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!!!

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

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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 assays3

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

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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

The Why and How of Potency Assays?

A “Phase-Driven” Approach to Bioassay Lifecycle Management

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• 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

•Target Binding (All mAbs)•ELISA•SPR

•Functional Cell-based Assays•MOA(s)

•Functional Cell-based Assays•MOA(s)

•Fc Effector Functions•SPR

•FcγR•C1q•Functional Assays

•ADCC•CDC•ADCP

•FcRn≥ 14 methods may be applied based on MOA

Bioassay Strategy is Guided by mAb Design

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Select Bioassay Based on mAb Design

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Primary MOA1 Description

Bioassay Selection Strategy

ELISA(FIH to P2)

Introduce Cell BasedEarly

(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 TrapBinds 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)

Case Study: Evolution of a Bioassay

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• 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 Design: IgG2 (low/no effector function) Cell A

MOA

mAbCell B

Cell B

Cell B

• 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)

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Assay Parameter Non Cell-Based (ELISA) Cell-Based

Development Time 1-3 months 6-12+ months

Reagents Readily available (often commercial) Require time to develop (eg. engineered cell lines)

Assay Performance (Target) Precision < 5% Precision < 10%

Robustness Less variable/easily transferable Requires more time to optimize/control variability

Throughput Single plate assay Often multi-plate assay

Reflects Drugs MOA Fab binding (essential MOA but may not be complete)

Likely more reflective of complete MOA

Stability Indicating Fab binding to antigen (may be influenced by assay design) Evaluates entire molecule

Non Cell- Based

Cell-Based

Regulatory expectations for registration

Considerations for Early Stage Bioassay Selection

Early Stage Bioassay Strategy (Pre-Clinical Through Phase II)

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• Non Cell-Based Ligand Binding Assay (ELISA)– Target antigen is receptor on Cell A

– Qualified method

ELISA

Release/Stability Comparability Forced Degradation

Product/ProcessDevelopment

ELISA

Characterization FIO

• Characterization Assays– Surface Plasmon Resonance (SPR) Affinity and

Kinetics (ligand binding, FcRn binding)

– Cell-based ELISA

• Non-qualified method

Cell-based Competitive ELISA

B B

B

Cell A

MOA

mAb Cell B

Cell B

Cell B

Bioassay Drives Product Knowledge and Understanding of Method Capabilities

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• 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

ELISA vs. Cell-Based ELISA: Forced Degradation Study

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Forced Degradation Method ELISA Cell-BasedELISA

Deamidation(pH 9.0, 37ºC, 4 days) 82% 92%

Oxidation (H2O2 30-120 min) 70% 73%

Control Acidic = 18%

Deamidated, Acidic = 71%

Abso

rban

ce

pI

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

Thermal Stress

Weeks @ 50ºC ELISA Cell-Based ELISA

2 101 96

4 85 83

6 82 68

16 Non bio-equivalent Non bio-equivalent

iCE

Summary Comparison of ELISA vs. Cell-Based ELISA

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• 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

Advancements in mAb design and formulation increase stability and make assessment of bioassay stability-indicating capabilities more challenging

Cell-based ELISA more sensitive to changes

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

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Develop and Qualify Cell-Based Assay(Phase 2)

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Design Mimics MOA

Assay PerformanceParameter ELISA1 Cell-Based2

Precision (historical) 4.9% (n = 123) 9.2%(n = 210)

Accuracy (historical) 100% 97%

Linearity at Range (50-150%), r2 0.992 0.994

1. ELISA is single plate assay (failure rate < 2%)

2. Cell-Based is two plate assay (failure rate ~10-20%)

Cell A Cell A Cell A Cell A Cell A

Cell Based Assay

B B B B

Cell A

MOA

mAb Cell B

Cell B

Cell B

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

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ELISA vs Cell-Based: On-going Clinical Stability (FIO)

• Condition = 5ºC

• 4 stability programs (2x DS/2x DP)

• 48 Months

• ELISA and Cell Based comparable

– No drop in potency

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5ºCELISACell-Based

• Condition = 25ºC

• 2 stability programs (1x DS/1x DP)

• 6 Months

• ELISA and Cell Based comparable

– No drop in potency ELISACell-Based

25ºC

70

130

70

130

ELISA vs. Cell Based: Differences detected at 40°C

• 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

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DS 6 months at 40°C/75%RH

Non-bioequivalent

No change in potency

Cell-Based vs. ELISA: Forced Degradation Studies Compare Stability-Indicating Potential of Bioassays

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Forced degradation studies

• Photostability

• Forced deamidation

• Oxidation

• Thermal Stress - only condition that impacts potency

Potency: ELISA vs Cell-Based

Bioassay is not a stand alone analytical tool

All methods show evidence of product degradation after 8 weeks @ 40ºC except the ELISA

ELISA is not a stability-indicating method

Analytical Methods

• Charge heterogeneity (iCE)

• Aggregates (SEC-HPLC)

• Oxidation (RP-HPLC)

• Fragments (r/nr CGE)

• Potency (Bioassay)

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)

Data Driven Bioassay Strategy (Pivotal Study to Commercial)

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Further evaluate and compare

Prepare for Validation

Cell A Cell A Cell A Cell A Cell A

Cell Based Assay

B B B B

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ELISA vs Cell-Based: Phase 3 Clinical Stability (DS)

ELISA Cell-Based

0 1 3 60 1 3 6 0 1 3 60 1 3 60 1 3 6 0 1 3 6

-40°C -20°C5°C

-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

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ELISA vs. Cell-Based: Phase 3 Clinical Stability (DP)

0

20

40

60

80

100

120

Rela

tive

Pote

ncy

(%)

Months on Stability

0

20

40

60

80

100

120

Rela

tive

Pote

ncy

(%)

Months on Stability

ELISA Cell-Based

0 1 3 60 1 3 6 0 1 3 60 1 3 60 1 3 6 0 1 3 6

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 potency @ 40°C for 6 months and trend @ 30°C

– Biochemical methods detect DP degradation @ 40°C for 6 months

Data Driven Bioassay Strategy: Commercial

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• 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

Cell A Cell A Cell A Cell A Cell A

Cell Based Assay

B B B

B

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)

– 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

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Case Study:Application of Pfizer’s Bioassay Strategy for a Combination Monoclonal Antibody

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

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Acknowledgements

Richard CornellDavid CirelliSara HanscomJennifer SmithDenise KwokAparna DeoraRichard JeromeNed Mozier