(

o Ps aeru"inosa "ram ne"ative non fermentin" rods

associated ith opportunistic infection

o Staphylococcus aureus is "ram positive cocci usuallyassociated ith s%in and "astrointestinal infections(

opportunistic in4ectionn.)n infection by a microor"anismthat normally does not cause disease but becomespatho"enic hen the bodyAs immune system is impairedand unable to fi"ht off infection(

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Microbioloical )i%it (estUSP recommendations and limits

+( Plant animal mineral based formula ((absence ofsalmonella

*( -rally administered product (( absence of 6 coli/( Topical pharmaceutical preparations absence ofS(aureus

and Ps( aeru"inosa

( Da"inalrectal and urethral ((absence of molds andyeasts

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Microbioloical )i%it (estUSP recommendations and limits

+( Plant animal mineral based formula ((absence ofsalmonella

*( -rally administered product (( absence of 6 coli/( Topical pharmaceutical preparations absence ofS(aureus

and Ps( aeru"inosa

( Da"inalrectal and urethral ((absence of molds andyeasts

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Microbioloical )i%it (estPreparatory testin"

It is done to assure that the sample itself do not inhibitmultiplication of microor"anisms under test conditions(

It includes the inoculation of diluted samples of the product ith

separate viable cultures of Staphylococcus aureus Ps(aeru"inosa 6(coli and Salmonella (

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Microbioloical )i%it (est!esult of the preparatory test 2preliminary test3

Presence of "roth((substance can be assayed by

this methodolo"y under test conditions

)bsence of "roth ((invalidate the MLT and0ecessitates modification of the procedure by

+( )n increase in the volume of the diluent ith the 'uantity oftest material remainin" the same e$1 +"m >+EEml

*( )ddition of suitable inactivatin" a"ent in the diluents 2E(Fsoy lecithin and polysorbate *E are used to neutrali#einhibitory substances present in the sample

/( Combination of + and *


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Microbioloical )i%it (est

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Microbioloical )i%it (estAntimicrobial Efectiveness Test (AET) - USPAntimicrobial preservatives are often added to compendialproducts for the following reasons:

1. To prevent proliferation or limit microbial contamination thatmay occur subsequent to the manufacturing process.2. To inhibit growth of microorganisms during normal

conditions of storage and use when microorganisms might beintroduced inadvertently from repeated withdrawing ofindividual doses from containment.. To prolong the life span of the product.

The Antimicrobial Efectiveness Test is performed to provethat the added preservative provides adequate protection fromadverse e!ects that may arise from microbial contamination or

proliferation during storage and use. This test is not to be used for routine control purposes but itdesigned to put a challenge on a compendial preparation in its"nal container with a prescribed inoculums of suitablemicroorganisms.

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Microbioloical )i%it (estTesting sterile pharmaceutical preparations#terile pharmaceutical preparations are those intended forin$ections and those intended for ophthalmic %sterile oticand nasal preparations

Sterility testsThese tests are e&clusively based upon the principle that

in case the bacteria are strategically placed in a speci"cmedium that contains requisite nutritive material andwater% and maintained at a favourable temperature '( )2*+,% the microbes have a tendency to grow% and theirlegitimate presence may be clearly indicated by theappearance of a turbidity in the originally clear medium.

Please notice that Compliance ith the tests !or

sterility individually cannot certi!y absoluteassurance o! !reedom !rom microbial contamination"

Tests for sterility are adequately designed to reveal the presence of

microorganisms in the 'samples' used in the tests.

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Sterility testin• )ll products labeled sterile must pass the sterility test as they

have ben subHected to an effective process of sterili#ation asper International Pharmacopoeia and USP

• These tests are suitable to reveal the presence of viable formsof bacteria fun"ai and yeasts in a pharmaceutical products or

devices


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6$traneous microor"anisms should be e$cludedthrou"hout the test procedure and period

The sterility testin" of human and veterinary productsis conducted by specific procedures

Test is based on assumptions that Microor"anisms

"ro on the provided culture medium

Limitations 2different or"anism have differentnutritional re'uirements temp( for "roth sporesta%e more time to "ro3


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'nti%icrobial precautions

To avoid any accidental contaminationuse Laminar ,lo ?ood

)lready present Microor"anisms on the

product must be %illed

8or%in" area should be monitoredperiodically both

)ir Surface


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Microbioloical )i%it (est

Tests for sterility are adequately designed to reveal the presence

of microorganisms in the 'samples' used in the tests.

-owever% the interpretation of results is based upon

the assumption that the contents of each andevery container in the batch% had they beentested actually% would have complied ith thetests"

As it is not practically possible to test every

container% a sucient number of containers mustbe e&amined to give a suitable degree o!con#dence in the ultimate results obtained ofthe tests.

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Microbioloical )i%it (est$atch % &a homogeneous collection o! sealedcontainers prepared in such a manner' that theris o! contamination is the same !or each o!the units present in it&"

Sampling

n*ectable Preparations

'a, /ot more than 100 containers ither 10 or 3containers whichever is greater.

