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
2
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
4
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
5
• 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
6
Select Bioassay Based on mAb Design
7
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
8
• 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)
9
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 variabilityThroughput 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)
10
• Non Cell-Based Ligand Binding Assay (ELISA)– Target antigen is receptor on Cell A
– Qualified method
ELISA
Release/Stability Comparability Forced DegradationProduct/Process
Development
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
11
• 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
12
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
13
• 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
14
Develop and Qualify Cell-Based Assay(Phase 2)
15
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
16
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
17
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
18
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
19
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)
20
Further evaluate and compare
Prepare for Validation
Cell A Cell A Cell A Cell A Cell A
Cell Based Assay
B B B B
21
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
22
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
23
• 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
24
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
25
26
Acknowledgements
Richard CornellDavid CirelliSara HanscomJennifer SmithDenise KwokAparna DeoraRichard JeromeNed Mozier
Case Study: A Phase-Driven Approach to the Development and Lifecycle Management of Potency AssaysTalk OutlineWhat is Potency?The Why and How of Potency Assays?A “Phase-Driven” Approach to Bioassay Lifecycle ManagementBioassay Strategy is Guided by mAb DesignSelect Bioassay Based on mAb DesignCase Study: Evolution of a BioassayConsiderations for Early Stage Bioassay SelectionEarly Stage Bioassay Strategy �(Pre-Clinical Through Phase II)Bioassay Drives Product Knowledge and Understanding of Method CapabilitiesELISA vs. Cell-Based ELISA: Forced Degradation StudySummary Comparison of ELISA vs. Cell-Based ELISALater Stage Bioassay Development�(Pivotal Study to Commercial) Develop and Qualify Cell-Based Assay�(Phase 2)Introduce Cell-Based Potency Assay Early as Characterization AssayELISA vs Cell-Based: On-going Clinical Stability (FIO)ELISA vs. Cell Based: Differences detected at 40°CCell-Based vs. ELISA: Forced Degradation Studies Compare Stability-Indicating Potential of BioassaysData Driven Bioassay Strategy �(Pivotal Study to Commercial)ELISA vs Cell-Based: Phase 3 Clinical Stability (DS)ELISA vs. Cell-Based: Phase 3 Clinical Stability (DP)Data Driven Bioassay Strategy: CommercialTake-Away MessagesTake-Away Messages Continued..Acknowledgements
