Timothy S' Charlebois, Ph'D'

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Technology and Opportunities in Mammalian Cell
Culture

• Timothy S. Charlebois, Ph.D.
• Director, Cell and Molecular Sciences
• Wyeth BioPharma
• Andover, MA
• BIOMAN
• Portsmouth, NH
• 26 July 2006

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The Evolution of Wyeth BioPharma
3
Current Biopharmaceutical Products Driving 3
Billion in Revenue
Launched 1997
Launched 1998
Launched 1998
Launched 2000
Launched 2001
Launched 2002
4
From Gene to Drug Product
Purification
Cell Culture
Formulation
Thaw
Cell
Gene
5
Current Choices of Host Cells in Biotech
Yeast
Transgenic Animals
Bacteria Cells
Transgenic Plants
Animal Cells
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Currently transgenic systems do not present a
transforming advantage over CHO
Comparison of Monoclonal Antibody Produced from
CHO Transgenic Goats
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The Majority of Biotech Products on the Market
Are Made in Animal Cells
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5 of the Top 20 Products Globally Are
Biotechnology Products
Source IMS data, 2005
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Fastest Growing Classes of Drugs are Biotech Drugs
BIOTECHNOLOGY
Source IMS data, 2005 Q3 MAT
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Current and Future Products are Expected to Have
a Significant Impact On Healthcare Budgets
Duration of Therapy for Late Stage Products
70
60
50
40
of Biopharmaceuticals
30
20
10
0
Short -term
Intermediate /Intermittent
Acute
Chronic
Source Supplement to Journal of Managed Care
Pharmacy, May 2004 Percentage of
Biopharmaceuticals in Late-stage Development
(Excluding Cancer-related Agents)
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Mammalian Cell ExpressionEvolution .. to
Revolution

• Early products Replacement factors
• EPO, FVIII.
• Highly active, small doses
• 1980s expression levels OK
• Recent products mAbs soluble receptors
• Rituxan, Remicade, Enbrel .
• Higher doses
• 102 103 increases in titer needed for product
  viability

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Producing these Products has required huge
capital investment

• Design to approval gt 5 years
• gt1,000,000 liters worldwide
• Hard to match capacity to need

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Wyeth examples of biotech capital investments
Dublin, Ireland
Andover, MA
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Investment in Bench-Scale InnovationGreat
Potential Return
Application of Improved Process Technology
Platform Process Rapidly Developed
9.6 g/L
3 g/L
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Capital Expenditure Can Be Limited by Applying
Innovation
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Innovation and Penicillin Production
INNOVATION
Penicillin discovered
1928
0.001 g/L
Improved Medium
1939
0.01 g/L
1947
200,000 /B U
Improved Strain
1957
0.1 g/L
3,800 /B U
COST
1967
Semi-synthetic Synthesis
Improved Stability
52 /B U
10 g/L
22 /B U
20 /B U (25-12/BU)
1995

• Billion Units

From PenicillinA Paradigm for Biotechnology Ed.
R.I. Mateles, Candidia Corp., 1998
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Where Are We in the Biotech Continuum?
1978
Recombinant insulin produced
Industry Standard 1-2 g/L
2006
Improved strains
2015-2020
Future Leaders gt10 g/L
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Technologies in cell culture development
improving the building blocks

• Choice of molecule manufacturability assessment
• Expression vector design
• Host cell development
• Cell line development and screening

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Wyeth Biopharma Historical Experience Qp Range
for Clinical and Commercial Cell Lines
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Platform Process Development at Wyeth
Initiation of Clinical Mfg
Pilot
Transfection
Months

• Create production host
• Utilize standard production and purification
  conditions
• Limited selection of dosage forms

• Drivers
• Larger number of protein candidates in pipeline
• Speed to clinic
• Minimize investment before clinical POC

• Challenges
• Production in a biological system
• Each therapeutic protein is unique
• Just enough time to execute

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Platform Process Development at Wyeth
Initiation of Clinical Mfg
Pilot
Transfection
Months

• Manufacturability assessment
• Integrate with our research colleagues to assess
  several candidate therapeutics before one is
  selected for development

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Selection of Molecules for Development
Candidate therapeutics Target A
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Goals of the Manufacturability Assessment

