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Eukaryotic Cells Ride the Wave
Expanding the Drug Discovery Pipeline by Improving Protein
Production Technology
Joey M. Studts, Ph.D.
Boehringer Ingelheim, Ridgefield, CT
Keck Graduate Institute
October 24, 2006
Riding the Wave
Outline
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• Introduction to Boehringer Ingelheim
• Overview of Drug Discovery Process
• Introduction of protein production strategies at BI
• Expression Screening – Two methods for different problems
• Method to optimize time from DNA to Biomass (known protein targets)
• Protocols
• Overview of current data
• Parallelized method for screening and expression optimization (novel
protein targets)
• Protocol and protocol optimization
• Two case studies
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Boehringer Ingelheim
Worldwide
Finland
Canada
USA
Egypt
Mexico
Panama
Venezuela
Colombia
Ecuador
Peru
Chile
Belarus
Russia
Ukraine
Kazakhstan
Uzbekistan
Japan
Korea
Turkmenistan
China
Hong Kong Taiwan
United Arab Emirates
Philippines
Saudi Arabia
Vietnam
Thailand
India
Malaysia
Indonesia
Brazil
Uruguay
Argentina
South Africa
Boehringer Ingelheim companies
Minority interest, or agency
Research Sites
Australia
New
Zealand
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Boehringer Ingelheim
The Fastest Growing Company
Growth (%) in 2005
BI Growth
+23.3%
( 1) Boehringer Ingelheim
( 2) Amgen
( 3) Teva + Ivax
( 4) Novo Nordisk
( 5) Roche
( 6) Otsuka
( 7) Merck KGaA
( 8) Novartis + Chiron
( 9) AstraZeneca
(10) Abbott
(11) Sanofi-Aventis
(12) Daiichi Sankyo
(13) Takeda
(14) Schering AG
(15) Lilly
(16) Eisai
(17) GlaxoSmithKline
(18) Astellas Pharma
(19) Bayer
(20) Wyeth
(21) Schering Plough
(22) Johnson & Johnson
(23) Merck & Co.
(24) Bristol-Myers Squibb
(25) Pfizer
Global Market
Growth +6.2%
-10
-5
0
5
10
15
20
25
Boehringer Ingelheim
Ridgefield, Connecticut
Therapeutic Areas:
• Cardiovacular
• Immunology &
Inflammation
Size
• 400 Acres
• ~3500 People on
site
• >750 in R&D
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Drug Discovery
Key Players…
¾ Target: A protein involved in
a disease whose activity can
be modified to improve
health.
¾ Lead: A chemical entity that
can modulate a target’s
function and putatively
modify disease.
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Timelines for protein production
R&D vs. PD/Production
Average: 12 Years and $800 Million (in 2003)
2-3 Years
+
2 Years
+
+
5 -6Years
1Year
12
= Years
Discovery
Preclinical
Development
Clinical Trials
FDA
Rx
1 2 3
4 5
6 7 8 9 10 11
12
13
I
II
III
‰ Protein target required quickly to being drug discovery
process
‰ Process development starts in years 2-3, have delivery
target of year 5-7.
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Timelines for protein production
R&D vs. PD/Production
Average: 12 Years and $800 Million (in 2003)
2-3 Years
+
2 Years
+
+
5 -6Years
12
= Years
1Year
Discovery
Preclinical
Development
Clinical Trials
FDA
Rx
1 2 3
4 5
6 7 8 9 10 11
12
13
I
II
III
Research and
Discovery*
Process
Development*
Production*
12 -16 weeks
40 weeks
2-3 years
Biomass Scale
0.1 – 25 L
1 – 80 L
80 – 15,000 L
Product Scale
10 – 100 mg
1-10 g
> kg
Timelines
*Averages from literature references and CMO websites
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Protein Resource
Responsibilities
Screening,
LI, and LO
Structural
Research
Target
Validation
Protein
Characterization
Protein Resources
Cloning, Expression, Purification
Tox
Proteomics
Collaborations
Cellular
Genomics
Protein Production
The Critical Path…
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• Often the critical path to High Throughput Screening
• Always the critical path to getting structural information on a
protein target.
• Faster we can produce 10-100 mg of a target protein, the more
efficient the drug discovery process.
Keys Points to Take Away
• Look at the process
• Understand process: strategy and science!
• Identify bottlenecks in that process to improve efficiency and
effectiveness
• Expand bottlenecks via strategy changes and scientific changes.
