BioReliance's Approach To Mycoplasma Testing: Introduction Of United .

Biologics Safety TestingBioReliance’s Approach toMycoplasma Testing:Introduction of United StatesPharmacopoeia 63 RegulationO-0660810

About BioRelianceBioReliance Corporation is a leading provider of cost-effective contract services to the pharmaceutical and biopharmaceuticalindustries, offering more than 1,000 tests or services related to biologics safety testing, specialized toxicology and animalhealth diagnostics. Founded in 1947, BioReliance is headquartered in Rockville, Maryland, with laboratory operations inRockville and Scotland and offices in Tokyo, Japan, and Mumbai, India. The Company employs more than 650 peopleglobally. For more information, visit www.bioreliance.com.Key Services: Custom Assay Development to fulfill your exact requirements Biosafety Testing of biologicals for viruses, bacteria, mycoplasma, fungi 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 Validation Studies for all biological products Contract GMP Production and Testing of viral vectors and cell banks Veterinary Vaccine Services including characterization/identity, extraneous agent testing Regulatory and Consulting ServicesAll work undertaken by BioReliance is in compliance with appropriate GLP or GMP standards.Further information is available by visiting our website at www.bioreliance.comwww.bioreliance.com

BioReliance’s Approach to Mycoplasma Testing: Introduction of USP 63 1IntroductionMycoplasma contamination of cell culture (both of primaryThe recently published USP chapter on “Mycoplasma Tests “and continuous eukaryotic cell lines) is common and rep-represents a step forward in bringing requirements in theresents an issue of importance in the basic research, de-US closer to those outlined in the EP. BioReliance is updat-velopment and production of biologicals. Contaminationing its mycoplasma detection assay range to include thecan alter virtually every physical and chemical property ofUSP method for our clients who are filing in the US. Ourcells (depending on the contaminating species and the cellnew, cGMP compliant assay will meet or exceed USP, EP, andtype) causing them to yield unreliable results and perhapsPTC requirements. In addition to the combined USP/EP/unsafe biologicals, biopharmaceutical drugs or viral vac-PTC test, a 21 CFR 610.30 compliant assay will be availablecines. In fact, mycoplasma contamination can be presentseparately for viral vaccines, as well as a JP-compliant testwith no obvious change in the host culture, even when thefor those clients who need to meet Japanese regulations.concentration of mycoplasma exceeds that of the host cellsby 10-100 fold. Thus, testing for mycoplasma contamina-Comparison of the EP, USP, and PTC methodstion during development and manufacturing of biologi-While the regulatory documents describe methods thatcals is required by the worldwide regulatory authorities inare mostly harmonized, there are still some differences be-the United States, Europe and Japan. Key regulations aretween United States3–7 and European1 regulatory guide-defined under FDA Points to Consider (PTC, 19933; 19974lines. These differences include:562010 ), 21 CFR 610.30 (CFR) , European Pharmacopoeia (EP)section 2.6.71, Japanese Pharmacopoeia section 14 (JP)2 The acceptance criteria for quantitative recovery ofand the recently announced United States Pharmacopoeiapositive controls in the test for nutritive properties and7 63 (USP) monograph which will become effective inqualification test for inhibitory substances. The incubation conditions and number of positiveOctober 2010.controls included during testing.All biologics produced for clinical investigation and as licensed therapeutics produced via cell substrates (e.g., vi- The number of subcultures performed during brothevaluation.ral vaccines, monoclonal antibodies and similar products)must be tested to ensure the absence of mycoplasma con-Qualification Testing: The pharmacopoeias (EP and USP)tamination. Tests for the presence of mycoplasma contami-require testing for inhibitory properties once, while the PTCnation in Master Cell Banks (MCB) and Working Cell Banksdoes not stipulate this testing requirement. This qualifica-(WCB) originating from metazoan cells should also be con-tion (“mycoplasmastasis”) testing entails the recovery ofducted as part of purity testing. Guidance for this testing isspiked control organisms in the presence of test product.detailed in the PTC3, 4, EP1, and USP7 publications referenced.The acceptance criteria for the test for nutritive propertiesLive viral vaccines produced from in vitro living cell culturesof media and qualification testing in the EP and USP meth-prior to clarification or filtration and inactivated viral vac-ods are aligned for broth culture (Table 1). The growth ofcines produced from living cell cultures prior to inactivationmycoplasma in the presence of test product must be withinmust also be tested to ensure the absence of mycoplasmaone subculture of that in the absence of test product. For6per 21 CFR 610.30 .the direct agar portion of the test, however, the quantitativewww.bioreliance.com