(b) 4ore than 100% but not more than 500 containers10 containers.

'c, 4ore than 500 containers. ither 2 or 20containers whichever is less.


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Microbioloical )i%it (estSteri,it test cou,d be per4ormed either b2a3 Membrane ,iltration2b3 ;irect Inoculation(

*embrane +i,tration

This method is applied to the sample hich contains antimicrobialsubstances ( The Membrane filtration sterility test is the re"ulatorymethod of choice for filterable pharmaceutical products( The test isparticularly suitable for samples containin" preservativebacteriostatic or fun"istatic compounds hich inhibit microbial"roth of potential contaminants( 8ith membrane filtrationmicroor"anisms are retained by a E(F


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Microbioloical )i%it (est+icroorganisms Tested !or Positive Control Tests %

(a)$acillus cerreus'b, Staphylococcus aureus'c, ,lebsiella aerogenes'd, Enterobacteria"

the membrane #ltration is to be preferred e&clusively in thecase of the following

(i) an oil or oil-based product'

(ii) an ointment that may be put into solution%

(iii) a non-bacteriostatic solid that does not become soluble inthe culture medium rapidly% and

'iv, a soluble poder or a liuid that essentially possesseseither inherent bacteriostatic or inherent fungistaticcharacteristic features


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Microbioloical )i%it (estThe test should be carried using(a) a sophisticated laminar sterile air.o cabinet 'having hepa6"lters,%

(b) necessary precautionary measures ta7en to be such so as toavoid contamination that they do not a!ect any microbes whichmust be revealed duly in the test.

'c, ensuing environment (i.e., wor7ing conditions, of the laboratorywhere the &tests !or sterility& is performed must always bemonitored at a de"nite periodical interval by :

8 sampling the air of the wor7ing area%8 sampling the sur!ace of the wor7ing area% and8 perforing the stipulated control tests"

Once the "ltration is completed% wash the membrane "lter withpeptone water and then clise the device to introduce the liquidculture medium &/luid Soyabean-Casein 0igest +edium&1' andincubate at 23-245C !or a duration o! seven days" And /luidThioglycollate +edium&'11 and incubate at 63-645 C !or sevendays"


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Microbioloical )i%it (est0irect noculation 7or 0irect noculation o! Culture+edia8The three usual methods being used for performing the 'tests for sterility' areas enumerated under :(a) Nutrient Broth, for the 'aerobic microorganisms'.(b) Cooed !eat !edium for clostridium and Thioglycollate !edium, for

anaerobic(c) "abouraud !edium for moulds and fungiThis test is suitable for non filterable solutions lie oily or viscous solutionsterile ointments "terile containers , gloves and gau#es$. %iquid from the 'test containers' must be removed carefully &ith a &ith asterile syringe.

'. Transfer aseptically the requistite prescribed volume of the substance fromeach container to a vessel of the culture medium.. !i the liquid &ith the medium carefully taing care not to aerateecessively.*. +ncubate the 'inoculated media' for not less than $* days


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Microbioloical )i%it (estLimulus Amebocyte Lysate test. (pyrogen test )

+t is done to detect the presence of pyrogen in any parental pharmaceutical

preparation

pyrogen is frequently described as a fever producing substance. The most

potent pyrogens originate from gram negative bacteria, &hich are common &ater-

borne organisms.

Limulus amebocyte lysate (LAL) is an aqueous etract of blood cells

(amoebocytes) from the horseshoe crab, Limulus polyphemus.

%% reacts &ith bacterial endotoin or lipopolysaccharide (%"), &hich is a

membrane component of /ram negative bacteria. This reaction is the basis of the LAL test, &hich is used for the detection and quantification of bacterial

endotoins.


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Microbioloical )i%it (estLimulus Amebocyte Lysate test. (pyrogen test )The primary application for %% is the testing of parenteral pharmaceuticals

and medical devices that contact blood or cerebrospinal fluid

For pharmaceutical preparations

- $ ml of diluted solution using %% &ater is in0ected into %% test tube andincubated at 12 C for $ hr

3ormation of gel indicates presence of pyrogen

3ormerly rabbit test &as used &here dilution &as in0ected into a rabbit andrabbit &atched for pyreia


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Microbioloical )i%it (estAntibiotic assay

The activity 'potency, of antibiotics may be demonstrated under

suitable conditions by their inhibitory e!ect on microorganisms.

To general methods are employed'

1. the cylinder-plate or 9plate9 assay difusion of theantibiotic from a vertical cylinder through a solidi"ed agar layer ina petri dish such that growth of the added microorganism isprevented entirely in a circular area or 9one9 around holecontaining a solution of the antibiotic.

2. the turbidimetric or 9tube9 assay" the inhibition of growthof a microbial culture in a uniform solution of the antibiotic in a

;uid medium that is favorable to its rapid growth in the absenceof the antibiotic.


