Bioassays Through the Product

Lifecycle : Perspectives of
CDER and CBER Reviewers
Chana Fuchs, Ph.D.
Division of Monoclonal Antibodies
Office of Biotechnology Products
Center for Drug Evaluation and Research
U.S. Food and Drug Administration
Deborah Hursh, Ph.D.
Division of Cell and Gene Therapy
Office of Cellular, Tissue and Gene Therapies
Center for Biologics Evaluation and Research
U.S. Food and Drug Administration

Disclaimer: some of the views expressed in this

presentation are my own and may not necessarily
represent FDA policy.

Bioassays 2009: Scientific Approaches & Regulatory Strategies

Lister Hill, Bethesda, MD
Nov. 2, 2009
 Potency – The Regulations
“The word potency is interpreted to mean the
specific ability or capacity of the product (...) to
effect a given result.”
result 21 CFR 600.3(s)

“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 in a manner adequate to
satisfy the interpretation of potency given by
the definition in § 600.3 of this chapter.”
21 CFR 610.10
 Potency – ICH Q6B
“…for complex molecules, the physicochemical
information may be extensive but unable to confirm the
higher-order structure which, however, can be inferred
from the biological activity”
activity
“The measure of the biological activity using a suitably
quantitative biological assay (also called a potency
assay or bioassay), based on the attribute of the
product which is linked to the relevant biological
properties. (Glossary)”

“Other procedures such as ligand and receptor binding

assays may be acceptable”
So a well designed bioassay will reflect the mechanism
off action
ti off the
th prod
d ctt
 Application of a Bioassay
• Indicates biological activities specific/ relevant
to the product (mechanism of action) (“…to
effect a given result”)
• Clinical correlate (if possible)
• Product release (“…to confirm higher order
structure”/activity)
• Stability program
• Manufacturing changes – Comparability
• QbD – one would want an appropriate bioassay to
allow better understanding of changes as one moves
within the design space.
 Types of potency assays:
•Binding assays
•Bioassays
–Animal based
–Organ or tissue based
–Cell culture based
•Early response (signaling pathway)
•Late response (proliferation, cytokines)
•Multiple cell types (cell-cell adhesion)
–Biochemical
•Enzyme activity assays
CDER Products regulated under BLA
• Division of Monoclonal Antibodies
– Monoclonal Antibodies
– Monoclonal Antibody Fragments e.g. Fab, sFv, diabodies
– mAb conjugates: radionuclide, drug, toxin, bispecific
– Fc Fusion proteins

• Division of Therapeutic Proteins

– Enzymes
– Cytokines
– Growth factors
– toxins

Wide range of biological products necessitates

specific considerations of requirements for each.
One size does not fit all.
Having said that…
Timeline for Bioassay development: Phase I
• A potency assay is needed for phase 1
• Ideally based on intended mechanism of action of drug
product but…
– many monoclonal antibodies do not require a functional
bioassay for phase 1 (can use a binding assay), but many
recombinant proteins or antibody-drug conjugates may
require a bioassay.
– Can use multiple assays for protein activity(s) characterization
• Bioassay requirements depend on protein product,
indication, therapeutic window, etc. Potency assay for
products with a narrow therapeutic window, e.g.
toxins, may require a validated bioassay for phase 1.
• There should be provisional preliminary specification
established for the potency assay on release and
stability. Depends on therapeutic index and dosing.
Timeline for Bioassay Development
• 21 CFR 312.22: For Phase 2 and 3,
FDA’s primary objectives include:
“to help assure the quality of the
scientific evaluation of drugs is
adequate to permit an evaluation of the
drug’s effectiveness and safety”.
safety
• Therefore product characterization
assays (e.g. potency) should be
“adequately qualified”.
 Timeline for Bioassay Development
Phase 2:
• Potency assay(s) further developed. Does your bioassay relate to what is
being done in the clinic?
• Bioassay may be added when binding assay was used for phase I
potency.
•Additional data or assay issues may impact on assays one uses, e.g. It
may not be feasible to develop a single, quantitative potency assay for a
product so it may be necessary to develop a correlation between one or more
analytical assays and biological activity.
• Refine acceptance criteria
Phase 3:
•Bioassay may be changed to a better one. Achieve appropriate process
control/consistency
•Assay is well-characterized
•Specifications tightened and justified
• (Nearly) validated
Timeline for Bioassay development: Phase
2/3
• Often, a new assay may be developed and transitioned
in during this time period
• More than one potency assay may be required as the
mechanism of action is further defined. consider using
multiple bioassays early in development for better understanding of
product attributes and MoA;
• Potency assays can be stability-indicating (ideal but not
always possible)

• When planning manufacturing changes or scale-up

programs, product comparability may have to be
demonstrated. A well developed potency assay is very
useful at such times.
 Timeline for Bioassay development: Post-
approval

• Change may be necessary!

– Reagents are no longer available
– Science (knowledge of the product) and technology
(assay capabilities) have significantly progressed.
– Market demand is so much better than expected that
one needs a more QC friendly assay (e.g. one that
can be easily transferred to many manufacturing
sites).

• Change is possible!!!
– Even after the BLA has been approved.
 Changing Potency Assays
• The degree of data needed to support a change will
depend upon
– The development stage of the product
– The type(s) of assay(s) being exchanged

• A new assay should have a justifiable advantage

over the old assay, e.g.
– Stability indicating
– Product variants
– Degradation products
– Accuracy, precision, sensitivity, etc.

