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  • Strategic and scientific approaches to developing bioassays to support biologics

    Darren Kamikura, Ph.D.Bioassay DevelopmentEli Lilly and CompanyIndianapolis, IN 46221

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  • Why do we care about Potency?

    For startersits the law!!!

    PHS Act: 42USC262

    (B) The Secretary shall approve a biologics license application -(i) on the basis of a demonstration that -(I) the biological product that is the subject of the application is safe, pure, and potent;

    21CFR601.2

    To obtain a biologics license the manufacturershall submit datawhich demonstrate that the manufactured product meets prescribed requirements of safety, purity, and potency

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    Adapted from Susan Kirchner (FDA)-USP-Bioassay Workshop 2008

    Company ConfidentialCopyright 2012 Eli Lilly and Company

  • What is potency?

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    Potency:

    21CFR600.3(s) The word potency is interpreted to mean the specific ability or capacity of the product() to effect a given result.

    Potency is a measure of the ability of a drug to elicit its function.

    If that ability is to induce (or abrogate) a biological response, then a potency assay should be a bioassay.

    Company Confidential Copyright 2012 Eli Lilly and Company

  • What is a Bioassay?

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    A bioassay is defined as an analytical procedure measuring a biological activity of a test substance based on a specific, functional, biological response of a test system. (WHO/NIBSC, J. Immunol. Methods (1998), 216, 103-116. International consensus, Dev. Biol. Standard. (1999) vol 97)

    Company Confidential Copyright 2012 Eli Lilly and Company

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  • Why do we need Bioassays?

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    Biologics are complex and heterogeneous in composition and can exist in a number of physical and chemical conformations that can impact product safety and efficacy (eg. glycosylation, deamidation, conformational changes, polymer). By measuring the potency, we can infer the structural integrity of complex biologicals. Thus, bioassays are a measure of quality of the therapeutic. ICH Q6B (Specifications: Test Procedures and acceptance criteria for Biotechnological/Biological Products)

    Company Confidential Copyright 2012 Eli Lilly and Company

  • What a Bioassay can and cannot tell us

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    A bioassay CANindicate product qualitybe relevant to mechanism of action, and give insights into the way a therapeutic functions at a molecular level be reproducible and suitable for use in a QC environment A bioassay CANNOT directly predict clinical efficacy/outcome (eg. Biodistribution, PK, non-specific interactions, anti-drug antibodies, etc)

    Company Confidential Copyright 2012 Eli Lilly and Company

  • Types of Bioassays

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    Cell based bioassays can be both quantitative and physiologically relevant

    Company Confidential Copyright 2012 Eli Lilly and Company

  • Cell based assays

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    Cell based bioassays represent a space between drug composition and the clinic (ie. Part way between chemistry and biology)

    Company Confidential Copyright 2012 Eli Lilly and Company

  • Where do we begin?

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    Design of a relevant bioassay begins with understanding how a drug affects the target biology eg. Growth factor/cytokine neutralization by NAb What are the critical biological pathways and can they be exploited to develop a bioassay? Acquire the right reagents choice of cell type relevant to disease downstream read-out (reporter gene, proliferation, etc)

    Company Confidential Copyright 2012 Eli Lilly and Company

  • Company Confidential

    Copyright 2012 Eli Lilly and Company

    Cells, being part way between chemistry and the clinic are more complex than a defined set of chemicals but less complex than an organism The cells are the foundation of a cell-based assay cells represent the most critical and difficult to control reagent for bioassay performance (large contributor of assay variability) Two critical, but difficult to answer questions: Why can cells be difficult to control? What defines The happy/well behaving cell?

