Baolin Zhang, Ph.D. Senior Investigator/Product Quality Reviewer

Division of Therapeutic Proteins

Office of Biotechnology Products

Center for Drug Evaluation and Research

CASSS Bioassays 2014: Scientific Approaches & Regulatory Strategies Washington D.C., March 24-25, 2014

Issues to Consider When Developing Potency Assays for Biologic Products

Disclaimers

The views and opinions expressed in this presentation should not be used in place of regulations, published FDA guidances or discussions with the Agency.

Outline

• Regulatory expectations

• Applications

• Issues to consider • Relevance to the mechanism(s) of action (MoA)

• Acceptance limits

• Comparability when making changes

• Case study

Potency (21 CFR 600.3(s))

“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.”

Potency Tests (21 CFR 610.10)

“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(s) of this chapter.”

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”

Examples of complex molecules: •Biological products (e.g. therapeutic proteins, mAbs)

•Mixture products wherein the proportion of “active” ingredients could not be determined by typical physicochemical/biochemical testing

Why a Potency Test?

Complexity of Therapeutic Proteins

• Heterogeneity • post-translational modifications (e.g. glycosylation) • aggregates/degraded products • charged variants • misfolded species • oxidized species • host cell residuals (Host cell proteins, DNA) • leachables (heavy metals, resin)

• Large molecular sizes: 6 – 300 kDa • Higher-order structures (1, 2, 3) • Less defined structure-function relationships

(Compared to small molecule drugs) • Complex manufacturing processes

Regulatory Requirements for Potency of Biologics License

• 21 CFR 601.2 & FDC Act

All biological products regulated under section 351 of the PHS Act must meet prescribed requirements of safety, purity and potency for Biologic License Application (BLA) approval.

• 21 CFR 610.1

“No lot of any licensed product shall be released by the manufacturer prior to the completion of tests for conformity with standards applicable to such product,” which include tests for potency, sterility, purity, and identity (21 CFR Part 610, Subpart B).

•For all phases of IND clinical studies, data are required to assure product

•Identity, quality, purity and strength (21 CFR 312.23(a)(7)

•Stability (21 CFR 312.23(a)(7)(ii)

• ICH Q6B Specifications: Test procedures and acceptance criteria for biotechnological/biological products

Regulatory Expectations for Potency Testing - Investigational Protein Products

Potency Tests: Applications

• Required for characterization, lot release, in-process and stability testing.

• Demonstrate product activity, quality and consistency throughout product development

• Provide a basis for assessing product comparability before and after manufacturing changes

• Evaluate product stability (expiry dating)

• Control clinical dosing consistency

Relationship Between Potency and Clinical Efficacy

21 CFR 314.126(d)

•Potency is a measure of the bioactivity of a drug product that produces a defined clinical effect.

•Potency tests are used to establish that a consistently manufactured product is administered during all phases of a clinical investigation.

•Clinical efficacy is demonstrated by “substantial evidence” from adequate and well-controlled investigations with a consistently manufactured product. Other determining factors include: – PK/PD profile

– Patient population

– Clinical end-point (e.g. overall survival in cancer treatment)

• Cell-based assays • cellular responses - proliferation, growth arrest, cell death, cytokine release • signal transduction - phosphorylation of signaling components • gene transcription (reporter genes) • ligand-receptor binding

• Animal-based assays • eg Lethal Dose 50 (LD50)

• Biochemical assays • eg enzyme activity

• Multiple assays (array matrix) • For products that have complex and/or not fully characterized mechanism of action

Typical Potency Assays

Potency Assays for Products Targeting Cell Death or Cell Growth Pathways

A large number of protein therapeutics function through modulation of cell death or cell growth pathways in the target cells.

• Induce cell death or growth arrest – Cancer – Pain syndromes – Viral infection

• Promote cell growth or cell survival – Wound healing – Organ transplantation – Chronic heart failure – Neurodegenerative diseases

• Readouts: • Cell viability/cell proliferation • Apoptosis • Signal molecules, such as:

- Caspase activation - MAPK phosphorylation - Receptor binding

• Correlation between the readouts and the intended biological activity –Cancer drugs: Cell death vs. growth inhibition

Joslyn Brunelle and Baolin Zhang (2010) Drug Resistance Updates, 13:172-179.

Potency Assays for Products Targeting Cell Death or Cell Growth Pathways (cont’d)

Issues to Consider

• Relevance to MoA • Acceptance limits • Validation • Changes to bioassays

– Comparability

Mechanism of Action (MoA)

21 CFR 201.57©(13)(i) • Clinical Pharmacology section of the labeling, which

states the following: • “This section must contain information relating to

the human clinical pharmacology and actions of the drug in humans.”