Project Flow – Protein Resources
~4 months to protein delivery
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Cloning
Gene Synthesis
• Collaborators Deliver
• Sequence verified DONOR
vector
• Raw Sequencing Data
• Advantages
• Paralleled process
• Guaranteed/reproducible
timelines
• 3 weeks to DONOR
• 4 weeks to Expression vector
• For HT Strategy used to
obtain wild-type construct
• FTE Focus on expression
screening, not cloning
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Target Protein Sequence (in silico)
Reverse transcription
Codon optimization
Target DNA Sequence
Oligo Synthesis
PCR primer extension
Target DNA Sequence
Sequence verification
Subcloning into vector
Target DNA Sequence
Introduction to BEVS
Benefits of BV for R&D production
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• Eukaryotic cell lines
• Cells fairly robust
• Species fairly well defined.
• Eukaryotic protein processing & post-translational
modifications.
• Expression protocols fairly rapid and highly reproducible
• Timelines fairly short relative to other eukaryotic expression
technologies.
• Once virus produced, bench scale fermentation highly
reproducible
• Yields 1-50 mg/L, consistent with research needs.
Introduction to BEVS
Infection Progression
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• Viral RNA can be detected 30 minutes after inoculation – Viral DNA
arriving in the nucleus and initiation of early transcription is a
rapid process.
• DNA replication begins 6 hours post infection
• Early Phase: First 6 hours of infection – Cytoskeletal
rearrangement, nucleus enlargement, host chromatin disperses
• Late Phase: 6 – 24 hpi - Characterized by viral DNA replication, late
gene expression, and BV production – Virogenic Stroma form in
nucleus
• Very Late Phase: Begins around 20-24 hpi – Production of BV
greatly reduced, p10 accumulation, by 72hpi cells are no longer
synthesizing proteins
Baculovirus Expression Screening
Two Pronged Approach
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Known Target
Design 5-10
Constructs
Synthesize &
Subclone
Transfect, Screen
and Scale-up Virus
Target
Approval
Design 10-100
Constructs
< 10
Construct &
Subclone
Novel Target
Transfect &
Screen
Select Optimal < 5
Construct(s)*
Biomass
Production
< 10
Identify Optimal >10 Small Scale Expression
Constructs*
and Characterization
*optimal construct selected
based on expression levels
Production Process
Goals
Baculovirus Expression Group:
Alycia Shoultz Terry Wilson
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Nene Kalu
Goals
• Develop process to rapidly screen and scale up construct for
baculovirus expression
• Optimized for time to protein
• Process designed to be reproducible and to supply protein for
long term projects
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Virus Scale-up to Production
Standard Strategy
Expression
Screen
Standard Strategy
1
5
Transfection
10
Plaque
P1
Purification.
15
19 20
P2
P3
Virus
Stock
25
27 Plaque Assay
Express. Opt.
P4
• Initial expression screening via SDS-PAGE and Western analysis at
~3 weeks, very slow feedback times
• 48 days to biomass with titered virus bank
• MOI optimization studies added 1-2 weeks and often not
reproducible in bioreactor
48 Days to
Biomass
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Viral Scale-up to Production
Screening and Production Process
Expression
Screen
Standard Strategy
1
5
10
Plaque
P1
Purification.
Transfection
15
19 20
P2
P3
Virus
Stock
25
Virus
Stock
Expression
Screen
20
P3
1
5
Transfection
Plaque
Purification.
10
P1
Express. Opt.
P4
Current Process
Expression
Screen
27 Plaque Assay
Plaque Assay
15
P2
20
Wave
Reactor
20 Days to
Biomass
• Expression Screening at 4 days
•
Earlier read on expression helps with project decisions
48 Days to
Biomass
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Viral Scale-up to Production
Screening and Production Process
Expression
Screen
Standard Strategy
1
5
Transfection
10
P1
Plaque
Purification.
15
19 20
P2
P3
Virus
Stock
25
P4
Virus
Stock
Current Process
Expression
Screen
Expression
Screen
20
P3
1
5
Transfection
Plaque
Purification.
10
P1
Plaque Assay
15
P2
• Biomass in 20 Days!
•
•
•
27 Plaque Assay
20
Wave
Reactor
20 Days to
Biomass
Directly from P2 to fermentation
Carry-out plaque purification maintain reproducibility
Viral titer after initial fermentation
• Not in critical path
Express. Opt.
