Current Trends and Strategies in

Analytical Method Validation, applied to Biologicals and Cell Therapy products

R. JansMobile 0492 593602Email ralph.jans@businessdecision.com

Sint-Lambertusstraat 141 Rue Saint-Lambert1200 Brussels

www.businessdecision-lifesciences.com

Agenda

1 Analytical Method Validation

2 Biologicals

3 Cell therapy products

4 Strategies & examples

5 Conclusions

Analytical Method Validation for Beginners

• Validation ?– GMP: Validation is the documented act of demonstrating that a

procedure, process, and activity will consistently lead to the expected results.

• Validation of analytical methods for QC?– Validation of an analytical method is the process by which it is

established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application

• Main Message: Method Validation ...– ... for ‘classical’ pharmaceutical compounds = ‘easy’ & straightforward

• chemistry-based

– ... for biologicals = scientific creativity required, constant exchange of ideas with authorities• biochemistry- and biology-based

– ... for cell therapy products = biologicals3

A handbook for method validation?

Analytical Method Validation for Beginners

• For a qualitative assay:– test result = pass or fail, no quantitation– validate specificity and detection limit

• The “10 commandments” for a quantitative assay:1. specificity2. linearity3. accuracy4. precision5. range6. detection limit7. quantitation limit8. robustness9. stability-indicating10.system suitability test

Biologicals & Cell therapy products

• Biologicals– washing powder– typically pharmaceutical products• of biological origin

– vaccines» live or fixed viruses or bacteria (rota,

polio, hepatitis A, whole cell pertussis, ...)

• bio-engineered– vaccines

» virus-specific proteins produced by genetically engineered bacteria (HBsAg)

» virus- or bacteria-specific polysaccharides produced by genetically engineered bacteria

– humanized antibodies» anti-psoriatics, Enbrel or Remicade

(anti-TNFa)

Cell therapy products

• Cellular and Gene Therapy products (FDA)• Advanced Therapies Medicinal Products (EMA)

• Pharmaceutical product = Cells– Haematopoietic stem cells for restoring bone marrow function after

cancer treatment – minimally manipulated– Regenerative Medicine• Stem Cells, autologous• Stem Cells, allogeneic

– Anti-cancer cell therapy• Allogeneic Lymphocytes• Allogeneic Dendritic cells

Methods required for QC release of biologicals & cell therapy products

• Microbiological Testing– straightforward

• Identity• Purity / Content• Potency• Stability

– uses ID/content and potency assays

Ref: FDA Guidance for Industry: Content and Format of Chemistry, Manufacturing and Controls Information and Establishment Description Information for a Vaccine or Related Product (1999)

Ref: FDA Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy (1998)

Ref: Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs) (2008)Ref: FDA Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products (2011)

Example: validation of ID/Content ELISA assay for vaccine X

• ELISA– specificity (matrix + antigen)– linearity-precision-accuracy-range:• 2 ops, n=2, 4 days-> n=16 par level• 8 levels of validation standard, 50%-150% of specification: diluted FC,

FC, FC+spikes with standard, spike only• data n=128• ANOVA

– robustness– stability-indicating

Differences in QC release testing methods for biologicals and cell therapy products

Test Type Biologicals Cell therapy product

Identity(What is it?)

Physico-chemical and biochemical methods: NMR, HPLC, ELISA,

Western blot, ...

Biomarkers by biological methods: flow cytometry, qPCR, ...

Purity(How pure is it?)

HPLC, ...

Non-cell impurities: Physico-chemical and biochemical

methodsCell population purity: Biomarkers by

biological methods

Potency(How efficacious

is it?)

In vivo or surrogate biological or biochemical methods (content)

In vivo or surrogate biological or biochemical methods

Challenges for identity/content method validation, biologicals vs.cell therapy products: WHAT IS IT?

• biologicals, for example protein vaccine– identity/content by ELISA– specific GMP antibodies– GMP conform reagents– GMP reference standard– straightforward validation

• cell therapy products, for example MSC-type product– identity by multicolor Flow Cytometry– limit-type assay– desired cell type = MSC primed for specific differentiation pathway– biomarkers? – antibodies/reagents? (GMP)– validation of a flow cytometry method?

The nemesis of validating QC release methods for cell therapy products

Challenges for purity testing, biologicals vs.cell therapy products

• biologicals, for example protein vaccine– impurity dosage by physico-

chemical testing– quantitative assays– GMP conform reagents– GMP reference standard– straightforward validation

• cell therapy products, for example MSC-type product– purity by qPCR– limit-type assay– undesired cell types = mesenchymal stem cells differentiating towards

bone, fat or cartilage– biomarkers? – qPCR primers?– validation of a qPCR method?

Challenges for potency testing, biologicals vs.cell therapy products

• biologicals, for example protein vaccine– potency testing in vivo

• inject vaccine in vivo anddose serological response using ELISA

• « quantitative « assay• variability = nightmare

– potency testing in vitro• ELISA• relatively straightforward validation

• cell therapy products, for example MSC-type– desired effect: tissue regeneration – potency testing in vivo

• technically not feasible for autologous batches

– potency testing in vitro• surrogate markers for biological activity - bioassays

– engraftment and differentiation into functional tissue– paracrine secretion of regenerative proteins

Conclusions

• QC method validation for biologicals & cell therapy products– evolving field – requiring scientific creativity– involves latest technology– constant exchange of ideas with regulatory

authorities