Davinder Gill, PhDAsst Vice President, Biologic TherapeuticsWyeth Research

Biotherapeutic Drug Development: Progress and Challenges in the new Regulatory Environment

Fact: It takes an average 10-15 years from initiation of research to FDA approval for a single drug

Fact: It typically costs $802 million to develop a single drug from concept to market

Fact: Of every 5,000 drugs in research today only 5 will ever be tested in clinical trials

Fact: Of every five drugs now in clinical trials only 1 will ever reach patients

Pharmaceutical Drug Development

Biologics are becoming a key growth driver for Pharma

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xBiotherapeutic approvals are on the rise

Biotherapeutics are being tested in unconventional areas e.g. Alzheimer’s Disease

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18-monthvehicle

18-monthAN 1792

Schenk et al 1999, Nature

Autopsy case report: reduction of Aβplaques in cortex after Immunotherapy

James Nicoll et al 2003, Nature Med

The regulatory environment has changed

Agencies are putting new demands on safety and tolerability of Biotherapeutic drugs

n Mode of actionn Nature of targetn Relevance of animal model

General philosophy behind Protein screening

nFocus on function, not just bindingnDesign screening for broad functional

coveragen Identify a broad set of binding proteins in

addition for later use nWe can always optimize (so affinity is less

important than function)nCompound safety is often left for the pre-IND

stage

The clichéd “needle in the haystack” problem

What would we do if we could analyze1000

10,000100,000

1,000,00010,000,000

Proteins in parallel?

1011

phage scFv

antibody genes from 70 human donors

washing

elution

amplification

target

binding

2-3 selection cycles

target

The “upstream” steps in Protein HTS have largely been solved

QPix first takes digital images of the colonies on a bioassay plate

cameraplate with colonies

illuminated from below

pin sterilization bathsdestination plates

picking head

Image analysis software then selects well-separated colonies of correct size and shape

Cells from identified colonies are picked up on each of 96 sterile steel pins and inoculated into growth medium

@3000 colonies/hour

Plates are incubated in microplate shaker

•humidified•20 stacks per chamber

•2 chambers for microplates

•After overnight growth, glycerol can be added with Multidrop and plates stored at –80.

Making protein to analyze: Phage

E. coli

+ helper phage

phagemid

1. Inoculate 150 μl.2. Grow 5-7 hr.3. Infect with helper phage4. Replace with Amp/Kan medium

to select infected cells5. Grow overnight6. Spin plate and collect supt.

Pros: •Not much work on the day of assay.•Strong signal in ELISA.

Cons:•ScFv is attached to huge particle•Not monovalent•Relatively low concentration.

Plate replication using QPix

•Start from fresh growth plates or glycerol stocks.

•All 96 pins transfer at the same time.

•Transfer to 1 or more standard or deep-well plates.

•@60 plates/hour

QC criteria to decide which pools of clones to analyze by HTS

1. How many clones bind to the target?• standard ELISA

2. How many clones neutralize the target (if applicable)?• multiple formats

3. How much sequence diversity is there among the hits?• sequence PCR products from bacterial cultures

Analyze 24-96 clones per pool (n=6-24 pools)

HTS capacity

n 64x 96-well plates/runn 2 days/runn Most of the work can be handled by one person.n 2 people required on the morning of the second day (1

to make peri-preps, 1 to prepare screening assays)n Multiple assay formats run simultaneously (e.g.,

binding ELISA, neutralization HTRF assay, cell based binding)

Limitations of our current methods: Antibody Screening

n IgG conversion is labor-intensive, so we only convert limited numbers

n Rank order of potency of scFv does not always carry over to IgG

n scFv might not be an appropriate format for finding certain types of hits

cell-based assays that are sensitive to endotoxinneed for crosslinking (e.g., for agonists)scFv affinity is generally too low for assay to detect hits

Would converting 1000 clones to IgG help us?

n Yes, if the functional assay cannot be done with scFv. Rare functional clones that might have been unremarkable in a binding assay can be found with a functional assay.

n Yes, even if a functional assay can be done with scFv. Borderline scFv might be clearer hits in an IgG-based assay

n IgG format might make epitope grouping more robust.Variation: convert pool to murine IgG as well as human IgG

This is a worthwhile goal.

Technical challenges of making 10,000 IgG

n Need to increase throughput of DNA preps and transfections by 10-25x above what is currently available

Very small-scale plasmid preps—need to move to magnetic-bead based prepsTransfection with linear PCR products?

n Need robotics for transfectionLiquid Handler in TC hood or laminar flow hoodEnclosed robot/incubator combination

n Is this level of throughput necessary?

Technical requirements

n HT sequencingCurrent PCR capacity: 4 x 96 at a time, 8 or 12 x 96 in a day

n HT plasmid prepsPrep robot—4 x 96 at a time, 8 or 12 x 96 in a day96 well benchtop Prep RobotMacConnell 96 machine

n 6-well scale transfections (@ 1 ug DNA)

High-throughput plasmid purification: spin or filtration plates

nAutomated system (sequencing group): 4 x 96 well plates in @2 hr

nOther productsnInvitrogen PureLink HQnAxygen Axyprep

nIssue: stacks of filters are tall, so these might not work on all robots.

Need to combine HT transfection with HT purification?

nAntibody concentration might be too low for detection in functional assay

nHigh-throughput IgG purification methods can provide concentrated IgG

Protein A products analogous to high-throughput His6 purification products in use

- 96 at a time is currently feasible with Phynexus tips- Plate-based methods could raise throughput to 1000 per day

Requires buffer exchange (centrifugal method available—tedious but limit per day = number of centrifuge positions)

High-Throughput Protein PurificationWhen assays are not compatible with crude periplasmic extracts

• His Select 96-well filter plates:Purification is achieved usingvacuum manifold or centrifugation

• Phynexus prepacked IMAC tips:Purification can be achievedusing walk-away robot protocol

• His Trap 96-well plates:Purification is achieved as perhis select plates above

High-Throughput Protein Purification

His Select

Phynexus tips

His Multitrap

After purification, samples arebuffer-exchanged into PBS using96-well filter plates

Level of purity is high for all 3methods as judged by SDS-PAGE & analytical SEC

Yields range from 30-50 μg/10 mlstarter culture

from Cummins et al, JIM, 2008

High-Throughput Protein PurificationSmall-scale material compatible with primary T-cell proliferation assay

• His Select purified scFv wastested alongside scFv purified using a 2-step affinity & SECprotocol

• The scFvs were tested for theirability to inhibit stimulation ofprimary T-cell proliferation

• scFvs purified using differentmethods behaved in a similarfashion with no obvious assayinterference with His Selectmaterial

from Cummins et al, JIM, 2008

High-Throughput Protein PurificationA similar method has been used for IgG & scFv-Fc purification

Protein A Multitrap 96-well Plates:

Yields ranged from 5-15 μg/ml CMfor IgGs and scFv-Fc fusion proteins

Purity was high by SDS-PAGE &analytical SEC

Quality & quantity of material wassufficient to carry out kineticanalysis of clones by BIAcore

from Cummins et al, JIM, 2008

n Biotherapeutic Screening and OptimizationRules for protein SAR do not existStructural information instructive but difficult to obtain

n ManufacturingProduct quality, cost of goods

n Limited knowledge-base relative to SMn Robust Protein HTS capbility downstream that

enables best drug candidate selection

How does this lead to discovery of better and safer drugs?

Davinder Gill, PhDAsst Vice President, Biologic TherapeuticsWyeth Research

Thank You