48 Days to
Biomass
Current Process Protocol…
Optimized for time to biomass
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Step
Description
Transfection
Plaque
Purification
1 mL of viral supernatant, 4 days, 6 well plate, adherent cells
60 mm tissue culture plates, 5 days, adherent cells
Screen
P1
P2
Expression screen with Western
5 mL viral supernatant, 5 days, T-25 flask, adherent cells
100 mL viral supernatant, 3 days, 250 mL shake flask
suspension cells
Expression screen with Western
5L Wave BioReactor, 48-72 hour infection
500 mL viral supernatant, 3 days
Master virus bank
Titered (normally 10 fold increase between P2 and P3)
Archived
Screen
Fermentation
P3
Protein Expression- Baculovirus
Cedex Information
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Following the Course of Infection
Cedex Instrument
• Cedex Data
• Cell viability
• Cell density
• Cell diameter
100
Cell Density
28
90
27
Cell Viability
80
Cell Size
26
Infected
25
Harvest
135.9 grams
50
40
% Viable
Cells / ml
60
24
23
30
22
20
10
5
0
10
0
1
2
Day
3
21
20
Cell Diameter (uM)
70
Viral Scale-up Monitoring
Key Statistics for Volume Based MOI
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Infect Wave Bags with the P2 Virus. (The amount of virus used to infect is
determined by the cell data and the SDS-PAGE/Western Blot results.)
If at the P2 stage:
Viability
Cell Diameter Cell Density
Expression
5L Infection
Range
Range
Range
Level @ P2
Volume
<70%
<24 μm
No significant Fair
0.5-1.0 mL/L
increase
>80% but
>26 μm
No significant Good to
2.0 mL/L
<90%
increase
Excellent
< 28 μm
>95%
>24 μm
Slight
Decent to
5 mL/L
increase
Excellent
<26 μm
>90%
<24 μm
Significant
Fair to Decent 10-20 mL/L
increase
Expression Level Rating System:
1. Poor 2. Fair 3. Decent 4. Good 5. Excellent
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Expression Screening
Example Western Rankings
MW 567
A
100kD ►
75kD ►
1
567
634 634
697 697 703 703
718 718
B
A
A
A
B
5
B
4
A
B
3
Neg
B
2
50kD ►
Subjective Rating System:
1. Poor 2. Fair 3. Decent 4. Good 5. Excellent
MW
Protein Expression – Eukaryotic
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Large Scale Expression
Stir Tank Bioreactors
3x25L, 1x15L
Wave Bioreactors
4x25L, or 4x10L, or 8x5L
(6x25, or 6x10L, or 12x5L)
Large Scale Expression
Wave BioReactor
• Greatly increased capacity
and decreased timelines!!!
• 1 reactor = 2x5L or 1x10L
• 2 rounds/reactor/week
• No cleaning/sterilization
• Turn-around time a matter of
minutes
• FTE costs are very low!!!
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Optimized Throughput System
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Statistics on Success
Key:
This is not an Expression Optimization Strategy
Success based: on
reproducing
deliveringscreening
protein toresults
project
•
•
•
•
•
95% - Successful on 1st run
+ 4% - Successful on 2nd run
1% - Follow-up optimization required
Constructs labeled as “poor expressers” not included
Purified Protein Yields
•
•
•
•
Average Production Level – 6.8 mg/L
Median Production Level – 3.4 mg/L
High is 69.0 mg/L
Low is 0.11 mg/L
• 80% of proteins going for Structural Research efforts
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Purified Protein Yields
Optimized for time to biomass…
80
Protein Yield (mg/L
70
60
50
40
30
20
10
0
1
8
15
22
29
36
43
50
57
64
71
Fermentation Number
78
85
92
99 106
Parallelized Expression Screening
Goals
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HTC&E Group - Jim King, Ph.D.
Mike DiCandido
Jonathan Hill
Xiaoyan Yang
Alycia Shoultz
Goals
• Develop protocols for a paralleled approach for cloning and
expression screening
• Develop automated processes for executing these protocols
• Provide highly paralleled expression screening data to quickly
and efficiently identify highly expressing clones.
