1. Technical Report on Alternative Methods for Mycoplasma Testing Status update- Sept 2008 Kurt Brorson, Ph.D. CDER/FDA

2. Views expressed in this talk constitute my professional opinion and not official FDA policy Statements in this talk should not be construed as official approval of particular test methods by the FDA or the US Government

3. Perspectives among PDA membership Advances have been made in testing and screening methods for mycoplasma –PCR methods –Others (enzyme, etc.) New methods: –More rapid and high throughput than culture methods –Detect components, not growth –Many appear to have similar sensitivity and species range as existing assays –Allow go/no-go forward processing decisions that are highly advantageous in a biotech facility

4. Perspectives among PDA membership- 2 Need greater clarity concerning: –Method applicability, strengths and weaknesses –Validation approaches –Appropriate reference standards –Demonstration of comparability of new methods to existing methods –Regulatory pathway for implementation –Implementation EU vs. USA

5. Task Force (TF) leadership PDA formed Mycoplasma TF to address these & other needs Mycoplasma Committee leader: –Barbara Potts, Ph.D., Genentech Alternative methods subcommittee leadership –Kurt Brorson, Ph.D., CDER/FDA –Thomas Haemmerlae, Ph.D., Baxter –Mark Kaiser, Ph.D., Lancaster Labs

6. Scope: Alternative Mycoplasma testing methods (i.e. not current culture methods) including NAT and others (e.g. flow cytometry, enzyme activity based) Purpose: Describe procedures, validation, demonstration of comparability, appropriate reference standards, potential applications for alternative mycoplasma test.

7. Not goals of TF Broad discussion of existing culture methods Harmonization of existing compendial methods Recommend one method over others Write a regulatory guidance –PDA is not a regulatory organization –However… PDA documents often cover highly technical areas efficiently and in great detail PDA can promote consensus on difficult technical issues in a fair, reasoned and timely manner

8. Status TF subgroup for alternative method technical report formed –~15 members from CTOs, biotech manufacturers, regulatory authorities, vendors of enabling technologies Complete draft in place Areas for discussion identified –LOD –Sample volume –GC:CFU ratio

9. Issues for discussion Limit of detection –Existing assays vs. new methods General perception that new method should have comparable or lower LOD –CFUs vs. genome copies (GC) Assays have different readouts GC:CFU ratio in –Typical reference standard preparations –Actual contaminant samples Sample volume Regulatory expectations –EU vs. USA –Biotech products vs. vaccine products

10. What determines GC:CFU ratio? Mycoplasma clumping –One clump of 100+ mycoplasma will grow as one colony –Mycoplasma trapped in a dead mammalian cell may grow as one colony % culture viability –Dead mycoplasma still have DNA but won’t form colony Culture length & growth conditions –Free DNA released from disintegrated mycoplasma will amplify in PCR assays With all of these variables, can LOD of PCR and culture methods really be compared? –For the purposes of go/no-go, forward processing decisions, is an assay LOD based on GC suitable for intended use

11. Risk based approach -assay “suitable for intended use” Biotechs Grown in highly characterized, transformed cell lines Mycoplasma kill/removal post- culture –chromatography steps –Inactivation steps –filtration Risk to facility is as great as risk to the product Rapid go/no-go forward processing decisions highly advantageous in biotech manufacturing Vaccines Often grown in primary cell cultures Often minimal processing & filtration past culture phase Vaccines are given to healthy individuals, mostly children Large volume assays minimizes LOD –21 CFR 610.30 applies to lvirus vaccines

12. Table of Contents Glossary Introduction- Historical background of existing assays NAT methods –Overview –Applicability & Points of use in cell culture manufacturing and development –Targeted species –Primer and probe selection –Methods overview –Sample volume –Nucleic acid extraction & purification

13. Table of Contents, cont. NAT methods, cont. –Assay set-up –Data interpretation & follow-up –Reference materials –Method validation –Comparability studies to previous assays Cross-industry comparability CFU: NA sequence copy ratio Non-NAT alternative methods –Enzyme-based methods –Non-amplified nucleic acid hybridization assays –Mycoplasma testing using a recombinant cell line.

14. Final steps Resolve inconsistencies within TF subgroup –“minority report” approach Continues to smooth out document to minimize overlaps and gaps PDA BioAB & board review Publish as new PDA TR –Target Q1 or Q2 2009

15. Thank you for your attention! Kurt Brorson, Ph.D. Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration E-mail: kurt.brorson@fda.hhs.gov