O06304102MediaSterilityWhitePaperfinal

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Meeting USP/EP/CFRsterility testing requirements

with a two media assay

Biologics Safety Testing

You Can Rely on Us

The World Relies on You


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About BioReliance

BioReliance Corporation is a leading provider o cost-eective contract services to the pharmaceutical and

biopharmaceutical industries, oering more than 1,000 tests or services related to biologics saety testing, specializedtoxicology and animal health diagnostics. Founded in 1947, BioReliance is headquartered in Rockville, Maryland,

with laboratory operations in Rockville and Scotland and oces in Tokyo, Japan, and Bangalore, India. The Company

employs more than 600 people globally. For more inormation, visit www.bioreliance.com.

Key Services: Custom Assay Development to ulfll your exact requirements

Biosaety Testing o biologicals or viruses, bacteria, mycoplasma, ungi

Cell Line Characterization including identity testing, genetic stability, EM, sequencing

Final Product Testing including biopotency testing, residual DNA, host cell proteins, cross-reactivity

Virus/TSE ValidationStudies or all biological products

Contract GMP Production and Testingo viral vectors and cell banks

Veterinary Vaccine Services including characterization/identity, extraneous agent testing

Regulatory and Consulting Services

All work undertaken by BioReliance is in compliance with appropriate GLP or GMP standards.

Further inormation is available by visiting our website at www.bioreliance.com


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Sterility Testing Media Review 1

www.bioreliance.com

Abstract

Historical data rom BioReliance presented herein sup-

ports that only 2 media, Fluid Thioglycollate (FTM(THIO))

and Soybean-Casein Digest (SCDM(TSB)) when used in a

standard USP/EP/CFR sterility test is sucient to detectmicrobial contaminants.

Introduction

BioReliance currently oers a panel o standard sterility as-

says that comply with US and EU requirements (Table 1).

The two media outlined in the USP, FDA, EP and JP require-

ments are Fluid Thioglycollate (FTM(THIO)) and Soybean-

Casein Digest (SCDM(TSB)) (USP, 21CFR 610.12, EP

2.6.1, and JP 4.06 respectively). These media detect anaer-

obic and aerobic bacteria as well as ungal contaminants.

An additional series o assays that employ the use o our

media as opposed to the standard two defned in the

compendial methods have also been oered by BioReli-

ance (Table 1). Sabouraud Dextrose Agar (SDA or SAB),

designed to support ungal growth, and Pre-reduced Pep

tone Yeast Glucose broth (PYG), designed or anaerobic

bacteria have been used in select sterility assays. These

media were added to the compendial media with the in-

tent o oering broader coverage o microbial contami-nants in sterility tests.

Historical data rom the past hal decade (20052009) o

BioReliance sterility testing were analyzed and assessed in

the context o the two versus our media assay ormulations

and are presented in this report. The data demonstrate

that microorganisms detected in the fve year data set

are capable o growth on the two media FTM(THIO) and

SCDM(TSB). From this we conclude that the addition o

SDA(SAB) and PYG to the testing scheme does not oer

any competitive advantage or additional inormation

to the compendial Sterility Tests currently available at

BioReliance.

Table 1. BioReliance Sterility Tests currently available.

GLP Assay Number GMP Assay Number Assay Description Media

510011 510811Direct Inoculation Sterility or Final

Bulk, 2 MediaSCDM(TSB)/FTM(THIO)

510036 510836Direct Inoculation Sterility or Final

Vials, 2 MediaSCDM(TSB)/FTM(THIO)

510006 510806 Membrane Filtration Sterility SCDM(TSB)/FTM(THIO)

5100001 5108881Direct Inoculation Sterility or

Prebanking or Cells at Limit,

4 Media

SCDM(TSB)/FTM(THIO)/SDA(SAB)/PYG

510D00

1

510D88

1 Direct Inoculation Sterility or

Unprocessed Bulk, 4 Media SCDM(TSB)/FTM(THIO)/SDA(SAB)/PYG

510R002 510R882Direct Inoculation Sterility or Cell

and Virus Banks, 4 MediaSCDM(TSB)/FTM(THIO)/SDA(SAB)/PYG

1 Replaced by 510011 and 510811.2 Replaced by 510036 and 510836.


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2 Sterility Testing Media Review

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Fluid Thioglycollate Medium (FTM(THIO))

The intended use o THIO medium is the cultivation o

anaerobic, microaerophillic and aerobic microorganisms

and or detecting the presence o bacteria in normally

sterile materials (Dico manual, 10th edition). FTM(THIO)

conorms to the requirements o USP and AOAC or

testing the sterility o antibiotics, biologics, and ood. The

ormulation o THIO supports the growth o a wide variety

o astidious microorganisms having a range o growth

requirements, including anaerobic bacteria (Table 2).

