Case Study: A Phase-Driven Approach to the Development and Lifecycle Management of Potency Assays. Spring in New England!!!

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1 Case Study: A Phase-Driven Approach to the Development and Lifecycle Management of Potency Assays CASSS Bioassays 2016: Scientific Approaches & Regulatory Strategies Session Potency Assays: Cell-based versus Non Cell-based Formats KATHLEEN SHIELDS, ANALYTICAL R&D, BIOTHERAPEUTICS PHARM. SCI., PFIZER INC., ANDOVER, MA. Spring in New England!!!

2 Talk Outline Potency: The What, Why and How of Potency Testing Choosing a Fit-for-Purpose Bioassay Case Study: Evolution of a Bioassay (Phase I to Commercial) Take-Away Messages 2

3 What is Potency? Definition of Potency (21 CFR 600.3) The word potency is interpreted to mean the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result. Guidance (ICHQ6B) A valid biological assay to measure the biological activity should be provided by the manufacturer. Examples can include: Animal-based biological assays, which measure an organism's biological response to the product Cell culture-based biological assays, which measure biochemical or physiological response at the cellular level Biochemical assays, which measure biological activities such as enzymatic reaction rates or biological responses induced by immunological interactions Other procedures such as ligand and receptor binding assays 3

4 The Why and How of Potency Assays? Why is Potency Required? Size and complexity of large bio-molecules necessitate assays beyond traditional physicochemical techniques Some changes (chemical or physical) may/or may not impact potency Changes in tertiary/quaternary structure are sometimes difficult to detect with other assays Complex (often multiple) mechanism of action can only be evaluated using well-designed potency assay How is Potency/Bioassay Data Used? Pre-clinical (Guides design and development of candidate drugs) Clinical to Commercial (Increase product and process knowledge) Structure/function studies (identification of critical quality attributes) Formulation development (guides stability/storage decisions) Manufacturing process (clipping, N-Glycans etc) Release/Stability (specification), lot-to-lot comparability 4

5 A Phase-Driven Approach to Bioassay Lifecycle Management Multiple bioassays are used throughout the product development life-cycle Bioassay strategy is continually evolving and driven by increased product knowledge and critical method performance evaluation 5

6 Bioassay Strategy is Guided by mab Design Target Binding (All mabs) ELISA SPR Functional Cell-based Assays MOA(s) 14 methods may be applied based on MOA Functional Cell-based Assays MOA(s) Fc Effector Functions SPR FcγR C1q Functional Assays ADCC CDC ADCP FcRn 6

7 Select Bioassay Based on mab Design Bioassay Selection Strategy Primary MOA 1 Description ELISA (FIH to P2) Introduce Cell Based Early (When developed) Later (P2) Bind and Block (Antagonist) Binds to receptor on cell surface and blocks binding to its cognate ligand Bind and Signal (Agonist) Binds to receptor on cell surface and transduces an intracellular signal Ligand Trap Binds to soluble ligand and prevents (traps) it from binding to cognate receptor on cell surface 1. Assumes low/no effector function (mabs with effector function require early introduction of Cell-based assay) 7

8 Case Study: Evolution of a Bioassay Mechanism of Action (MOA) Binds to receptor on surface of cell A and blocks binding of co-receptor on surface of cell B thereby preventing cell-cell interaction mab Cell B mab Design: IgG2 (low/no effector function) Cell A Cell B Cell B MOA Early Stage Bioassay Selection (Pre-FIH) Early engagement of Research Unit Review bioassays used for clinical candidate selection and delineation of MOA Evaluate bioassay readiness (Phase 1 enabling) 8

9 9 Considerations for Early Stage Bioassay Selection Assay Parameter Non Cell-Based (ELISA) Cell-Based Development Time 1-3 months months Reagents Assay Performance (Target) Robustness Readily available (often commercial) Require time to develop (eg. engineered cell lines) Precision < 5% Precision < 10% Less variable/easily transferable Requires more time to optimize/control variability Throughput Single plate assay Often multi-plate assay Reflects Drugs MOA Stability Indicating Fab binding (essential MOA but may not be complete) Fab binding to antigen (may be influenced by assay design) Likely more reflective of complete MOA Evaluates entire molecule Regulatory expectations for registration Non Cell- Based Cell-Based

10 Early Stage Bioassay Strategy (Pre-Clinical Through Phase II) Non Cell-Based Ligand Binding Assay (ELISA) Target antigen is receptor on Cell A Qualified method Cell B mab Cell A MOA Cell B Cell B ELISA Characterization Assays Surface Plasmon Resonance (SPR) Affinity and Kinetics (ligand binding, FcRn binding) Cell-based ELISA Non-qualified method B B B Cell-based Competitive ELISA Release/Stability Comparability Forced Degradation Product/Process Development ELISA Characterization FIO 10

11 Bioassay Drives Product Knowledge and Understanding of Method Capabilities Sample Testing Development Structure-Function studies: identify potential quality attributes impacting potency Increase process understanding Forced degradation Guide formulation development Relevant degradation pathways that impact potency Early evaluation stability-indicating capabilities of bioassay Clinical Stability DS/DP Final formulation/presentation (most relevant) Makes bioassay suitability/selection a data-driven decision 11

12 ELISA vs. Cell-Based ELISA: Forced Degradation Study Forced Degradation Method ELISA Cell-Based ELISA Control Acidic = 18% Deamidation (ph 9.0, 37ºC, 4 days) 82% 92% Oxidation (H 2 O min) 70% 73% Thermal Stress Absorbance ice 50ºC ELISA Cell-Based ELISA Deamidated, Acidic = 71% pi Non bio-equivalent Non bio-equivalent ELISA and Cell-Based ELISA Results are Comparable Deamidation does not impact to potency Oxidation (100%) marginally impacts potency Thermal stress significant loss of potency at 16 weeks Cell-based ELISA detects changes resulting from thermal stress earlier than ELISA 12