• Determines fit with platform process development
  paradigm
• Not intended to reject molecules from development
• Establishes expectations with regard to timing
  and material production
• Identifies unique attributes of molecule prior to
  the initiation of process development
• Applied to all candidate biotherapeutics
• Antibodies
• Fc fusions
• Recombinant proteins

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Key Steps in the Manufacturability Assessment

• Expression level
• Stable CHO pools
• Biochemical attributes
• Levels of aggregate (ability to remove)
• Product heterogeneity
• Modifications (some that you want, some that you
  dont)
• Solubility/ stability
• Compatible with clinical dose and delivery
  expectations
• Completed prior to the initiation of process
  development
• 2-4 candidates evaluated for each target
• 1 candidate selected for platform process
  development

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Establishing Relevant Expression Assessment Tools

• Rapid and robust
• Predictive of performance trends observed during
  process development
• Ability to compare to historical programs
• CHO stable pools

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Expression System Impacts Expression Assessment
Antibody
2-fold difference
20-fold difference
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Expression Assessment is Predictive
Note expression assessment and process
development cell lines are derived from
independent efforts and methods
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Variation in Expression of Different Antibodies
in CHO Pools
Same host, vector, gene configuration, MTX
selection level, media, culture format
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Variation in Expression of Different Antibodies
in CHO Clones
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Expression Assessment Experience at Wyeth
Antibodies
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Collaborative Assessment Balances Risk
5 candidate therapeutics (humanized
variants) Target A
Manufacturability Assessment mAb x.1 gt20 fold
lower expression

• x.5 selected for development
• Production of gt2 kg in one clinical batch
  (platform process)
• Program met the platform expectations

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Technologies in cell culture development
improving the building blocks

• Choice of molecule manufacturability assessment
• Expression vector design
• Host cell development
• Cell line development and screening

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High RNA Expression per Integrated Gene Copy
Without Formal Gene Amplification
CHO stable pools expressing a soluble Receptor-Fc
fusion protein
- Improves timelines - Enables HTS strategy
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Technologies in cell culture development
improving the building blocks

• Choice of molecule manufacturability assessment
• Expression vector design
• Host cell development
• Cell line development and screening

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Wyeth NICB/Dublin City University
CollaborationProject Scope Potential Outcomes

• Project Scope Development of a deeper
  understanding of CHO cell biology (potential
  development of a rationalized cell engineering
  strategy)
• Integrated use of Microarry Proteomics tools to
  interrogate selected CHO cell samples
• Identification of gene protein targets
  influencing phenotypes of interest
• Potential Practical Outcome Move the CHO
  manufacturing platform to a new level of
  capability (1-3g Mabs /L? gt10g /L)
• Better meet capacity demand for successful
  biopharma products
• Improved utilization of existing manufacturing
  capacity

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Wyeth NICB/Dublin City University
Collaboration Benefits to Project Success

• Intellectual Partnership
• Resource/critical mass increase
• Complementary expertise
• Wyeth
• Large library of CHO cell lines (20 years of PD
  experience)
• Cell culture technology infrastructure
• Affymetrix technology infrastructure
• First generation Affymetrix custom CHO chip
• NICB/DCU
• Bioinformatics expertise
• Proteomics technology
• Affymetrix technology

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Intended Path From Target IdentificationTo Cell
Engineering
Y0
Y1
Y2
Y3
Y4
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Omics Technology Platforms

• Genomics Platform (Wyeth)
• Affymetrix Custom CHO chip (M. Melville, et al.
  CCE-IX. 2004)
• 3500 CHO sequences (15 20 coverage of the CHO
  transcriptome)
• Proteomics Platform (DCU/NICB)
• 2-D DIGE (Differential In-Gel Electrophoresis)
• Typhoon variable mode imager
• DeCyder Differential Analysis Software
• Automated gel spot picker
• Identification of protein spots via MALDI-TOF
  analysis of tryptic peptides

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Industrially Relevant Phenotypes Generation of
the CHO Sample Plan

• Industrially relevant cell phenotypes -
  phenotypes that enable highly productive
  fed-batch CC processes
• Phenotypic categories were populated with the
  appropriate CHO cell samples taken from shake
  flask bench top bioreactor cultures
• 375 individual samples (including biological
  triplicates or quadruplicates multiple cell
  culture time points )
• 29 different CHO lines expressing Mab,
  cytokines, coagulation factors Fcreceptor
  fusion molecules.

