Title: Timothy S' Charlebois, Ph'D'
1Technology 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
2The Evolution of Wyeth BioPharma
3Current Biopharmaceutical Products Driving 3
Billion in Revenue
Launched 1997
Launched 1998
Launched 1998
Launched 2000
Launched 2001
Launched 2002
4From Gene to Drug Product
Purification
Cell Culture
Formulation
Thaw
Cell
Gene
5Current Choices of Host Cells in Biotech
Yeast
Transgenic Animals
Bacteria Cells
Transgenic Plants
Animal Cells
6Currently transgenic systems do not present a
transforming advantage over CHO
Comparison of Monoclonal Antibody Produced from
CHO Transgenic Goats
7The Majority of Biotech Products on the Market
Are Made in Animal Cells
85 of the Top 20 Products Globally Are
Biotechnology Products
Source IMS data, 2005
9Fastest Growing Classes of Drugs are Biotech Drugs
BIOTECHNOLOGY
Source IMS data, 2005 Q3 MAT
10Current 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)
11Mammalian 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
12Producing these Products has required huge
capital investment
- Design to approval gt 5 years
- gt1,000,000 liters worldwide
- Hard to match capacity to need
13Wyeth examples of biotech capital investments
Dublin, Ireland
Andover, MA
14Investment in Bench-Scale InnovationGreat
Potential Return
Application of Improved Process Technology
Platform Process Rapidly Developed
9.6 g/L
3 g/L
15Capital Expenditure Can Be Limited by Applying
Innovation
16Innovation 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
From PenicillinA Paradigm for Biotechnology Ed.
R.I. Mateles, Candidia Corp., 1998
17Where 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
18Technologies in cell culture development
improving the building blocks
- Choice of molecule manufacturability assessment
- Expression vector design
- Host cell development
- Cell line development and screening
19Wyeth Biopharma Historical Experience Qp Range
for Clinical and Commercial Cell Lines
20Platform 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
21Platform 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
22Selection of Molecules for Development
Candidate therapeutics Target A
23Goals 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
24Key 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
25Establishing Relevant Expression Assessment Tools
- Rapid and robust
- Predictive of performance trends observed during
process development - Ability to compare to historical programs
- CHO stable pools
26Expression System Impacts Expression Assessment
Antibody
2-fold difference
20-fold difference
27Expression Assessment is Predictive
Note expression assessment and process
development cell lines are derived from
independent efforts and methods
28Variation in Expression of Different Antibodies
in CHO Pools
Same host, vector, gene configuration, MTX
selection level, media, culture format
29Variation in Expression of Different Antibodies
in CHO Clones
30Expression Assessment Experience at Wyeth
Antibodies
31Collaborative 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
32Technologies in cell culture development
improving the building blocks
- Choice of molecule manufacturability assessment
- Expression vector design
- Host cell development
- Cell line development and screening
33High 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
34Technologies in cell culture development
improving the building blocks
- Choice of molecule manufacturability assessment
- Expression vector design
- Host cell development
- Cell line development and screening
35Wyeth 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
36Wyeth 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
37Intended Path From Target IdentificationTo Cell
Engineering
Y0
Y1
Y2
Y3
Y4
38Omics 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
39Industrially 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.
40Industrially Relevant Phenotypes Generation of
the CHO Sample Plan
An eighth category of samples taken from
representative platform fed-batch processes were
also included
41Sample Preparation
Total RNA
RNA Purified
QC on Bioanalyzer
A260
Biological Replicate Cultures
42Technologies in cell culture development
improving the building blocks
- Choice of molecule manufacturability assessment
- Expression vector design
- Host cell development
- Cell line development and screening
43Drivers 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
44Wyeth Biopharma Phase 1 Cell Line Development
Platform
- Aggressive but balanced cell line development
window (6 months)
45Transfectant 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
46High 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.
47Automation/ 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
48Implementation 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
50Reproducible 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.
53Options 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
54Increased Cellular Productivity Following
Reselection in Higher MTX
HTS/ subcloning of Phase 1 Ab cell line in
original or increased levels of MTX
55Further Development of CHO Production Cell Lines
Has Yielded Significant Benefits
56Summary
Innovations provide faster speed to market and
dramatic cost reductions
i
57Acknowlegements
- Mark Leonard
- Marty Sinacore
- Haley Laken
- Judy Chou
- Mark Melville
- Yen-Tung Luan
- Bob Adamson
- Louane Hann
- Steve Max
58tcharlebois_at_wyeth.com