Soybean-Casein Digest Media or Tryptic

Soy Broth (SCDM(TSB))

Tryptic Soy Broth is used or cultivating a wide variety o

microorganisms and conorms to the Harmonized USP/

EP/JP requirements or microbiological testing, including

sterility. This medium is also recommended by the Clinical

and Laboratory Standards Institute (CLSI) or inoculum

preparation in disk diusion sensitivity tests or testing

bacterial contaminants in non-sterile pharmaceutical

and cosmetics. TSB complies with established standards

in the ood industry and is the media o choice by theUSDA APHIS or detecting viable bacteria in live vaccines

(9CFR:113.26).

Commonly reerred to as Soybean-Casein Digest

Medium, Tryptic Soy Broth (TSB) is a general purpose

medium with a rich nutritional base used or isolating

astidious and non-astidious microorganisms, including

ungi (Table 3).

Table 2. Growth characteristics of control organisms for

growth promotion in Fluid Thioglycollate medium after

18-48 h incubation at 35C incubated under ambient

(aerobic) conditions

(Dico 10th ed.; Dico and BBL Manual online).

Organism Growth

Staphylococcus aureus ATCC 6538 Good to excellent

Pseudomonas aeruginosa ATCC9027

Good to excellent

Bacteroides vulgatus ATCC 8482 Poor to air*

Bacillus subtilis ATCC 6633 Good to excellent

Bacteroides vulgatus ATCC 8482 Poor to air*

Candida albicans ATCC 10231 Good to excellent

Clostridium sporogenes ATCC

11437Good to excellent

Kocuria. rhizophila ATCC 9341

(ormerly Micrococcus luteus)Good to excellent

Neisseria meningitidis ATCC 13090 Good to excellentStreptococcus pyogenes ATCC 19615 Good to excellent

* Growth good to excellent ater 3 days incubation.

Table 3. Growth characteristics o control organisms in TSB (Dico and BBL Manual online).

Organism Incubation temperature Recovery

Escherichia coliATCC 25922 3035C Growth by 48 hStreptococcus pneumoniae ATCC 6305 3035C Growth by 24 h

Staphylococcus aureus ATCC 25923 3035C Growth by 48 h

Bacillus subtilis ATCC 6633 2025C Growth by 3 days

Candida albicans ATCC 10231 2025C Growth by 5 days

Aspergillus brasiliensis ATCC 16404 2025C Growth by 5 days


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Sabouraud Dextrose Agar (SDA or SAB)

SAB is a general-purpose medium devised by Sabouraud

or culturing o yeasts, molds, and aciduric microorgan-

isms and is used or the growth o ungi, particularly

those associated with skin inections (dermatophytes)(Sabouraud, 1892). The low pH o approximately 5.6 is

avorable or the growth o ungi, especially dermato-

phytes, and slightly inhibitory to contaminating bacteria

in clinical specimens. This medium is recommended in

the USPor use in perorming total combined mold and

yeast counts (Microbial Limit Tests).

Pre-reduced Peptone Yeast Glucose Broth (PYG)

PYG was developed by the Virginia Polytechnic Institute

(VPI) anaerobe laboratory or the cultivation o anaer-

obes or downstream identifcation tests (Holdeman,

et al., 1977). The anaerobic bacteria cultivated in the

PYG broth are also ound in the anaerobic phase o the

FTM(THIO) media employed in the standard two media

sterility tests. Sodium thioglycollate lowers the oxida-

tionreduction potential o the FTM(THIO) media creat-

ing anaerobic conditions in this medium and the small

amount o agar assists in the maintenance o a low redox

potential by stabilizing the medium against convection

currents, thereby maintaining anaerobiosis in the lower

depths o the medium (McFaddin, 1985) rendering the

inclusion o PYG redundant in the sterility assays.

Historical Data AnalysisThe data rom Sterility Assays perormed by BioReliance

over the past 5 years (Figure 1) indicate that Bacillus spe

cies (19%), especially Bacillus circulans, was the most

commonly identifed contaminant in non-sterile biolog-

ics tested. The second most common cause o ailure

was Proprionibacterium spp. (17%), ollowed distantly by

Paenibacillus spp. (8%) and Staphylococcus spp. (7%). The

remaining contaminants identifed in test articles were

ound at a requency o 1-3%. The ungal contaminant

Penicillium (5%) was ound as inrequently as Staphylcoc-

cus spp., even when SDA(SAB) was included in the tests

In comparison, an analysis o FDA product recall data o

134 non-sterile pharmaceutical products rom 1998 to

September 2006 demonstrated that 48% o recalls were

due to contamination by Burkholderia cepacia, Pseudomo

nas spp., or Ralstonia picketti, while yeast and mold con

tamination were ound in 23% o recalls. Gram-negative

bacteria accounted or 60% o recalls, but only 4% were

associated with Gram-positive bacteria (Jimenez, 2007).