• Overlapping data using both assays is required to

support assay change and link between assays, non-
clinical and clinical data.
 CBER Products: Biologics
• OVRR
– Preventive vaccines
– Therapeutic vaccines for infectious disease indications (i.e. HIV)
– Toxins & allergenic products
• OBRR
– Blood Products, (i.e. whole blood, and testing kits)
– Blood-Related Products (Antibodies, coagulation factors, hemostatic
agents)
• OCTGT
– Gene Therapy Products
– Cellular Therapy Products
– Therapeutic Vaccines (Cancer, Alzheimer’s)
– Tissues, Tissue Engineered Products

Complex biological products raise

challenges in bioassay design
 Potency Assay Attributes for
Licensed Biological Products
• Indicate biological activity (s) specific/relevant to
the product
• Results available for lot release
• Provide quantitative readout
• Meet predefined acceptance and/or rejection
criteria (demonstrate lot to lot consistency)
• Include appropriate reference material/controls
• Measure activity of all components deemed
necessary for activity
• Indicate product stability
• Validated for licensure
 A Challenge For Complex Products
• Complex mechanism of action
– e.g. Multiple steps involved in vector transduction
• Multiple active components with multiple activities
– e.g. multiple cell types, vector types, multiple gene
products
– Potential for interference or synergy
• Product variability due to variability in starting cells or
tissue
– e.g. patient specific tumor vaccine
• Limited material to test
– e.g. patient specific cellular therapy
• Product stability
– Many products administered within hours of harvest
– Storage/holding may effect viability, potency, etc.
Approaches to measure potency of cell
and gene therapy products.
• Direct measure of biological activity (bioassays)
– Biological assay methods: unique and specific product characteristics
• In vivo:
– Animal models
» e.g. structural repair, gene function, immune response and/or protection, cell survival
• In vitro:
– Cell & tissue culture
» e.g. signaling pathways, proliferation, immunogenicity, enzymatic

• Indirect measure of biological activity

– Analytical assay methods: non-bioassay method directly correlated to a unique and
specific activity of the product
• Immunochemical Procedures
– e.g. ELISA, ELISPOT, Q-flow cytometry, quantitative western blots
• Molecular and Biochemical Procedure
– e.g. Q-PCR, RT-PCR, microarray/genomics, proteomics
– Activity

What is necessary depends on the product and

indication: case-by-case
Another approach : Assay Matrix
It may not be possible or feasible to develop a single
assay that encompasses all elements of an
acceptable potency assay:
• Limited knowledge of product and mechanism of action
• Product has multiple components with multiple biological
activities
• Time constraints due to limited product stability (e.g. cellular
therapy)
• Biological assay is not quantitative
‹ Multiple Assay Approach (Assay Matrix)
‹ Combination of assays where the combined results constitute an
acceptable potency assay
‹e.g. a quantitative physical assay along with a qualitative
bioassay
Assay Development Timeline: Potency
CLINICAL DEVELOPMENT
Preclinical BLA
Develop- Submission
ment
Phase 1 Phase 2 Phase 3

Product characterization, investigate

mechanism of action to justify critical
biological activity as target of potency Validated
assay development. Potency
Assay
Potency assay (s) development,
refinement and qualification.
Potency assay in place, ongoing
validation studies.
 Progressive Assay Implementation

• During preclinical and early clinical development:

– Characterize biological activity of the product
• Wide variances and acceptance criteria
• One assay may not capture all critical attributes
• Determination of product consistency
• Phase II:
– Potency assay further developed with relationship to
biological activity
– As product development proceeds -- assays evolve:
• Assays added, deleted, refined
• Acceptance criteria modified as needed
 Examples
• Cytokine-producing viral vector
– Viral titer (genomes/particles and infectious)
– ELISA: measure cytokine quantitatively relative to titer
• Functional activity by Phase 3 investigation
– e.g. Cell proliferation assay

• T-cell product: Tumor Infiltrating Lymphocytes (TIL)

– Potential Potency Assay Matrix:
• Viable cell number
• Phenotype characterization (e.g. Flow cytometry)
• Tumor specific cytotoxicity assay
– Tumor specific cytotoxicity assay. -- necessary to measure
the relevant anti-tumor activity in a heterogeneous
population of T cells.
 Progressive Assay Implementation
• By Phase III
– Assay well-characterized
– Acceptance criteria should be defined and justified
– Generally fully qualified, validation on-going

• For BLA: validated potency assay

– Used for release of DS/DP and drug product and
stability. May be used for in process testing.
 Link between Assay Development
and Product Development
• Assay development should at a minimum keep pace
with product development
• Generate data to inform manufacturing process
• Generate data to inform pre-clinical testing
• Generate data to design clinical studies

• Progressive assay refinement

– Start Potency Assay Development Early!
• Recognize challenges to meeting requirements
• Evaluate more than one assay
• Collect correlation data
• A well characterized product is important when
interpreting clinical data!
 Summary Points
• Regarding bioassay development, CBER and CDER
perspectives are comparable.
• Regulations on potency are the same
• Bioassay should reflect the biological activity (s)
specific/relevant to the product.
• Progressive assay development and implementation is
common
• Assay Matrix - Combination of assays when:
– MoA is too complex to run a single bioassay
– Multiple activities of the product call for multiple assays
(e.g. IFN-alpha has both anti-proliferative and anti-viral activities)
• Any differences reflect the heterogeneity of the products