The Role of Bioassays in the Determination of Critical Quality Attributes

Transcription

1 The Role of Bioassays in the Determination of Critical Quality Attributes Aparna Deora, Ph.D. Associate Research Fellow Pfizer BioTherapeutics January 25, 2010

2 Outline of Talk What is a Critical Quality Attribute? Bioassays Examples of Bioassays in Determination of Critical Quality Attributes Pre-Phase 1 Phase 1-2 Phase 2-3 Phase 3 and Beyond Limitations of Bioassays New Bioassays and their role in CQAs

3 Disclaimer mab-centric view of potency and QAs Combined examples from multiple projects Overall goal is to provide strengths and weaknesses in bioassay testing approaches for determination of CQAs Will not discuss process for determining criticality scores and factors Will not discuss control strategies for CQAs

4 Quality Attributes Attributes arise from various sources Process-related * Product-related * Contaminants * Excipients Packaging

5 Quality Attributes Not all-inclusive

6 Critical Quality Attributes ICHQ8: A physical, biological or microbiological property or characteristic that should be within appropriate limit, range or distribution to ensure the desired product quality Determination of criticality driven by, Safety Immunogenicity Impurities Efficacy Biological activity PK

7 Determination of CQAs Regulations Literature Prior/platform knowledge from other products in vitro and in vivo assessments of products and purified degradants Clinical experience

8 Determination of CQA s Regulations Literature Prior/platform knowledge from other products in vitro and in vivo assessments of products and purified degradants Clinical experience BIOASSAYS

9 Bioassay Background Required to do bioassay A valid biological assay to measure the biological activity should be provided by the manufacturer. (ICHQ6B) Tests for potency shall consist of either in vitro or in vivo tests, or both, which have been specifically designed for each product so as to indicate its potency. (21 CFR ) Good science Size and complexity of large bio-molecules necessitate assays beyond traditional physicochemical techniques Changes in tertiary/quaternary structure difficult to detect

10 Toolbox for Biological Characterization ELISA Indirect Competition SPR Direct Competition Functional Cell-based Assays MOA Primary cells Cell lines in vivo assays ELISA Conformational Abs SPR FcγR FcRn Functional Assays Effector Function

11 Pre-Phase 1 Pre-clinical Phase 1 Phase 2 Phase 3 Filing Literature Platform knowledge Establish and qualify potency assay Look for the unusual

12 Literature, Prior/Platform Knowledge and Guidances Safety Bioburden Viral Testing Endotoxin Mycoplasma Residual Process-related Impurities Aggregation Efficacy Potency Concentration Identity Critical Quality Attributes

13 Quality Attributes Not all-inclusive

14 mab X (Pre-Phase 1) First look at product Platform methods A fully human IgG2ĸ molecule run on SEC A Pre-peak elutes as a partially resolved shoulder on the leading edge of the monomer peak. Typical IgG2 SEC Profile mab1 SEC Profile

15 Pre-peak Different from platform/prior knowledge Other physicochemical tests indicate it is not HMMS, clip or break-down (Mass intact) or a major charge variant Change in molecule is unknown. Need to determine if there is a change in biological activity Indirect binding ELISA

16 Isolated pre-peak fraction is subpotent Data by Tom Schomogy

17 Summary An unexpected peak seen by a physicochemical method during preclinical development Isolate peak from production lots and stressed samples and test for impact on biological activity (characterization) Pre-peak has reduced activity (ELISA and SPR) Critical Quality Attribute

18 Phase 1 Phase 2 Pre-clinical Phase 1 Phase 2 Phase 3 Filing Literature Platform knowledge Establish and quality potency assay Tox study for safety Look for the unusual Develop potency assay(s) Release and stability testing Literature and platform knowledge Comparability Look for the unusual

19 Trending Examine Release Data Outlier lots Process deviations Mean + 3xstdev Mean + 2xstdev Grand-Mean Mean Mean - 2xstdev Mean - 3xstdev Jun Jan-04 1-Aug Feb-05 5-Sep Mar Oct Apr Nov-07 1-Jun Dec-08 None Examine Stability data Ensure potency assay is stability-indicating (stressed samples) Correlate stability profiles with loss of potency (e.g., clips)

20 maby FIH Study -Understanding of product profile crucial in understanding safety profile in Tox and Clinical studies -First chance to look at any clinical outcomes of product -See lower than expected in vivo ½ life for an IgG1 mab (maby) - Product profile showed no unusual properties - Glycosylation profiles were normal - Examine other biological properties

21 FcRn Binding FcRn transfers IgG from mother to neonate FcRn expressed on vascular endothelial cells and regulates serum IgG levels in adults Recent studies show FcRn is responsible for maintaining the long plasma half-life of IgG s. Protects Abs from degradation in lysosome. Plays important role in half-life of therapeutic mabs: higher affinities for FcRn slower turnover and higher serum IgG level Nature Biotechnology 23, (2005) Does mab 2 bind FcRn with reduced affinity?

22 FcRn SPR Assay Control IgG1

23 FcRn SPR Assay maby appear to bind to human FcRn with 20X greater affinity compared to assay control IgG1 (400pM vs. 8nM) No significant difference seen between the two maby lots. mab2 Lot 1 mab2 Lot 2 Control IgG1 13nM 13nM 4.4nM 1.5nM 4.4nM 1.5nM 4.4nM 13nM Data by Jim Zobel

24 maby Summary Shorter than expected half-life observed with mab2 Typically decreased FcRn binding affinity correlates with shorter ½ life maby molecules appear to bind to human FcRn with greater affinity maby molecules bind with higher affinity than control IgG1 to human FcRn at neutral ph and has slower off rate Less efficient recycling could decrease ½ life in vivo

25 Summary of Phase 1-2 Phase 1-2 generally is mainly routine testing Track potency data Try to maintain contact with clinical programs Can gain insights into efficacy beyond in vitro potency Complex!

