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Timothy S' Charlebois, Ph'D'

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Technology and Opportunities in Mammalian Cell Culture. The Evolution of Wyeth ... BioMek workstation (Beckman Coulter) plus Bioveris Meso-Scale Discovery assay ... – PowerPoint PPT presentation

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Title: Timothy S' Charlebois, Ph'D'


1
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

2
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
6
Currently transgenic systems do not present a
transforming advantage over CHO
Comparison of Monoclonal Antibody Produced from
CHO Transgenic Goats
7
The Majority of Biotech Products on the Market
Are Made in Animal Cells
8
5 of the Top 20 Products Globally Are
Biotechnology Products
Source IMS data, 2005
9
Fastest Growing Classes of Drugs are Biotech Drugs
BIOTECHNOLOGY
Source IMS data, 2005 Q3 MAT
10
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)
11
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

12
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

13
Wyeth examples of biotech capital investments
Dublin, Ireland
Andover, MA
14
Investment in Bench-Scale InnovationGreat
Potential Return
Application of Improved Process Technology
Platform Process Rapidly Developed
9.6 g/L
3 g/L
15
Capital Expenditure Can Be Limited by Applying
Innovation
16
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
17
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
18
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

19
Wyeth Biopharma Historical Experience Qp Range
for Clinical and Commercial Cell Lines
20
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

21
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

22
Selection of Molecules for Development
Candidate therapeutics Target A
23
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

24
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

25
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

26
Expression System Impacts Expression Assessment
Antibody
2-fold difference
20-fold difference
27
Expression Assessment is Predictive
Note expression assessment and process
development cell lines are derived from
independent efforts and methods
28
Variation in Expression of Different Antibodies
in CHO Pools
Same host, vector, gene configuration, MTX
selection level, media, culture format
29
Variation in Expression of Different Antibodies
in CHO Clones
30
Expression Assessment Experience at Wyeth
Antibodies
31
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

32
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

33
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
34
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

35
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

36
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

37
Intended Path From Target IdentificationTo Cell
Engineering
Y0
Y1
Y2
Y3
Y4
38
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

39
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.

40
Industrially Relevant Phenotypes Generation of
the CHO Sample Plan
An eighth category of samples taken from
representative platform fed-batch processes were
also included
41
Sample Preparation
Total RNA
RNA Purified
QC on Bioanalyzer
A260
Biological Replicate Cultures
42
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

43
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

44
Wyeth Biopharma Phase 1 Cell Line Development
Platform
- Aggressive but balanced cell line development
window (6 months)
45
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

46
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.

47
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

48
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.

49
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
50
Reproducible Outcome from HTS Process
Comparison of limiting dilution and automated
CLONEPIX cloning in primary HTS
51
High Throughput Purification (HTP)
52
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.
53
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

54
Increased Cellular Productivity Following
Reselection in Higher MTX
HTS/ subcloning of Phase 1 Ab cell line in
original or increased levels of MTX
55
Further Development of CHO Production Cell Lines
Has Yielded Significant Benefits
56
Summary
Innovations provide faster speed to market and
dramatic cost reductions
i
57
Acknowlegements
  • Mark Leonard
  • Marty Sinacore
  • Haley Laken
  • Judy Chou
  • Mark Melville
  • Yen-Tung Luan
  • Bob Adamson
  • Louane Hann
  • Steve Max

58
tcharlebois_at_wyeth.com
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