• This section must include the following subsections: • Mechanism of action… • Pharmacodynamics… • Pharmacokinetics…

• The MoA should be discussed at various levels, including the cellular, receptor (selectivity), target organ, and the whole body level, depending on what is known.

• Only reasonably well-characterized mechanisms should be described (21 CFR 201.56(a)(2)).

• Speculation on the mechanism of drug action must be avoided (21 CFR 201.56(a)(2)).

• “How Therapeutic and Adverse Effects Occur” – Guidance for Industry (2009): Clinical Pharmacology Section of Labeling for Human

Prescription Drug and Biological Products— Content and Format

MoA: What is it?

MoA in Human Body: A Learning Process in the Product Lifecycle

Relevant disease models

Cell-based assays

Enzyme assays

• A bioassay may not capture all the functional attributes of a product:

– e.g. glycosylation, pegylation, ADCC, CDC, etc.

• Bioassays are performed in combination with physicochemical/biochemical tests to support product quality.

e.g. LD50

1) Phenotypic changes

2) Signal transduction

In vitro enzymatic reactions

What Should Be Assessed for Potency?

• Relevance to the MoA(s).

– Desired MoA vs. well-characterized MoA

– Assay Matrix - Complex or unknown MoA

• Correlation between the surrogate assay(s) and the biological activity related to potency

- Signal transduction assays

• Ability to discriminate between an active and inactive product or degraded form of the product.

Issues to Consider

• Relevance • Acceptance limits • Validation • Changes to bioassays

• Comparability

Acceptance Criteria for Potency – Biologics License

• A validated potency assay or assay matrix with defined acceptance criteria must be described and justified in the BLA (21 CFR 601.2(a) and 211.165(e))

• “… should reflect the capacity of manufacturing process, and the potency limits established for product lots used in the pivotal clinical studies demonstrating clinical effectiveness” (FDC Act, Section 505(d), 21 U.S.C. 351).

Acceptance Criteria for Potency – Early Phases

• In early development phases, potency acceptance criteria can be difficult to set:

- Limited manufacturing experience - Limited lots of drug substance (DS) and drug product (DP) - Assays not fully validated

• Broad acceptance criteria

– Evaluated with physicochemical and biochemical testing data

• As development proceeds, the acceptance criteria should be tightened to reflect the actual manufacturing capacity, clinical experience and assay performance.

Issues to Consider

• Relevance • Acceptance limits • Validation • Changes to bioassays

• Comparability

Assay Parameters that are Usually Validated

• Robustness to assess sources of variability – reference standards (ICH Q6B) – instruments – reagents (e.g. stable cell lines)

• System suitability • Accuracy • Linearity & Range • Precision (Repeatability, Reproducibility) • Intermediate Precision (analysts, days, laboratories if more

than one will be used)

• Specificity

Stable Cell Lines Used in Potency Assays

• Selection of cell lines – a lineage close to the cell/tissue type targeted by the drug

– a surrogate cell line if an appropriate cell surface receptor is expressed (either endogenously or via stable transfection).

– growth characteristics (e.g. homogeneity, robust growth, functional stability)

• Two-tiered cell banking system (MCB & WCB) – plasmid copy number

– cell passage number

• Standard operating procedures (SOPs) for handling the cell line, including the cell culture conditions, passage number, and procedures for detecting microbial contaminants (e.g. mycoplasma).

Dose Titration Curve

• The dose titration curve should be optimized so that the dilutions are appropriately distributed throughout the entire dose response curve with sufficient coverage in the linear portion of the curve.

• Appropriate statistical analysis (e.g. parallel line analysis) needs to be applied when generating final relative potency results.

• Once a bioassay is validated, it is important to monitor its performance over time. • trending chart for suitable parameters of the ref standard

(RS) response curve and potency of analyzed QC samples

Issues to Consider

• Relevance • Acceptance limits • Validation • Changes to bioassays

• Comparability

Comparability Exercise

• Demonstrate that changes to the potency assay do not interfere with the suitability of the analytical procedures for their intended purposes in terms of

– Accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, robustness

– Pre-defined acceptance criteria

• Same test samples should be assayed using both the original bioassay and the proposed bioassay

– DS, DP, RS, stress and accelerated stability samples

• Appropriate statistical analysis

• In an IND Phase 1 study, a cell-based signal transduction assay (MAPK phosphorylation) was used as a potency assay for a protein product intended to treat Type 2 diabetics.