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Industrially Relevant Phenotypes Generation of
the CHO Sample Plan
An eighth category of samples taken from
representative platform fed-batch processes were
also included
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Sample Preparation
Total RNA
RNA Purified
QC on Bioanalyzer
A260
Biological Replicate Cultures
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Technologies in cell culture development
improving the building blocks

• Choice of molecule manufacturability assessment
• Expression vector design
• Host cell development
• Cell line development and screening

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Drivers for a Platform Approach

• Expectations for increased pipeline throughput
• Increased clinical uncertainty for many/ most
  candidates
• Speed to clinical evaluation critical
• Ensure best use of Development resources and
  facilities
• Minimize investment in manufacturing process
  before clinical POC
• Create capacity - more projects and/or
  Technology Platform improvements
• Improve efficiency and predictability of outcome
• Standard approach and practices
• Defined options and expectations for each
  development area
• Consistent metrics for platform assessment and
  cross-project comparisons
• Trend towards antibodies
• May better fit a standard work process

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Wyeth Biopharma Phase 1 Cell Line Development
Platform
- Aggressive but balanced cell line development
window (6 months)
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Transfectant and Screen Large Number of
Cells(t0-2 months)

• Each transfected cell is unique
• Genotype - number and chromosomal location of
  integrated product genes
• Phenotype cell metabolism, growth rate, ceiling
  density
• Robotics and HT technologies to pick and screen
  hundreds of cell clones

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High Throughput Titer Screening

• Results of up to 1500 samples in one day
• The High-throughput Titer Assays shortened the
• development timeline and enable us to select
  high
• producer.
• The fast turnaround of results also enable
  technology
• development.

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Automation/ High Throughput Tools

• CLONEPIX (Genetix) HT clone picking
• More than 300 clones/hour
• Alamar Blue HT cell counting
• Guava (Guava Technologies) HT cell counting/
  viability
• BioMek workstation (Beckman Coulter) plus
  Bioveris Meso-Scale Discovery assay platform HT
  titer assessments
• Up to 720 samples in 3 hours
• Titer and binding activity
• BioMek workstation - HT PrA purification and
  analytics
• Up to 336 samples in 2 hours for further
  characterization
• Charge, size/ aggregate, glycosylation

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Implementation of Automation Systems to High
Throughput Titer Screening

• Biomek FX Workstation
• 96-probe automation system
• high throughput
• Application on our HTS
• Sample dilution and reagent distribution

• TECAN Genesis
• 8-tip automation system
• moderate throughput
• Application on our HTS
• Standard and control prep.

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High Throughput Titer Screening Assay
720 Conditioned Media Samples
Sample Dilution and Reagent Distribution
Biomek FX TECAN 3 hours
Incubation
2 hours
Instrument Reading
Bioveris4hrs
Data Process
2 hours
Results
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Reproducible Outcome from HTS Process
Comparison of limiting dilution and automated
CLONEPIX cloning in primary HTS
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High Throughput Purification (HTP)
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SEC HPLC Data (HMW) of Small Scale Fed-batch
Studies
24 clones, stdev 0.1-0.2
The methods enabled us to obtain product
characterization information of the evaluated
clones within the project time frame.
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Options for further cell line development

• Recloning/ reselection same cell line but new
  cell bank
• Increased material needs
• Improved stability
• New transfection - new cell line and cell bank
• Increased material needs/ instability
• Improved product quality
• Host cell engineering
• Ideally done pre-Phase 3
• Regulatory path more straightforward
• May improve efficiency of Phase 2 re-supply

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Increased Cellular Productivity Following
Reselection in Higher MTX
HTS/ subcloning of Phase 1 Ab cell line in
original or increased levels of MTX
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Further Development of CHO Production Cell Lines
Has Yielded Significant Benefits
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Summary
Innovations provide faster speed to market and
dramatic cost reductions
i
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Acknowlegements

• Mark Leonard
• Marty Sinacore
• Haley Laken
• Judy Chou
• Mark Melville
• Yen-Tung Luan
• Bob Adamson
• Louane Hann
• Steve Max

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tcharlebois_at_wyeth.com