expectations and limitations of bioassays: an eu regulator`s view

Austrian Agency for Health and Food Safety www.ages.at

Christian MayerAGES - Austrian Agency for Health and Food Safety

CASSS Bioassays 2015: Scientific Approaches & Regulatory StrategiesSilver Spring, Maryland, March 23-24, 2015

Expectations and Limitations of Bioassays:An EU Regulator`s View

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The views and opinions expressed in the following PowerPoint slides are those of the individual presenter and do not necessarily reflect the view of the AGES and/or the EMA

Disclosure and Disclaimer

Austrian Agency for Health and Food Safety

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• AGES – Austrian Agency for Health and Food Safety:

- Expert organization for risk minimization in all matters related to health, food safety, and consumer protection.

• AGES MEA - Austrian Medicines and Medical Devices Agency:

- One of five divisions of the AGES- National competent authority for regulation of medicinal

products and medical devices - A partner to competent authorities and agencies throughout

Europe and pharmaceutical industry

Organization


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• Activities of AGES MEA according to lifecycle of a medicine

Organization


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• Relevance of quality data

- 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.

Why bioassays?


ATMPs Vaccines Therapeutic proteins Peptides Pharmaceuticals

analytical comprehensive full analyticalcharacterization limited analytical characterization characterization

increasing complexity, increasing size

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• Complex molecules – manufactured by complex manufacturing processes

• Microheterogeneity in structure:- Deamidation- Oxidation- Glycation- Glycosylation profile- Charged variant profile- C-terminal lysine- Fragmentation- Aggregation…

>> Impact on biological activity??

Why bioassays?


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• Life-cycle changes of the manufacturing process

Rituximab: Change occurred in several physico-chemical quality attributes

>> Impact on biological activity??

Why bioassays?


Schiestl et al. 2011

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• Measurement of the biological activity using a reliable bioassay is an essential requirement throughout a biological medicinal product’s life-cycle:

- R & D activities and process development- Product characterization- In-process & release control testing- Dosing- Formulation studies- Stability- Post-marketing process changes

Why bioassays?


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• Measure of activity- Link to the Mode of Action- Calibration against a reference standard

• High variability- Appropriateness for QC control

• Resource intensive- Time- Materials

• Performed on samples in a complex matrix- Vaccines produced in living systems- Recombinant therapeutic proteins purified from cell culture

Challenges with bioassays


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• Relevance to Mode of Action

- An appropriate bioassay should establish a clear, unambiguous link between the measured relevant biological activity and the MoA of the drug molecule:

o Based on a sufficient understanding of structure-function relationships

- Some molecules have more than one biological activity which contribute to MoA

- Bioassays should be performed in combination with physicochemical/biochemical tests to get a picture of the product quality

The ideal bioassay?


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• Example - Rituximab: Fc-related biological activities contribute to the Mode of Action

- ADCC via binding of macrophages, monocytes or NK-cells- Complement activation (CDC) - Apoptosis

- Serum half-life (recycling via binding to FcRn)

• More than one bioassay needed to address the relevant biological activities

The ideal bioassay?


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• ICH Q6B: - Potency is the measure of the biological activity using a suitably

quantitative biological assay, based on the attribute of the product which is linked to the relevant biological properties.

• Another interpretation: - The potency is a measure of drug activity expressed in terms of

the amount required to produce an effect to given intensity.- The more potent the drug, the lower the amount required to

produce the effect.

Potency


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• The bioassay for quantitative measurement of potency(„potency assay“)

- Required for release and stability testing- Validation according to ICH Q2(R1)

Potency assay


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• Standardization of potency assays requires biological potency international / national standards - calibrated in units (e.g. IU)

• Where no standard exists:… establish an appropriately characterized in-house primary reference material, prepared from lot(s) representative of production and clinical materials. In-house working reference material(s) used in the testing of production lots should be calibrated against this primary reference material. ICH Q6B

Potency assay


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• A good potency assay should be set up as early aspossible

- Potency may evolve during development- Functional in vitro assay more meaningful as it integrates

specific receptor interaction with measurable response – in vitro cell based preferred

- On the other side: Receptor binding assays have less variability but may not reflect the cascade of events resulting in the biological effect

Potency assay


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• Potency assays for vaccines

Vaccines:- Often produced by or derived from living micro-organisms- Complex molecular structure of their active components- Presence of excipients in their final formulations (e.g.

