Biological activity testing...Biological activity testing Regulatory considerations Marcel Hoefnagel Senior Assessor Biopharmaceuticals Medicines Evaluation Board 1 DISCLAIMER: Personal

Biological activity testing

Regulatory considerations

Marcel Hoefnagel

Senior Assessor Biopharmaceuticals

Medicines Evaluation Board

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DISCLAIMER: Personal views only, meant to initiate further discussion; may not necessarily reflect views/opinions of MEB, EMA or EDQM.

Outline

• Definitions & Guidelines Biological Activity

• Infliximab biological activities

• Other Pivotal Activities

• (In vivo) bio-assays are inherently variable

• Cell-based medicinal products

• CAR-T cells

• Take home considerations

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Definition Biological EC Directive 2001/83

Biological [active] substance is a substance that is produced by or extracted from a biological source and that needs for its characterisation and the determination of its quality a combination of physico-chemical-biological testing, together with the production process and its control ().

CASSS_NL_Leiden ©Hoefnagel 2018

ICH Q6B 2.1.2 Biological activity

• A valid biological assay to measure the biological activity should be provided by the manufacturer.

• Examples of procedures used to measure biological activity 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, may be acceptable.

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Recombinant proteins

• Which parameters?

– AA sequence

– Di-sulfide bridges

– Glycosylation

– Molecular Weight

– Oxidation /De-amidation

– Potency


From https://commons.wikimedia.org

• potency is the quantitative measure of biological activity based on the attribute of the product, which is linked to the relevant biological properties.

• The assay demonstrating the biological activity should be based on the intended biological effect which should ideally be related to the clinical response.

ICH Q6B Definition Potency


“based on the intended biological effect”: MoA

• Sometimes exact Mode of Action (MoA) not fully known

• Developing representative in vitro assay not always straightforward

• If not practically feasible: sometimes surrogate assays as potency tests for release.

• Often >1 MoA: Difficult to capture in a single potency assay


Modes of Action: Infliximab

Primary MoA

• Infliximab binds to both soluble and transmembrane TNFα and TNFα receptor activation is prevented

• Potency test: ability to block TNF-induced inhibition of (WEHI-164) cell proliferation (Ph.Eur. monograph)


OmicsOnlineOrg:

Secondary Mode of Action Infliximab

• Binding Fc receptors (FcγRI, FcγRIIa, FcγRIIb & FcRn) on effector cells

• ADCC (antibody-dependent cellular cytotoxicity)

• Binding C1q

• CDC (Complement-dependent cytotoxicity)

• All affected by glycosylation

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From: https://openi.nlm.nih.gov


Biosimilarity Inflectra/Remsima vs Remicade • Comparable binding activity to both monomeric and

trimeric TNFα

• Comparable result in TNFα neutralisation assay

• Relative binding affinities to Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb and FcRn) were comparable

• Differences in relative in vitro binding affinity of FcγRIIIa and FcγRIIIb

• Differences Ex vivo binding assay with isolated neutrophils and NK cells for Crohn’s Disease patients

• Genotype dependent difference in NK binding

• In presence of diluted CD patient serum all differences in binding were abrogated


How about other pivotal functional aspects?

• Delivery: e.g. homing and uptake in cell (binding)

• For enzyme-replacement products uptake in the cells is also an pivotal aspect for their efficacy.

• Potency testing is often based on testing MoA (e.g. enzyme activity) only.


Inherent variation of bioassays

• Example: Well-defined biological (rDNA protein)

• In vivo potency testing in rodents

• High variation: % RSD (intermediate precision) 59%

• Range Clinical batches 92-362%

• Proposed limits (95% confidence): 20-569%

• Limits tightened

• Risk of rejecting suitable batches

• In vivo assays generally high variability

• Bioactivity assays for well-defined products?

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Not accepted!


Cell-based medicinal products: the new biologicals

Potency is a key parameter for complex products which are difficult to characterise.

A combination of multiple methods may be needed to adequately define the potency of these products during the development. Certain assays may be needed to control process changes, whereas others are more suitable for release testing.

Preferably, the potency assay should reflect the clinical Mechanism of Action.


Cell-based medicinal products: the new biologicals • Often exact MoA unknown (consequence: e.g. no surrogate

markers available)

• Sometimes in vitro assay does not correlate with in vivo situation

– Assay conditions are insufficient (e.g. presence of immune suppressiva in vivo)

– Surrogate markers etc. are not appropriate read-out for biological activity

• Assay qualitative instead of quantitative

• Reference standard difficult to obtain

• Not up-to-date with most recent scientific knowledge (fast evolving field)


CAR-T cell therapy

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T cell cytolytic activity

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Zaritskaya et al. Expert Rev Vaccines 2010


(CAR) T cell potency tests

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Assay type Responder cells

Stimuli Read out

Phenotyping assay

T cells CD markers, antigen (e.g. CD19) specific receptor

Avidity assay T cells MHC multimers loaded with antigens

Amount/duration of TCR-MHC-peptide binding (EC50)

Proliferation assay

Effector T cells

Irradiated (peptide-pulsed) tumour cells

Proliferation (via CFSE dilution)

Cytokine production assay

Effector T cells


Intracellular cytokines (e.g. IFN-γ and IL-2)


(CAR) T cell potency tests (2)

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Assay type Responder cells

Stimuli Read out

Effector molecule release assay

Effector T cells

Irradiated (peptide-pulsed) tumour cells/ peptides

- Secretion of cytokines (e.g. IFN-γ, TNF-α, IL-2) - Secretion of cytotoxic factors (e.g. granzyme B)

Degranulation assay

Effector T cells


Surface expression CD107a/b

Growth inhibition assay

(Patient specific) Tumour cells

Effector T cells

Tumor cell proliferation (3H incorporation)

Cytotoxicity assay (various release assays)

(Patient specific) Tumour cells (transduced)

Effector T cells

- Release intracellular proteins (e.g. LDH, β-gal or luciferase), or 51Cr - Intracellular 3H thymidine, GFP


Rossi, Blood 2018

Hoefnagel, PDA 2017

Correlation potency and clinical response


• Anticipated MoA involves tumour recognition & cell death

• Potency: cytotoxic potential of antigen-specific T cells

• Release assay based on surrogates due to practical limitations (time, sample size)

• Establish correlation with clinical response

• Autologous product: inherent variability between batches

• How to set specifications?

Avoid rejection of good batches

Can detect clinically relevant defects & sub-potent batches

• Post-approval: Evaluation Specifications/ Appropriateness test

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Considerations for (CAR-)T cell potency tests


Take home considerations

• Analytical methods for rDNA proteins generally adequate

• mAb: Relation heterogeneity-biological activities-clinical relevance

• Ensure non-MoA Biological Activities (e.g. homing)

• In vivo bio-assays highly variable not suitable as release assay

• In vitro bioassays; de facto obligatory as release assay but limited usefulness

• Cell-based medicinal products: The new biologicals

• Analytical methods still being developed

• Relevant potency tests in infancy


The Everyday Benefit of Philosophy Is That It Helps You Live with Uncertainty

Bertrand Russell

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Biological activity testing...Biological activity testing Regulatory considerations Marcel Hoefnagel Senior Assessor Biopharmaceuticals Medicines Evaluation Board 1 DISCLAIMER: Personal