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Erik Familial Hypercholesterolemia USA

Validation of Rapid Mycoplasma Testing using Real-Time PCR

John Duguid, Principal Process/Analytical Scientist

June 8, 2012

Agenda

• Background

− Autologous Cell Therapy Products

• Past

− Selection and Risk Assessment

• Present

− Validation

• Future

− Automation

Our Products

Statement of Problem

• Testing is required at various stages in the production

process for therapeutic biological products intended for

human use.

• Standard culture methods take at least 28 days to

complete.

• Lot release tests for cell therapy products having

abbreviated shelf-lives are ideally completed in 1 day.

Potential Mycoplasma Test Technology

Potential

Technology

Principle

of

Operation Sensitivity

Agar and Broth Culture Growth 10 CFU/mL

Indicator Cell Line/Hoescht Stain Growth/DNA Stain 100 CFU/mL

PCR DNA Amplification/Endpoint 10 CFU/mL

qPCR DNA Amplification/Kinetic 10 CFU/mL

RT-PCR RNA Reverse Transcription <1 CFU/mL (theor.)

ELISA Enyzme/Antibody 104 CFU/mL

PCR ELISA DNA Amplification/ELISA 103 CFU/mL

Non-amplified nucleic acid hybridization assays DNA:RNA Hybrid 105 CFU/mL

NASBA Isothermal RNA Amplification R&D

TMA Isothermal DNA/RNA Amplification <10 CFU/mL (theor.)

Mycoplasmal Enzymes Bioluminescence 50 CFU/mL

Biochemical Reaction 6-MPDR Mammalian Cell Toxicity 100 CFU/mL

Recombinant Cell Line TLR-2 Activation 103 CFU/mL

Immunobinding Fluorescent Antibody Not Documented

Immunoblotting (Western Blot) Protein Detection R&D

Immunoperoxidase Immunohistochemical Stain R&D

FACS Immunofluorescence R&D

Microarray Oligonucleotide Genotyping 10 CFU/mL

Preliminary Risk Assessment

0 1 2

All Species 1 >1 All

Live Only N/A Live/Dead Live Only

Bacterial X-Reactivity Yes N/A No

103 CFU/mL 10

2 CFU/mL 10

0 CFU/mL

N/A Manual Instrument

2+ Days Overnight Same Day

N/A No Yes

Live Controls N/A Dead Controls

No Not Recommended Yes

N/A Undefined Yes

Unweighted Product

ParameterRating

Lot Release Method

Commercially Available

No Live Mycoplasma Controls

Antibiotics OK

Sensitivity

Specificity

Ease of Use

Regulatory Acceptance

Score

• Reviewed the literature for 20 commercially available Mycoplasma tests

• Ranked Critical Risk Attributes

− 0 = Unacceptable

− 1 = May be acceptable

− 2 = Acceptable

• Scored each test based on the product of the rankings

• Selected 3 tests with the highest scores for evaluation at our facility

Kit Evaluation

qPCR Kit 1 qPCR Kit 2 qPCR Kit 3

Negative Negative Negative

Positive Mixed Mixed

Wide Range Wide Range 8

None Detected S. pyogenes N/A

4 copies

(0.004 µL PC)0.016 µL PC

50 copies

(0.25 µL PC)

8 copies

(0.008 µL PC)0.03 µL PC

50 copies

(0.25 µL PC)

Optimized None <5% Recovery

> 1 month > 1 month < 1 month

Least Most N/A

Yes Yes No

TBD 2 Wk - 2+ Mo 1 Day

None Difficult NoneLogistics

Criteria

Kit Stability

Complexity

Optimized for Various Instruments

Delivery

Specificity

Sensitivity

Other

Minimum Reproduced

Sample Preparation

Un-spiked Samples

PC-spiked Samples

Organisms Detected

Minimum Detected

Cross-reactivity

• Selectivity

− False Positives (Un-spiked samples)

− False Negatives (Positive Control-spiked samples)

− Cross-Reactivity (Bacteria-spiked samples: S. pyogenes, C. sporogenes, L. acidophilus)

• Sensitivity

− Serial Dilutions of Positive Control

Failure Mode and Effects Analysis (FMEA)

Frequency Detectability Severity

Failure

Cause

Failure

Mode

Failure

Effect

Refe

rs to

Risk Priority Number (F x D x S = RPN)

xx

Refe

rs to

Refe

rs to

Design FMEA

• Mitigation Activities

− Choose appropriate sample

configuration.

− Optimize sample preparation for

recovery.

− Qualify kit vendor.

Process FMEA

• Mitigation Activities

− Develop clear SOP’s to address

reagent preparation, sample

handling, and verification of

instrument settings.

− Provide molecular biology

training.

− Obtain feedback from regulatory

agencies on validation plan (prior

to validation).

Regulations and Compendia

• Accepted Methodology

− 21 CFR §610.30

− US FDA’s Points to Consider in the Characterization of Cell Lines Used to

Produce Biologicals

− USP <63>*

− EP 2.6.7*

− JP XV General Information Chapter 14*

• Validation Guidance

− EP 2.6.7

− USP <1223>

− EP 5.1.6

− 21 CFR §610.9

− JP XV General Notice 13

* NAT are an option when properly validated

Validation Parameters

Validation

Parameter

Need to Address

for Qualitative Test

Accuracy No

Precision No

Specificity Yes

Detection limit Yes

Quantification limit No

Linearity No

Operational range No

Robustness Yes

Repeatability Yes

Ruggedness Yes

Specificity

• Range of microorganisms potentially present in the test article

− Broad range of Mycoplasma species

− Closely Related Bacteria

• Target nucleic acid detected in the presence of components expected to be present

