Technical Report on Alternative Methods for Mycoplasma Testing

Status update- Sept 2008

Kurt Brorson, Ph.D.

CDER/FDA

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

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

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

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

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.

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

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

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

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

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

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

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.

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

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