BioReliance’s Approach to Mycoplasma Testing: Introduction of USP 63 2 evaluation in the USP is more stringent than the EP. TheAll regulatory documents specify the number and types ofUSP states that the test is compliant if the recovery of themycoplasma positive controls to be included in the assays;spike organisms is within 0.5-log in the presence of the testfor PTC and USP at least two known mycoplasma species arematerial as compared to that in absence of test material.required, one being a dextrose fermentor and one an arginineBy contrast, the EP defines that the plates inoculated withhydrolyzer. The EP requires the use of at least one of sixspiked test product must be within one-fifth the number ofmycoplasma species listed in the chapter as a positive control.colonies of those inoculated without the test product. In thisinstance, the USP criterion is more stringent than the EP.Number of subcultures: All methods require sub-culturingfrom the broth bottle cultures throughout the 28 dayIncubation Conditions and Controls: There are alsoperiod (Table 2 and Figure 1). The number of subculturessubtle differences in the incubation conditions between theranges from 3 subcultures for PTC (Days 3, 7, and 14) to 4methods (Tables 1 and 2). The EP defines a temperature rangesubcultures for the USP and EP. The additional subcultureof 35-38 ºC for incubation, while the USP and PTC stipulateon day 21 in the USP and EP method is incubated for 7it as 36 1ºC. Similarly, the terminology for atmospheresdays. This fourth subculture gives added sensitivity withoutslightly varies between the methods. The PTC refers to it asincreasing the duration of the assay.“anaerobic” incubation while the EP and USP use the term“microaerophilic” (although the definition remains identicalIndicator cell culture methods. The use of indicator cellsacross the regulations at 5 – 10% CO2 in nitrogen).to detect non-cultivable mycoplasma is also includedTable 1. Differences between the EP, USP, and PTC methods in the agar culture conditions and ted StatesPharmacopoeiaFDA Points toConsider (1993)Inoculum volume0.2 ml on each solid mediaplate0.2 ml on each solid mediaplate0.2ml/2 or more platesPositive control recoveryNot more than 100 cfu perinoculumNot more than 100 cfu perinoculumNot more than 100 cfu perinoculumTemperature /Incubationcondition35-38 C Microaerophilic(nitrogen containing 5-10%CO2)36 1 CMicroaerophilic (hydrogenatmosphere containing 0.5%oxygen and/or nitrogencontaining 5-10% CO2 innitrogen)36 1 CHydrogen atmospherecontaining 0.5% oxygenand/or nitrogen containing5-10% CO2 in nitrogenInhibition assaySpike recoverySpike recoveryNo spike recovery requiredPlates inoculated with TAspiked with control must beMycoplasmastasis testingwithin 1/5 of the number ofcolonies without TAPlates inoculated with TA arewithin 0.5log range of numberof colonies without TAN/APositive control selectionBased on type of sampleOne dextrose fermentor andone arginine hydrolyzer. Othersmay be used based on sampletypeOne dextrose fermentorand one argininehydrolyzer.Test for nutritiveproperties of mediaEssential using all appropriatepositive control organismsEssential using all appropriatepositive control organismsNot requiredwww.bioreliance.com

BioReliance’s Approach to Mycoplasma Testing: Introduction of USP 63 3in all the three regulations (Table 3). Vero cells are mostadditional passage step allows for additional amplification ofcommonly used but another cell substrate may be used ifthe potential contaminants which enhances the detectionequivalence for detection of mycoplasma is demonstrated.of mycoplasma in the test product.In the PTC, the test article is added to the cells and incubatedfor 3-5 days before direct staining and examination byThe degree of alignment amongst these monographs (EP,epifluorescence microscopy. However, per both the EP andUSP, PTC) makes it more practicable to satisfy the regulatoryUSP methods, the test article is added to indicator cells inexpectations under a single assay system. However, furthera flask and allowed to grow for 3-5 days before passagingalignment in the monographs should aid in achieving aonto coverslips for a subsequent 3-5 day growth period. Thismore concise assay.Table 2. Differences between the EP, USP, and PTC methods in broth culture conditions and validity criteria.EuropeanPharmacopoeiaUnited StatesPharmacopoeiaInoculum volume10 ml/100 ml;subculture days: day 2-4,day 6-8, day 13-15, day 19-2110 ml/100 ml;subculture days: day 2-4,day 6-8, day 13-15, day 19-2110 ml/50 ml; subcultureday 3, 7,14Positive control recoveryNot more than 100 cfu perinoculumNot more than 100 cfu perinoculumNot more than 100 cfu perinoculumTemperature /Incubationcondition35-38 C Aerobic36 1 C Aerobic36 1 C AerobicInhibition assaySpike recoverySpike recoveryNot requiredValidity criteria ofinhibition testRecovery within 1 subculturein presence of TA comparedwith positive controlRecovery within 1 subculturein presence of TA comparedwith positive controlN/APositive control selectionBased on type of sampleOne dextrose fermenter andone arginine hydrolyzer.Other may be used based onsample typeOne dextrose fermenterand one argininehydrolyzer.Test for nutritiveproperties of mediaEssential using allappropriate positive controlorganismsEssential using all appropriateNot requiredpositive control organismsMediumBrothCultureFDA Points toConsider (1993)Table 3. Comparison of the EP, USP, and PTC cell culture ed StatesPharmacopoeiaFDA Points toConsider (1993)Seeding density2 104 cells to 2 105 cells/ml(4 103 to 2.5 104 cells/cm2)2 104 cells to 2 105 cells/mlNot defined(4 103 to 2.5 104 cells/cm2)Inoculum volume1 ml1 ml1 mlPositive control recoveryNot more than 100 cfu perinoculum; M. hyorhinis andM. oraleNot more than 100 cfu perinoculum; M. hyorhinis andM. orale100 cfu or less perinoculum; M. hyorhinisand M. oraleTemperature condition35-38 C36 1 C36 1 CInoculation of test articleCulture 3-5 days theninoculated onto coverslipsCulture 3-5 days theninoculated onto coverslipsTA inoculated directly oncells on coverslipsInhibition assaySpike recovery when usingneutralising antiserumSpike recovery when usingneutralising antiserumNo spike recoverywww.bioreliance.com