Construct Design
Parallelized Screening Process
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• Currently the major variable being tested in expression screening
• Two approaches
• N- and C-terminal truncations
• Mutation scanning
• Other expression variables tested
•
•
•
•
infection time
MOI
temperature
cell density at induction
Two Approaches
Domain Scouting by PCR Truncation Screening
Domain 1
Domain 2
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Target Domain
etc…
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Two Approaches
Mutation Scanning (single or multiple AA)
Mutation Scanning (single or multiple amino acids)
* * * * * * * * * *
Target Domain
*
*
*
*
*
*
*
*
*
*
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Paralleled Approach - Cloning
HTC&E Skill Center
Gateway Cloning™ efficiently switch expression systems
High-Throughput PCR
Construct Design
Qualify templates
Primer ordering
PCR
BP-LR recomb.
Sequence confirm clones
Bacmid trans.
Bacmid Prep
Bacmid QC
Transformation
O/N growth
Cloning Timeline
7
14
Design constructs, order supplies, qualify template
3 – 4 weeks
21
1
PCR & minipreps
3 weeks
7
14
Sequence Analysis
21
Bacmid Prep. & QC
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Protein Expression Screening
Expression Data in 7 Days
Virus
Stock
Viral Scale-up – 15 clone throughput
Expression
Screen
Expression
Screen
20
P3
1
5
10
Transfection Plaque Purif. P1
Plaque Assay
15
P2
20
HT Expression Testing - Scalable
Expression
Screen
1
Wave
Reactor
20 Days to
Biomass
5
Transfection P1
• Optimized timelines and protocols for small scale reproducibility
• Expression screen by magnetic beads purification and Caliper LC90 give
objective, quantifiable results
Parallelized Expression Screening
Initial Protocol
Step
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Description
Cloning
DNA Plate delivered in 96 well plate format.
Transfection 24 well plate, 2 mL, 5 Days, suspension cells.
P1
Expression
Screen
24 deep-well plate, 48 hrs, suspension cells, 5 mL culture
Transfect at two different volume based MOI (100 and 400 uL
virus)
Screen using affinity purification and Caliper LC90
Prioritize
Identify optimal expressing clones
If > 10
Constructs
100 ml, 48 hour expression test in shake flask for biomass
generation
Small scale purifications to access protein stability and solubility
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Case Study
Domain Scanning Project A
BD
1
Functional Domain
158
675
158
665
675
298
298
N-terminal GST – 13/13 constructs
N-6xHis – 10/13 constructs
C-6xHis – 12/13 constructs
BD = Binding Domain
675
665
309
675
309
665
318
675
318
665
328
675
328
665
675
338
665
338
352
352
675
665
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Expression Screening
MagneGST, SDS-PAGE, Western, LC90
1 2 3 4 5 6 7 8 9 10 11 12 13 14 N
1 2 3 4 5 6 7 8 9 10 11 12 13 14 N
1
2
3 4
5 6
7
8 9 10 11 12 13 14
1
BD-FD(158-675)-100
2
BD-FD(158-675)-400
3
FD(298-665)-100
4
FD(298-665)-400
5
FD(298-675)-100
6
FD(298-675)-400
7
FD(309-665)-100
8
FD(309-665)-400
9
FD(309-675)-100
10
FD(309-675)-400
11
FD(318-665)-100
12
FD(318-665)-400
13
FD(328-675)-100
14
FD(328-675)-400
15
Negative control
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Expression Trails – Project A
Reproducibility Lower with P1 Screening
Conc. 100 Inoc. (ng/ul) - clone #1
3000
Conc. 500 Inoc. (ng/ul) - clone #1
Conc. 100 Inoc. (ng/ul) - clone #2
2500
Conc. 500 Inoc. (ng/ul) - clone #3
Conc. 400 Inoc. (ng/ul) - clone #1
ng/ul
2000
Conc. 400 Inoc. (ng/ul) - clone #3
1500
1000
500
-
-
-
-
-
-
-
-
-
-
-
0
• Data from multiple screening tests at two different MOI levels.
• Variability seemed high
• Different MOI not as reproducible
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Data Collection
Caliper LC90
• Micro-fluidic implementation
of electrophoretic separation
of protein samples.
Instrument scans the fluid
path over time to detect
protein bound dye.
• Data is captured as a trace,
area of peaks are integrated to
give a protein concentration
measure. Software can
convert data to a “Gel View”.