− Host Cell DNA

− Sample Matrix

• Acceptance Criteria

− Un-spiked samples negative

− Spiked samples positive

− No cross-reactivity

Limit of Detection

• Lowest number of microorganisms in a sample detected

under the stated experimental conditions

• Lowest amount of target nucleic acid in a sample

detected but not necessarily quantitated as an exact

value

• Acceptance Criteria

− 10 CFU/mL or below

to replace culture method

− Equivalent Nucleic Acid

Copy Number

http://commons.wikimedia.org/wiki/User:Kkmurray

Precision

• Precision – agreement among individual test results for multiple samplings from a homogeneous sample

− Inter-day

− Inter-lab

• Repeatability – agreement among individual test results for multiple samplings within a laboratory over a short time period with the same analyst and equipment

− Intra-day

− Intra-lab

• Acceptance Criteria

− Qualitative Test

− Spiked Samples positive

− Un-spiked Samples Negative

− Quantitative Test

− Summary Statistics for Spiked Sample Results

− Un-spiked Samples Negative

Ruggedness

• Precision of test results obtained by analysis using the

same samples under a variety of normal test conditions

• Different

− Analysts

− Instruments

− Reagent Lots

− Laboratories

− etc.

• Acceptance Criteria

− Same as Precision

Equivalence

• Comparability study demonstrating equivalence required

to replace an official method with an alternative method

• Validation

− Perform the official method and the alternate method in parallel

using the same validation samples

− Compare validation results from the alternate method to previous

validation results from the official method

• Condition of Approval

− Analysis of routine test samples using the official method and the

alternate method run in parallel

• Acceptance Criteria

− Alternate method is equivalent or better

Sample Preparation

Sample PK Treatment, Lysis, and Purification: 150 min

PCR

compatible

NA solution

Lysis

Magnetic

Particles

+

Binding

Solution

Purification

steps

Magnetic

Separation

Magnetic

Separation

Elution

Load Plate: 30 min

PCR: 150 min

Prep: 3½-4 hr

Analysis: 2½ hr

Total: 6-6 ½ hr

Sample Preparation – Automated

Real-Time PCR Detection Positive Test Result

Tm

75-81°C

Ct

36

Real-Time PCR Detection Negative Test Result

No Tm

75-81°C

Ct

> 36

Automate Express Preliminary Results

Extraction Control

M. arginini

M. fermentans

M. hyorhinis

References

• Duguid J. Top Ten Validation Considerations when Implementing a Rapid Mycoplasma Test. Am. Pharm. Rev. 2010; 13:26-31.

• Test for Mycoplasma. In: Code of Federal Regulations. Title 21 §610.30; 2009.

• Recommended Procedures for Detection of Mycoplasma Contamination in Biological Products Produced in Cell Substrates. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals. Rockville, MD: The United States Food and Drug Administration Center for Biologics Evaluation and Research; 2003.

• <63> Mycoplasma Tests. In: United States Pharmacopeia. 33rd ed. Rockville, MD: The United States Pharmacopeial Convention, Inc.; 2010.

• 2.6.7. Mycoplasmas. In: European Pharmacopoeia. 6th ed. Strasbourg, FR: European Directorate for the Quality of Medicines; 2010.

• Mycoplasma Testing for Cell Substrates used for the Production of Biotechnological/Biological Products. In: Japanese Pharmacopoeia. 15th ed. Tokyo, JP: Ministry of Health, Labour and Welfare; 2006.

• <1223> Validation of Alternative Microbiological Methods. In: United States Pharmacopeia. 32nd ed. Rockville, MD: The United States Pharmacopeial Convention, Inc.; 2009.

• 5.1.6. Alternative Methods for Control of Microbiological Quality. In: European Pharmacopoeia. 6th ed. Strasbourg, FR: European Directorate for the Quality of Medicines; 2010.

• Equivalent methods and processes. In: Code of Federal Regulations. Title 21 §610.9; 2009.

• General Notices. In: Japanese Pharmacopoeia. 15th ed. Tokyo, JP: Ministry of Health, Labour and Welfare; 2006.

• <1225> Validation of Compendial Procedures. In: United States Pharmacopeia. 32nd ed. Rockville, MD: The United States Pharmacopeial Convention, Inc.; 2009.

• Chambers D, Kelly G, Limentani G, et al. Analytical Method Equivalency: An Acceptable Analytical Practice. Pharm. Technol. 2005; 29:64-80.

• Duguid J, Kielpinski G, Seymour B, du Moulin GC. Risk Assessment for a Rapid Mycoplasma Test Optimized for Cell Therapy Products. Am. Pharm. Rev. 2009; 12:100-104.

• Windsor H, Windsor D. NAT vs Microbial Culture: Comparing Chalk with Cheese? Gaithersburg, MD: FDA/CBER - Public Workshop: Rapid Methods for Detecting Mycoplasma Contamination in the Manufacture of Vaccines, Including Pandemic Influenza Vaccines, and Other Biological Products; 2008.

• Technical Report No. 33: Evaluation, Validation and Implementation of New Microbiological Testing Methods. PDA J. Pharm. Sci. Technol. 2000; 54:23.

• Bacterial Pathogens. Mycosafe Diagnostics GmbH Web site. 2008. Available at: http://www.mycosafe.at/mycosafe/mycosafe.nsf/alldocs/7C21DDE60B3859B3C125700E00576B02?OpenDocument. Accessed August 5, 2008.

• FAQ. Bionique® Testing Laboratories, Inc. Web site. 2008. Available at: http://www.bionique.com/. Accessed August 5, 2008.

• Q9. Quality Risk Management. ICH Harmonised Tripartite Guideline. Current Step 4 version. November 9, 2005.

Thank you for your attention!

Questions?

john.duguid@genzyme.com

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