BRG Presentation Template

Transcript BRG Presentation Template

Strategic and scientific approaches to developing bioassays to support biologics

Darren Kamikura, Ph.D.

Bioassay Development Eli Lilly and Company Indianapolis, IN 46221

Why do we care about Potency?

For starters…it’s 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 manufacturer…shall submit data…which demonstrate that the manufactured product meets prescribed requirements of

safety

, purity, and

potency…

Kamikura MBSW 2012

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

What is potency?

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.

What is a Bioassay?

• A

bioassay

measuring a biological activity of a test substance based on a is defined as an analytical procedure

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)

Why do we need Bioassays?

• 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)

What a Bioassay can and cannot tell us

• A bioassay CAN 1. indicate product quality 2. be relevant to mechanism of action, and give insights into the way a therapeutic functions at a molecular level 3. 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) Kamikura MBSW 2012 Company Confidential Copyright © 2012 Eli Lilly and Company

Types of Bioassays

Cell based assays

• Cell based bioassays represent a space between drug composition and the clinic (ie. Part way between “chemistry” and “biology”) Kamikura MBSW 2012 Potency assay Company Confidential Copyright © 2012 Eli Lilly and Company

Where do we begin?

• 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) Kamikura MBSW 2012 Company Confidential Copyright © 2012 Eli Lilly and Company

Cells are Complex Systems

• 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: 1) Why can cells be difficult to control? 2) What defines “The happy/well behaving cell”?

Cells are Complex Systems

• 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.

:324

Cells are Complex Systems

• 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)

:1) Antibiotic Kill curve EC50 = 50% dead •What was different about the cells that survived?

Cells are Complex Systems

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

(-) HGF (+) HGF

•

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

Cell based Bioassays can be sensitive

• In an average person ~50 x 10 12 cells (clinical trials) • In a cell based assay • ~25 x 10 3 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!

Cell based Bioassays

• 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.

Generic Bioassay Protocol – Manipulations and Considerations

1. Add Target cells to plate (Numbers?) • Rinse, trypsinize, resuspend, count, dilute, add to plate (6-7 steps) 2. Pre-incubate cells at 37 °C (Time?) • Plate position in incubator/heating effects 3. Add Ligand/Antibody/other biologic dilutions (Range?) • Each dilution step is an independent manipulation (8-11 steps for each dilution series) 4. Incubate at 37 °C (Time?) 5. Add detection reagents 6. Read in a luminometer  How do we know which factors are critical?

Considerations for Assay Optimization

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 .

DoE: Consult your local statistician

• 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!

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 Confidential Copyright © 2012 Eli Lilly and Company

Qualification design: 9 total plates

Plate #

1 2 3 4 5 6 7 8 9

Assay #/Set/Day

1 2 1 2 1 2 3 3 3

Analyst

A B A B A B A, B, or C A, B, or C A, B, or C

Experiment (UNK1, UNK2, UNK3) UNK1 %RP

50%, 100%, 150% 50%, 100%, 150% 50%, 100%, 150% 70%, 100%, 130% 70%, 100%, 130% 100%, Specificity (alternative molecule 1), control 52.3

48.6

55.6

68.4

82.0

87.4

100%, Specificity (alternative molecule 2), control 100%, Interference (heat-killed), Interference (Spike) 100%, control 95.9

100.9

100.6

UNK2 %RP UNK3 %RP

101.5

91.1

101.6

90.3

106.6

160.5

127.9

174.7

105.7

142.5

ND ND ND 75.7

70.6

76.1

94.4

88.4

Validation design: >100 plates

Example Drug Substance Experimental Design

Validation Study Analyst 50% 75%

A B A B A B L, A, I, R, P A, I, R A, I, R A, I, R A, I, R A, I, R 3 plates 1,2,5 3 plates 2 3 plates 2 3 plates 1

100%

3 plates 1,2,3,4,5 3 plates 2,3,4 3 plates 2,3,4 3 plates 2,3,4 3 plates 2,3,4 3 plates 2,3,4

125%

3 plates 1

150%

3 plates 1,2,5 3 plates 2 3 plates 2 Example Drug Product Experimental Design

Validation Study Analyst 50%

B A B A B A L, A, I, R, P A, I, R A, I, R A, I, R A, I, R A, I, R 3 plates 1,2,5 3 plates 2 3 plates 2

75%

3 plates 1

100%

3 plates 1,2,3,4,5 3 plates 2,3,4 3 plates 2,3,4 3 plates 2,3,4 3 plates 2,3,4 3 plates 2,3,4

125%

3 plates 1

150%

3 plates 1,2,5 3 plates 2 3 plates 2 L = Linearity 1 , A = Accuracy 2 , I = Intermediate Precision 3 , R = Reproducibility 4 , P = Repeatability Precision 5 Kamikura MBSW 2012 Company Confidential Copyright © 2012 Eli Lilly and Company

Conclusion

• Cell based assays represent a space between chemical assays and the clinic • Biological systems are extremely complex and sensitive to small variations in environment  During assay development, statistics are invaluable in being able to identify interacting biological factors that can impact a given readout  Post-development, statisticians are critical for the design and analysis of qualification/validations to ensure that an assay is suitable for its intended use Kamikura MBSW 2012 Company Confidential Copyright © 2012 Eli Lilly and Company

BRG Presentation Template

Transcript BRG Presentation Template

Strategic and scientific approaches to developing bioassays to support biologics

Why do we care about Potency?

What is potency?

What is a Bioassay?

Why do we need Bioassays?

What a Bioassay can and cannot tell us

Types of Bioassays

Cell based assays

Where do we begin?

Cells are Complex Systems

Cells are Complex Systems

Cells are Complex Systems

Cells are Complex Systems

Cell based Bioassays can be sensitive

Cell based Bioassays

Generic Bioassay Protocol – Manipulations and Considerations

Considerations for Assay Optimization

DoE: Consult your local statistician

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

Qualification design: 9 total plates

Validation design: >100 plates

Conclusion

Directory