26 Phase 2 Phase 3 Pre-clinical Phase 1 Phase 2 Phase 3 Filing Literature Platform knowledge Establish and qualify potency assay Look for the unusual Develop potency assay(s) Release and stability testing Literature and platform knowledge Comparability Look for the unusual Validate Potency Assay * Release and stability testing Test purified impurities * Biological Characterization * Literature and platform knowledge Comparability Look for the unusual

27 Potency and CQA s Greatest time and investment for Potency assay evaluation, development and validation Greatest time and investment for CQA determination 100 Time and Effort 50 Determination of CQA's Potency Assay Development 0 preclinical Phase 1-2 Phase 2-3 Phase 3 and beyond

28 Quality Attributes Non-CQA s TBD CQA s Not all-inclusive

29 Biological Characterization Characterization of the biological activity of product and impact of quality attributes on activity Go beyond Lot Release assays Multiple Bioassays evaluated Purify key degradants and impurities, and test for potency

30 Toolbox for Biological Characterization ELISA Indirect Competition SPR Direct Competition Functional Cell-based Assays MOA Primary cells Cell lines in vivo assays ELISA Conformational Abs SPR FcγR FcRn Functional Assays Effector Function

31 Strategy and Challenges Test remaining QA s with panel of appropriate assays Which samples tested with which assays Samples can be limiting Planning is key Sample quality (concentration, sterility, etc.) critical for meaningful testing results

32 Bioassay Testing of Quality Attributes for mabz Validated Bioassay 200% Primary Cell Based Cell-based Assay ELISA 150% SPR Kinetic SPR Secondary Receptor 100% 50% 0% Degly Stability-1 Glycated Deamidated Oxidized Data by Kun Zhang

33 Disulfide Isomers and Potency of mabz 150% Pool-3: Enriched IgG2-B Pool-4: Enriched IgG2-A/B 100% Relative Potency, % 50 Pool-2: IgG2-B - acidic Pool-1: species Unknown Pool-5: Enriched IgG2-A 50% 0 A2 A4 A6 F2 A9 A11 B12 B10 B8 B6 B4 B2 C1 C3 C5 C7 C9 C11 D12 D10 D8 D6 D4 D2 E1 E3 E5 E7 E9 E11 F12 F10 F8 F6 F4 F2 G1 G3 G Data by Kun Zhang and Mike Schlittler

34 in vivo Testing Purify or create enriched impurity samples Deamidated, Oxidized, Deglycosylated, etc. Test in animal potency models Look for statistically signifcant changes in potency Variability great Challenge to define what is different

35 Additional Tests (SPR for FcRn Binding) FcRn binding correlates to ½ life Oxidation of Met 255 has impact to binding KD (M) 4.00E E E E E+00 Reference Material deamidated oxidized deglycosylated Data by Xianzhi Zhou

36 Additional Tests (Conformational anti-hu Abs) Use in-house and commercial Abs for conformational differences Source 1 Source 2 Ref Std Lot 08 Fitted outliers T-3 Ref Std Lot 08 Fitted outliers T Concentration (ug/ml) Response (%) Concentration(ug/mL) Response (%) Clone HP6014 Clone HP6002 Data by Richard Jerome

37 Summary Significant work leading up to Phase 3 Evaluation and Validation of Potency Assay Utilization of a broad set of assays (SPR in vivo) to characterize molecule Determination of Potency of Enriched Samples and Isolated fraction Change in Potency CQA (likely) No change requires further evaluation (safety, PK,etc.)

38 Phase 3 and Beyond Pre-clinical Phase 1 Phase 2 Phase 3 Filing Literature Platform knowledge Establish and qualify potency assay Look for the unusual Develop potency assay(s) Release and stability testing Literature and platform knowledge Comparability Look for the unusual Validate Potency Assay * Release and Release and stability testing stability testing Clinical Test purified Experience impurities * Literature and Biological platform Characterization * knowledge Literature and Comparability platform Look for the knowledge unusual Comparability Look for the unusual

39 Phase 3 and beyond Clinical experience significant in Phase 3 CQAs for product defined for filing Use appropriate control strategy for CQAs Continue diligence around change and new potential quality attributes

40 Quality Attributes Non-CQA s CQA s Not all-inclusive

41 Limitations of Potency Assays Potency Assays relate to Biological Activity of molecule (Does the molecule work?) Assay tend to be more variable Require significant time to develop Several assays may be required based on MOA Do miss parameters affecting in vivo efficacy (immunogenicity or PK issues) Potency assays do not provide safety evaluation typically Prior knowledge Tox and Safety studies Clinical experience Development of strong impurity assays

42 New Potency Assays Better assays Decrease variability Increase robustness Robust assays fully reflecting complex MOAs Assays to understand PK and link product quality attributes to ½ life Need models that are better predictors for immunogenicity and link to product quality attributes

43 Acknowledgements Bioassay Group Adita Akbani, Jennifer Butler, Jim DuMontelle, Kim Flecke, Richard Jerome, Ted Kocot, Abigail Lee, Vess Mitaksov, Tom Schomogy, Bruce Thompson, Tammy Thurman, Kun Zhang, and Xianzhi Zhou Pfizer Colleagues Laura Bass, Mike Schlittler, and Jim Zobel Ned Mozier