    Cells are Complex Systems

    Kamikura MBSW 2012

    Company ConfidentialCopyright 2012 Eli Lilly and Company

  • Company Confidential

    Copyright 2012 Eli Lilly and Company

    Human cell ~23,000 protein coding genes/haploid (~1.5% of genome) Different proteins perform specific tasks and have different locations, based on that task. There are many events that culminate in a given response

    Cells are Complex Systems

    Kamikura MBSW 2012

    Kamikura and Cooper, Traffic (2006) 7:324

    Company ConfidentialCopyright 2012 Eli Lilly and Company

  • Company Confidential

    Copyright 2012 Eli Lilly and Company

    Decision making at a cellular level is stochastic Many genes contribute to the probability of a given response in any particular cell (Ansel et al, PLOS Genet (2008) 4:1)

    Cells are Complex Systems

    Kamikura MBSW 2012

    Company ConfidentialCopyright 2012 Eli Lilly and Company

  • Company Confidential

    Copyright 2012 Eli Lilly and Company

    The ultimate biological response is also influenced by the metabolic state and health of the cells

    Cells are Complex Systems

    How many events go into the decision of these cells to scatter in response to HGF?

    Kamikura MBSW 2012

    Company ConfidentialCopyright 2012 Eli Lilly and Company

  • Cell based Bioassays can be sensitive

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    In an average person ~50 x 1012 cells (clinical trials) In a cell based assay ~25 x 103 cells/well (2 billion times less cells) EC50s can be in the range of pg/mL (10-10 - 10-12 g/mL)! for an antibody (150kDa), 1pg/mL is ~600 molecules/uL in 100uL, this is ~ 60000 molecules! just a few molecules are enough to perform the function

    Company Confidential Copyright 2012 Eli Lilly and Company

  • Cell based Bioassays

    Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    High sensitivity allows us to detect how various species present contribute to activity and product quality glycosylation deamidation clipping But it also means that slight differences or modifications in assay set-up can affect the performance of the assay.

    Company Confidential Copyright 2012 Eli Lilly and Company

  • Kamikura MBSW 2012

    Company Confidential

    Copyright 2012 Eli Lilly and Company

    Generic Bioassay Protocol Manipulations and Considerations

    Add Target cells to plate (Numbers?)

    Rinse, trypsinize, resuspend, count, dilute, add to plate (6-7 steps)

    Pre-incubate cells at 37C (Time?)

    Plate position in incubator/heating effects

    Add Ligand/Antibody/other biologic dilutions (Range?)

    Each dilution step is an independent manipulation (8-11 steps for each dilution series)

    Incubate at 37C (Time?)Add detection reagentsRead in a luminometerHow do we know which factors are critical?

    Company Confidential Copyright 2012 Eli Lilly and Company

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  • Company Confidential

    Copyright 2012 Eli Lilly and Company

    Variables to consider include:

    temperature (growth, media for seeding & stimulation) time of cell growth prior to stimulation time of stimulation confluence of cells (seeding & stimulation) ligand incubation time ligand concentration Therapeutic dilution curve

    Brand of 96-well plates Plate sealers/evaporation controls Media composition (FBS, Defined Media, antibiotics, etc) eg. Cells may require FBS for survival, but FBS may interfere with the assay outcome. Plate Layout

    Considerations for Assay Optimization

    Kamikura MBSW 2012

    Company Confidential Copyright 2012 Eli Lilly and Company

  • Company Confidential

    Copyright 2012 Eli Lilly and Company

    Optimizing each variable independently and one at a time is inefficient

    Effects of some of the variables may not be mutually exclusive. Single factor optimization will not lead to truly optimal conditions

    Make friends with your local statistician for success!

    DoE: Consult your local statistician

    Kamikura MBSW 2012

    Company ConfidentialCopyright 2012 Eli Lilly and Company

  • Qualification/Validation of a Bioassay: A critical role for statisticians

    Post Development: Demonstration of method accuracy, repeatability, intermediate precision, linearity, range and specificity (optional: reproducibility)

    Early phase qualification is less rigorous

    Meet release specifications of 50%-150% relative potency Demonstration of stability indication via forced/thermally degraded reference standard

    Late phase validation is much more rigorous

    Meet tighter release specifications (eg., 80%-120% relative potency) Use of multiple DS and DP lots Capable for assessing primary degradation pathway Demonstrate in-use sample stability

    Kamikura MBSW 2012

    Company Confident