19 %

17 %

8%

7%

6%

5%

Bacil lus spp.

Propionibacterium spp.

Paenibacil lus spp.

Staphy lococcus spp.

Pseudomonas spp.

Penic i l l ium spp.

Figure 1. Distribution o the most requent contaminants ound in samples tested rom 2005-2009.


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4 Sterility Testing Media Review

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Taken together, these data indicate that aerobic

and microaerophilic bacteria constitute the primary

concern in sterility testing among pharmaceutical and

biologics manuacturers. These organisms isolated

rom contaminants o biologics introduced duringthe manuacturing process are capable o growth

on FTM(THIO) and SCDM(TSB). These two (2) media,

independently or together detect representatives rom

all genera picked up on PYG and/or SDA(SAB), including

aerobic and anaerobic bacteria and most ungal species

(Table 4). Thereore, the addition o PYG or SDA(SAB)

does not oer any additional detection advantage over

the two media assay. Furthermore, testing using two

media as recommended in the USP, EP, and CFR guidelines

oers greater sensitivity and accuracy than use o our

media or testing. Spreading the test article across ewe

media increases the likelihood that contaminants wil

be detected and that they will be detected in replicate

media. Perorming sterility testing using the two media

to compliance as outlined per USP, 21CFR 610.12and EP 2.6.1 is scientifcally suitable and will meet

regulatory standards worldwide or the development and

production o sae products intended or human use.

Notifcation

Eective October 1, 2010, BioReliance will be discontinu

ing the availability o all our media sterility assays. We

encourage that all clients contact their Account Manager o

Program Manager to discuss two media assay alternatives.

Table 4. Genera and species identifed in sterility testing at BioReliance in the past fve (5) years. The designation

spp. indicates more than one species o a given genus has been detected in the sterility assays.

Organism

Acholeplasma laidlawii Mesorhizobium huakuii

Acinetobacter johnsonii Methylobacterium extorquens

Acinetobacter radioresistens Micrococcus luteus

Actinomyces meyeri Paecilomyces variotii

Afpia genomospecies 4 Paenibacillus spp.

Arthrobacter spp. Patulibacter minatonensis

Bacillus spp. Penicillium spp.

Bifdobacterium bifdum Phyllobacteriaceace

Bradyrhizobiaceae Propionibacterium acnes

Candida parapsilosis Pseudomonas spp.

Cauolobacter vibrioides Ralstonia pickettii

Cellulosimicrobium unkei Serratia liqueaciens

Cryptococcus spp. Staphylococcus spp.

Cupriavidus metallidurans Stenotrophomonas maltophilia

Enterococcus Streptococcus spp.

Herminiimonas saxobsidens Tsukamurella ( ormerly Corynebacterium)

Leuconostoc gelidum


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ReerencesDico Manual, 10th edition.

Dico and BBL Manual, online, 2009.

Jimenez, L, Microbial diversity in pharmaceutical product

recalls and environments. PDA J Pharm Sci Technol. 2007.

61:383-399

MacFaddin, J.F. 1985. Media or isolation-cultivation-

identifcation-maintenance o medical bacteria, vol. I.

Williams & Wilkins, Baltimore.

Holdeman LV, Cato EP, Moore WEC, Eds. Anaerobe laboratory

manual. 4th ed. Blacksburg: Virginia Polytechnic Institute and

State University, 1977.

Sabouraud. 1892. Ann. Dermatol. Syphil. 3: 1061.


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BioReliance Corp.

14920 Broschart Road

Rockville, Maryland 20850

Tel: 800.553.5372

Fax: 301.610.2590

Email: [email protected]

BioReliance Ltd.

Todd Campus

West o Scotland Science Park

Glasgow, Scotland G20 0XATel: 44 (0) 141 946.9999

Fax: 44 (0) 141 946.0000


BioReliance Ltd.

Innovation Park

Hilloots Road

Stirling, Scotland FK9 4NF

Tel: 44 (0) 141 946.9999

Fax: 44 (0) 141 946.0000


BioReliance, K.K.

Imas Hamamatsu-cho bldg., 6F2-10-4 Hamamatsu-cho

Minato-ku, Tokyo 105-0013, Japan

Tel: 03 5425.8491

Fax: 03 5425.8495


www.bioreliance.comNorth America Toll Free: 800 553 5372 Tel: 301 738 1000

Europe & International Tel: +44 (0)141 946 9999 Japan Tel: +03 5425 8491


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