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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Agenda - Rapid Methods for Detecting Mycoplasma Contamination in the Manufacture of Vaccines, Including Pandemic Influenza Vaccines, and Other Biological Products

Hilton Washington DC North/Gaithersburg
620 Perry Pkwy.
Gaithersburg, MD 20877

September 22, 2008, 8:30 a.m. to 5 p.m.

September 23, 2008, 8:30 a.m. to 12 noon


Workshop Chair: Rajesh K. Gupta, Ph.D., CBER

Program Committee:

Ms. Christine Anderson, CBER
Dr. Kurt Brorson, CDER
Dr. Vladimir Chizhikov, CBER
Dr. Konstantin Chumakov, CBER
Dr. Rajesh K. Gupta, CBER
Dr. Arifa Khan, CBER
Dr. Philip Krause, CBER

Day 1, September 22, 2008
8:00 a.m.Registration
8:30 a.m.Welcome Remarks
8:40 a.m.Introduction to Workshop
Rajesh K. Gupta, Ph.D., CBER
8:50- 9:15 a.m.Mycoplasma contamination of biologics: Regulatory aspects of the mycoplasma testing.
Vladimir Chizhikov, Ph.D., CBER.
9:15-10:40 a.m.PCR Mycoplasma assay according to EP Chapter 2.6.7.
Sven M. Deutschmann, Ph.D., Roche Diagnostics GmbH, Germany.
9:40- 10:00 a.m.Status of the PDA Technical Report for Alternative Mycoplasma Tests Methods
Kurt Brorson, Ph.D. CDER
10:00 -10:20 a.m.Break
Session 1. Direct NAT Methods
(Chairs Dr. Konstantin Chumakov and Dr. Arifa Khan)
10:20 - 10:45 a.m.Development and Validation of a Touchdown PCR method for the detection of Mycoplasma.
Barbara J. Potts, Ph.D., Genentech, Inc.
10:45-11:10 a.m.Detection of Mycoplasma/Acholeplasma contamination by amplification of specific nucleic acids and fluorescent probe technology.
Michael J. Hantman, Ph.D., Charles River
11:10 - 11:35 a.m.Design and development of a high-throughput platform for rapid Mycoplasma identification and automatic data management. "
Dr. Bjorn Breth, Greiner Bio-One GmbH
11:35-12:00 p.m.Effects of Mycoplasma Infection in Cell Culture Systems and development of a PCR-based assay for its detection.
Pranvera Ikonomi,Ph.D., American Type Culture Collection
12:00- 1:00 p.m.Lunch Break
1:00-1:25 p.m.Detection of Mollicutes by a two-step RT-PCR method.
Gert Jan Haan, Ph.D., Centocor B.V.
1:25-2:00 p.m.

Discussion (Topics to be covered)

  1. Limitations of NAT methods associated with the small sample volume used for NAT testing.
  2. What unit of measure (genomic copies or CFU) should be used for adequate assessment of sensitivity of NAT methods?
  3. Potential application of other Mycoplasma genetic markers present in multiple copies in bacterial cells (e.g. 16S rRNA)
Session 2 NAT Methods Combined with Initial Enrichment Step
(Chairs Dr. Philip Krause and Dr. Vladimir Chizhikov)
2:00-2:25 p.m.Rapid Mycoplasma Testing: The HyMy TM Assay combines amplification of viable Mycoplasma in Broth Culture with signal detection by Quantitative Polymerase Chain Reaction (QPCR).
Jeri Ann Boose, Ph.D., BioReliance Corporation
2:25-2:50 p.m.Biological Enrichment of Mycoplasma Agents using Co-Cultivation with Permissive Cell Cultures.
Dmitriy V. Volokhov, Ph.D., CBER.
2:50- 3:15 p.m.Incorporation of an Alternative Detection Method reduces Mycoplasma testing time
Timothy A. Coleman, Ph.D., Lonza Bioscience
3:15-3:40 p.m.Break
3:40- 4:05 p.m.Design, Development and Qualification of the MicroSEQ TM Mycoplasma Detection Assay.
Michael T. Brewer, Applied Biosystems Inc.
4:05-4:30 p.m.Rapid Mycoplasma detection coupling a novel sample preparation device to molecular methods.
Martha S. Rook, Ph.D., Millipore Corporation
4:30-5:30 p.m.

Discussion (Topics to be covered)

  1. Advantages and disadvantages of initial enrichment step in Mycoplasma detection with regard to:
    • Sensitivity (LOD)
    • Viability
    • Turnaround time of analysis
    • Complexity of testing procedure
    • Cost and convenience
  2. Biological enrichment of mycoplasmas: agar/broth media vs. cell cultures: Advantages and disadvantages of each approach.
  3. Biological enrichment vs. concentration approach (cells or nucleic acids)
Day 2, September 23, 2008
Session 3: Comparative Evaluation of Rapid Methods and Other Practical Aspects in Developing Rapid Methods
(Chairs Dr. Kurt Brorson and Ms Christine Anderson)
8:30-8:55 a.m.Application of Risk-based approach to optimize a rapid Mycoplasma test for cell therapy and tissue-engineered products.
John Duguid, Genzyme
8:55- 9:15 a.m.NAT vs. Microbiological culture - comparing chalk with cheese?
Helena M. Windsor, Mycoplasma Experience
9:15 - 9:30 a.m.Discussion
9:30 - 10:00 a.m.Break
Session 4: Panel Discussion on Selection of Promising Alternative Method(s) for Further Evaluation and Planning of Collaborative Study
(Chair Dr. Rajesh K. Gupta)
10:00 - 10:10 a.m.Introduction Remarks,
Rajesh K. Gupta, Ph.D.
10:10 - 12:15 p.m.Discussion (All speakers and members of committee as panel)
12:15 - 12:30 p.m.Concluding Remarks and Plans for a Collaborative Study

Other topics for Discussion

  1. Development of criteria for preparation of reference Mycoplasma stocks for adequate assessment of the relative sensitivity of NAT and microbiological methods (the significance of Mycoplasma stock viability and GC/microorganism (CFU/CCU) ratio for the NAT tests).
  2. Factors affecting the GC/microorganism (CFU/CCU) ratio of Mycoplasma stocks.
  3. Is it correct to measure thesensitivity of PCR using CFU/ml units? Analysis of recommendations of EP chapter 2.6.7.
  4. Proposal of Recommendations for adequate comparison between NAT and Compendial Mycoplasma testing methods.
  5. Role of Risk Assessments for assay format applicability and choice
  6. Establishment of a collaboration in development and validation of new Mycoplasma testing methods.