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Microbioloical )i%it (estAntibiotic assay< The potency of antibiotics is designated in either 9=nits9 or946g9 of activity< The corresponding =#> ?eference #tandard is 4ach antibiotic istested against a specific microorganisms listed used in the pharmacopeia eg

5 !icrobe under test is inoculated in a concentration of $ 6 in muller hintonagar &hich do not interfere in activity of all no&n antibiotics the seeded agar isleft to solidify

5 7oles are formed using 8 mm cor borer

5 Then certain volume of serial dilutions of the test and standard is instilled into

the holes left for one hour to diffuse then incubated for '* hrs the potency iscalculated through no&n formula by using diameter of inhibition #ones


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Environmental monitoring

nvironmental 4onitoring '@4, is a program designedto demonstrate the control of viable 'livingmicroorganisms, and non6viable particles in criticalareas.

These areas include clean6rooms for drug "ll@ "nish%

formulation tan7 rooms% laminar ;ow hoods% moldingmachines% 7it assembly lines% ntravenous ',compounding areas and sterile pac7aging.

iable monitoring is :

Testing for the detection and enumeration of bacteria%yeast and mold. t includes the monitoring of personnel%air and area surfaces for microbial contamination.

:on-viable environmental monitoring is particlecounts measured by a laser counter.


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Environmental monitoring The items that are sampled in a manufacturerCs clean roominclude

1. Personel 2. Air 3. Surface

1. Personnel - ersonnel are the biggest source of contamination in clean areas.ersonnel harbor bacteria, carrying them &ith them every&here they go./o&ning is the most effective &ay to protect the cleanroom environment from

ourselves.. ersonnel monitoring employs contact plates to assess microbialcontamination of clean room personnel.

!o" #ersonnel are monitored in a $lean %oom

Contact Plates - ersonnel in critical areas may be monitored for microbialcontamination utili#ing contact plates. The contact plates monitor areas of the

body that may interact &ith the sterile field or product eposure areas. Thesemay include gloved hands, forearms, or other areas. ersonnel monitoring is agood indication of ho& &ell personnel are go&ning &hen they enter the cleanroom. .

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Microbioloical )i%it (estAI" the air in a clean room is controlled and !onitored on a regular basis (e.g., daily, &eely,quarterly) for particle counts, viable counts, temperature and humidity.

74 filters are used to control the viable and non-viable particulate counts &ithin the air.74 filters have the capability to filter out particulates do&n to 2.' 'm in si#e.

These filters run continuously at a calibrated flo& rate in order to maintain the required airquality &ithin the room. 7umidity is usually ept at a lo& level in order to help prevent the

proliferation of microbes &ithin the room such as bacteria and mold, &hich tend to prefer dampconditions in order to replicate.

T"o methods of Air sampling in a $lean %oom

1. Air Samplers (active air sampling) - ir samplers dra& in predetermined volumes of air. The

air is dra&n over a sterile media plate, &hich is later incubated to reveal the number of viableorganisms per cubic feet or liter. Currently agar impaction is the method of choice throughoutthe industries. 9sing a specially designed, and calibrated piece of equipment &hich holds themedia plate under a perforated lid and dra&s in a no&n amount of air one can accuratelymeasure the amount of viable bacteria &ithin the air.

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Environmental monitoring Settling plates (passive air sampling) etri dishes containing sterile gro&th media are eposed to the environment for aspecific period of time, usually bet&een 2-12 minutes but can be eposed up tofour hours before compromising the integrity of the media itself. ;iablemicroorganisms &hich settle onto the media surface &ill gro& after the plates areincubated. !o"ever, passive air sampling is tending to be phased out becauseit does not reflect microbial contamination "ith an accurately measured

volume of air.


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Environmental monitoring

6" Sur!aces 'including ;oors% walls% equipment% etc., are cleaned

and monitored on a regular basis for viable counts by using speciallydesigned contact plate

To methods !or sur!ace monitoring in a Clean ;oom1. Contact Plates 6are special >etri dishes which contain sterilegrowth medium prepared in a manner so the surface of the mediaprotrudes above the rim of the plate. These plates that contain a

growth medium called Trypticase #oy Agar 'T#A, and #abouraudDe&tros Agar '#DA% T#A at 065+ which is mainly the optimal growingtemperature for most environmental bacteria% and 20625 + which isthe optimal growing temperature for most mold and yeast species. The contact plate is pressed against any ;at surface that needs to besampled. Any viable microorganisms on the surface will stic7 to theagar surface and will grow upon proper incubation. This techniquereveals the number of viable microorganisms on a surface.2. Swabs 6 are sterile and stored in a suitable sterile liquid. The swabsare rubbed over the test surface. The microbiologist can determine thetype of microorganisms on the swab by subculturing it to media.#wabs are used for surfaces that are not ;at% and can be used tosample hard to reach areas of machinery that could not be sampledwith a contact plate. #wabbing is more qualitative than quantitative.

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174362793 Bioburden Control