Caliper claims
30% conc. CV
Average of 7 LC90 runs
120
100
ng/ul
80
60
40
20
0
1
800
LM
600
500
157 ng/μl
53.3 kDa
400
300
200
157.11
*
14.29
5.52
7.05
5.67
91.74
209.02
100
0
2
3
4
5
6
7
Samples
700
Fluorescence
Reproducibility of LC90
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
Time (seconds)
8
9
10
11
12
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Protein Expression Screening
Expression Data in 9 Days
Virus
Stock
Viral Scale-up – 15 clone throughput
Expression
Screen
Expression
Screen
20
P3
1
5
10
Transfection Plaque Purif. P1
Plaque Assay
15
P2
20
HT Expression Testing - Scalable
Expression
Screen
1
5
Transfection P1
Wave
Reactor
20 Days to
Biomass
9
P2
• Each passage results in ~ 10-fold increase in viral titer
• Increased virus titer increases reproducibility and protein production
Case Study
Project B Construct Design
Binding 1
Binding 2
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Disordered in crystal
Catalytic Domain
433-725
448-725
N-terminal GSTN-terminal 6HisC-terminal 6His-
433-718
433-725
433-735
436-718
436-725
439-718
439-725
442-718
442-725
445-718
445-725
448-718
448-725
462-718
462-725
0
433-718
436-718
439-718
442-718
445-718
448-718
462-718
433-725
436-725
439-725
442-725
445-725
448-725
462-725
433-735
433-718
436-718
439-718
442-718
445-718
448-718
462-718
433-725
436-725
439-725
442-725
445-725
448-725
462-725
433-735
433-718
436-718
439-718
442-718
445-718
448-718
462-718
433-725
436-725
439-725
442-725
445-725
448-725
433-735
ng/ul
Project B Construct Design
Expression Screening
GST
N-terminal 6xHis
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350
300
250
200
150
100
50
C-terminal 6xHis
• Expression Screening Carried out after P2 amplification
• Higher titers after P2 amplification but same volume
• Carry-out duplicate experiments with single MOI
• Increased reproducibility after P2
Parallelized Expression Screening
Protocol Overview
Step
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Description
Cloning
DNA Plate delivered in 96 well plate format.
Transfection 24 well plate, 5 Days, adherent cells.
P1
P2
Expression
Screen
24 deep-well plate, 48 hrs, suspension cells, 5 mL culture.
24 deep-well plate, 48 hrs, suspension cells, 5 mL culture.
Pellets frozen over week-end.
Screen using affinity purification and Caliper LC90
Prioritize
Identify optimal expressing clones
If > 10
Constructs
100 ml, 48 hour expression test in shake flask for biomass
generation
Small scale purifications to access protein stability and solubility
Overview…
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Expression Screening – Two methods for different problems
• Method to optimize time from DNA to Biomass (known protein targets)
• Parallelized method for screening and expression optimization (novel
protein targets)
• Understand a process
• Strategy
• Science
• Eliminate bottlenecks in process
• Strategy
• Technology
• Science
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Acknowledgements
Translational Sciences Department – Sheenah Mische, Ph.D.
John Miglietta
Gale Hansen
Biologics and Biomolecular Sciences Department - Christine Grygon, Ph.D.
Protein Resources - Joey Studts, Ph.D.
Kevin Barringer – cloning
Alycia Shoultz – expression
Terry Wilson – expression
Anthony Kronkaitis – expression
Xiang Zhu, Ph.D. - expression
Daniela Cazacu – exp./purif.
Elda Gautschi – purification
Teresa Roma – purification
Alistair Baptiste – expression
High Throughput Cloning and Expression – Jim King, Ph.D.
Michael Dicandido – cloning
Jonathan Hill – IT/cloning
Xiaoyan Yang – expression
Structural Research Department – Sandy Farmer, Ph.D.
Neil Farrow
Mariana Margarit
Kathleen Haverty
Boehringer Ingelheim Production
Biberach, Germany
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New Biopharmaceutical
Production Plant
Image: Boehringer Ingelheim GmbH
http://www.boehringer-ingelheim.com/biopharm/news/biberach.htm
Boehringer Ingelheim Production
Biberach, Germany
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New Biopharmaceutical
Production Plant
Image: Boehringer Ingelheim GmbH
http://www.boehringer-ingelheim.com/biopharm/news/biberach.htm
Boehringer Ingelheim Production
Biberach, Germany
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New Biopharmaceutical
Production Plant
Image: Boehringer Ingelheim GmbH
http://www.boehringer-ingelheim.com/biopharm/news/biberach.htm