13 Summary Comparison of ELISA vs. Cell-Based ELISA Antibody fairly stable Clinical stability shows no significant change in potency at -20ºC, 5ºC and 25ºC for over one year. Required stressed conditions to see differences in activity 50ºC for 4 months Cell-based ELISA more sensitive to changes Advancements in mab design and formulation increase stability and make assessment of bioassay stability-indicating capabilities more challenging 13

14 Later Stage Bioassay Development (Pivotal Study to Commercial) Regulatory Expectation: Potency assay reflective of drugs MOA is required before pivotal clinical trial Links potency assay to clinical efficacy Data driven bioassay design Stability indicating 14

15 15 Develop and Qualify Cell-Based Assay (Phase 2) Design Mimics MOA mab Cell B B B B Cell A Cell A Cell A Cell A Cell A Cell Based Assay B Cell B Cell A MOA Cell B Assay Performance Parameter ELISA 1 Cell-Based 2 Precision (historical) 4.9% (n = 123) 9.2%(n = 210) Accuracy (historical) 100% 97% Linearity at Range (50-150%), r ELISA is single plate assay (failure rate < 2%) 2. Cell-Based is two plate assay (failure rate ~10-20%)

16 Introduce Cell-Based Potency Assay Early as Characterization Assay Replace cell-based ELISA with more mechanistically relevant cell-based assay Increased biological relevance of assay format Include cell-based assay in on-going stability studies (FIO) Trend data and compare results with non cell-based (ELISA) Begin testing forced degradation samples Early assessment of stability-indicating capabilities Comparability testing Process change Testing of retains from early clinical development 16

17 ELISA vs Cell-Based: On-going Clinical Stability (FIO) Condition = 5ºC stability programs (2x DS/2x DP) 48 Months ELISA and Cell Based comparable No drop in potency 5ºC 70 ELISA Cell-Based 130 Condition = 25ºC 2 stability programs (1x DS/1x DP) 6 Months ELISA and Cell Based comparable No drop in potency 25ºC 70 ELISA Cell-Based 17

18 ELISA vs. Cell Based: Differences detected at 40 C DS 6 months at 40 C/75%RH No change in potency Non-bioequivalent ELISA shows no decrease in potency at 40ºC over 6 months Lacks stability-indicating properties Cell-based assay at 40ºC for 6 months is non-bioequivalent Demonstrates stability-indicating properties 18

19 Cell-Based vs. ELISA: Forced Degradation Studies Compare Stability-Indicating Potential of Bioassays Forced degradation studies Photostability Potency: ELISA vs Cell-Based Forced deamidation Oxidation Thermal Stress - only condition that impacts potency Analytical Methods Charge heterogeneity (ice) Aggregates (SEC-HPLC) Oxidation (RP-HPLC) Fragments (r/nr CGE) Potency (Bioassay) Bioassay is not a stand alone analytical tool All methods show evidence of product degradation after 8 40ºC except the ELISA ELISA is not a stability-indicating method 19

20 20 Data Driven Bioassay Strategy (Pivotal Study to Commercial) Introduce Cell Based Functional Assay Reflects drug MOA (Clinical data supports MOA) Is stability-indicating Two Potency Assays for Release/Stability (Bridge assays) ELISA Cell Based Increase experience with method Robustness (#analysts/labs,etc) Trend data (SPC) Further evaluate and compare Prepare for Validation B B B Cell A Cell A Cell A Cell A Cell A Cell Based Assay B

21 ELISA vs Cell-Based: Phase 3 Clinical Stability (DS) -40 C ELISA -20 C 5 C Cell-Based -40 C -20 C 5 C Summary Potency results are comparable between both assays No loss of potency at storage temps Biochemical methods support DS stability 21

22 ELISA vs. Cell-Based: Phase 3 Clinical Stability (DP) Relative Potency (%) ELISA Months on Stability Months on Stability Relative Potency (%) Cell-Based 5 C 30 C 40 C 5 C 30 C 40 C Summary Stressed samples show difference in potency between ELISA and cell-based assay ELISA shows no change in potency at storage temps Cell-based shows significant decrease in 40 C for 6 months and 30 C Biochemical methods detect DP 40 C for 6 months 22

23 Data Driven Bioassay Strategy: Commercial Potency Assay: Cell-Based Functional Assay One assay for release/stability Meets Regulatory Expectations for Potency Reflects the drug mechanism of action Proven to be stability-indicating B B B B Cell A Cell A Cell A Cell A Cell A Cell Based Assay 23

24 Take-Away Messages Bioassay strategy is an evolutionary process that changes as product knowledge increases and new methods are developed Initial potency assay is often a simple ligand binding assay Guided by mab design and proposed MOA Multiple assays/formats may be required (Jackie Gallant, Poster) Case Study:Application of Pfizer s Bioassay Strategy for a Combination Monoclonal Antibody May or may not reflect the complete MOA of the drug Stability-indicating potential not fully understood Early stage programs rely heavily on orthogonal methods to increase product/process knowledge and ensure product quality/safety 24

25 Take-Away Messages Continued.. Later in development when more is known about the product a functional assay (cell-based) is introduced Method is designed to reflect MOA Selection of potency assay(s) for Commercial release are a data driven decision Reflects the MOA of the drug Is stability-indicating Performance is acceptable/amenable to QC environment 25

26 Acknowledgements Richard Cornell David Cirelli Sara Hanscom Jennifer Smith Denise Kwok Aparna Deora Richard Jerome Ned Mozier 26