preservatives, adjuvants) that may interefere with non-animaltesting

Quality control of classical vaccines heavily relies on live animalmodels

Potency assay


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• The 3 R`s - Replacement, Reduction, Refinement

„In accordance with the provisions of the European Convention for theprotection of vertrebrate animals used for experimental and otherscientific purposes and Directive 2010/63/EU on protection of animalsused for scientific purposes, the 3R principles (replacement, reductionand refinement) should be applied to production and control testing ofpharmaceuticals.“

Potency assay


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TNF-α antagonist as biosimilar candidate

- Biological testing programme for comparison of research, pilot and commercial scale material with reference (innovator) product:

- Biological assays using different approaches- TNF-α neutralization- SPR using a Biacore system to determine binding affinity and

kinetics - ELISA to determine the binding affinity Problem: - No ADCC or CDC testing included

Case study 1


Casestudy

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Consequences/outcome:

- Approach for binding/neutralization of TNF-α endorsed- However: Clinical relevance of the Fc domain functionality under

discussion for different TNF-α inhibitors (Arora et al. 2009)- Bioassays for ADCC and CDC should be part of the comparability

exercise

Case study 1


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VEGF antagonist as biosimilar candidate

Problem:- HUVEC proliferation assay shows high variability, significant

donor to donor variability and insufficient accuracy for QC purposes

Case study 2


Casestudy

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Alternative:- Development of cell based VEGF-internalization bioassay for

potency testing in batch release and for comparability with reference product

- Cell line overexpresses VEGFR2 and does not express VEGF - Inhibitory effect on the binding of fluorescence labeled VEGF to

VEGFR2 and the subsequent internalization of the ligand-receptor complex is measured.

Assessor`s comment: - VEGF internalization assay may properly reflect the mechanism

of action, agreed

Case study 2


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Recombinant Factor IX

- Potency assay for Factor IX products in Europe:

- One-stage clotting assay according to Ph. Eur. 201711 (uses activated partial thromboplastin time (aPTT) reagent)

- EMA/EDQM Workshop on Characterisation of new clotting factor concentrates (FVIII, FIX) with respect to potency assays used for labelling and testing of post infusion samples, held on 28-29 November 2013:

Problem:- Applicant indicated that potency results can be affected by the

type of aPTT reagent used in the clotting assay

Case study 3


Casestudy

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Gritsch et al., from Poster shownat WFH 2014, Melbourne, Australia,May 2014

Case study 3


some reagents giving results 40% above the labelled potency

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Consequence/outcome:- “Concerning the possible consequences of the dependency of

assay results on the aPTT reagent on product information the applicable changes of the revised core SmPC for FIX products have been implemented which includes high level information that plasma factor IX activity results can be significantly affected by both the type of aPTT reagent and the reference standard used in the assay”… EPAR

Case study 3


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Rabies vaccine

- NIH-test:i.p immunization i.c. challenge withvirus determine 50% protective dose – PD50

Problem:- Use of up to 120 animals; invasive and painful procedure- Highly variable results with up to 400% differences in estimated

potency- High number of invalid tests (up to 42%, Kraemer et al. 2009)- Unnatural route of infection (intracerebral) and vaccination

(intraperitoneal)

Case study 4


Casestudy

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Drivers and barriers for replacing the NIH test

- Schiffelers et al. 2014

- Survey to assess the main drivers & barriers for addressing theNIH test

- ~ 50 participants from regulatory authorities, governmentalinstitutes, industry, academia

Case study 4


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Drivers and barriers for replacing the NIH test

Case study 4


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Solution:

• Alternative assay for replacement of the NIH test- Serum neutralisation assay (i.e. rapid fluorescent focus inhibition

test)- Krämer et al. 2010. Collaborative study for validation of a

serological potency assay for rabies vaccine (inactivated) forveterinary use.

• Implementation into Ph. Eur. 0451 „Rabies vaccine for veterinaryuse“:

- It is not necessary to carry out the NIH potency test for eachbatch of vaccine if it has been carried out using a batch ofvaccine with minimum potency.

- An alternative validated method (e.g. RFFIT) should preferablybe used for routine testing

Case study 4


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“Not everything that can be counted counts, and noteverything that counts can be counted.”

Albert Einstein

Thanks for listening


expectations and limitations of bioassays: an eu regulator`s view

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