• Reviewer comment: A potency assay should reflect as much as possible the intended mechanism of action of the drug product. In this case, this would be a measure of improved glucose tolerance or increased glucose uptake in adipocytes. You should provide data demonstrating the correlation between MAP kinase activation and glucose uptake in response to the drug. Alternatively, you may develop an assay that directly measures the uptake of glucose in adipocytes.

Case study # 1

• A recombinant growth factor is tested for treating chronic heart failure. The sponsor developed a potency assay measuring phosphorylation of erbB2 receptor in a cancer cell line.

• Reviewer comment: The potency assay should be optimized to provide a more reliable quantitation of the product’s bioactivity in order to assure consistent dosing. Data should be provided to demonstrate the correlation between phosphorylation of erbB2 and the intended bioactivity of the product i.e. inhibition of cell death of cardiomyocytes.

Case study # 2

• In a Phase 1 IND, a cell viability assay was used as a potency test with a proposed acceptance criteria: IC50 (0.1- 60 nM)

• Reviewer comment: The release specification for potency is not acceptable. The acceptance criterion is too broad to ensure lot-to-lot dosing consistency. From the data provided, it is difficult to assess whether the proposed potency range is wide due to inherent assay variability or whether there are considerable differences in lot-to-lot activity. The potency assay should either be controlled by using a suitable internal reference standard with an allowable range or the manufacturing process must be better controlled.

Case study # 3

• A potency assay was changed from a cell proliferation format to a caspase activation format

– Specification (relative to RS) was not changed

– Better assay precision

– Less plates to meet system suitability

• Validated new assay

– Accuracy (spike recovery), precision (repeatability), intermediate precision, linearity, range and robustness

• Comparability demonstrated by assessing

– Lot release, drug substance, drug product, stability and stressed samples (e.g. heat, light)

Case study # 4

• One applicant developed a cell-based assay to replace animal-based assays for lot release of a licensed protein toxin.

• Issues with the mLD50 assay - highly variable - high rate of assay failure - limited detection range - nonspecific to the product - suffering and death of animals

Case study # 5

mLD50 Bioassay Cell Based Assay Specificity for toxin No Yes, depend on cell lines Sensitivity LD50 1 U ≦ 0.5 U (LD50 equivalent) Range 0.5 – 2.0 U ≦ 0.5 - >100U Precision +/-20-30% <10% Validity 70 – 80 % > 90%

• Comparability demonstrated using a variety of samples (drug substance, drug product, reference standards, stressed samples).

• Improved performance

Case study # 5 (cont’d)

• The potency assays for a licensed recombinant growth factor product have evolved from animal-based assays to cell-based proliferation assay to cell-based gene expression assay.

Case study # 6

Pros Cons

Animal-based assays

• Direct manifestation of MoA • Manifestation of the active

glycosylated forms

• Requires many animals • Highly variable

Culture cell-based assays

• Less laborious • More sensitive • More robust • Fast

• Respond to non-glycosylated product

• Not suitable for measuring drug levels in plasma

Case study #7: Bioassays for complex small molecule drugs

• A bioassay was included as part of the release specifications for a mixture cancer drug, because typical physicochemical/biochemical assays could not determine the proportion of “active” ingredients in the product.

• The approved potency assay uses xenograft tumor mouse models implanted with a murine tumor cell line.

• The assay was used for > 15 years as a release test until recently when tumors failed to grow appropriately upon implantation.

• A cell viability assay using a human cancer cell line is under development:

• Is the selection of such a cell line acceptable?

• Can this new assay be used to replace the animal-based assay for lot release?

Progressive implementation of potency assays

•Limited data on relevant biological attributes

•Broader acceptance criteria

•Release and stability testing

Early phase: pre-clinical

phase 1 phase 2

Later phase: phase 3 pivotal

Biologics License

• A validated potency assay or assay matrix

• Defined acceptance criteria

• Multiple bioassays • Validation • Narrower limits to

ensure lot-to-lot consistency

• Stability testing of validation lots to establish expiry dating prior to licensure

Final thoughts on potency tests

• Potency tests are product-specific, and the adequacy of these assays is evaluated on a case-by-case basis.

• Potency tests may evolve and change significantly in the course of product development and in the product lifecycle.

• It is recommended that sponsors seek timely advice from the FDA on designing, evaluating and validating potency assays.

Acknowledgements

• Serge Beaucage

• Susan Kirshner

• Amy Rosenberg

• Ennan Guan

• Dov Pluznik