Recent Advances in Cell Culture Technology:

Improvements in Biopharmaceutical
Production and Cost

International Knowledge Millennium Conference

October 31 - November 2, 2004
Hyderabad, India

Howard L. Levine, Ph.D.

BioProcess Technology Consultants, Inc.
Acton, MA
Biopharmaceutical Products
¾ Products made by or composed of viable organisms and
biopolymer analogs
• Natural & rDNA Proteins, Hormones, Peptides
• Monoclonal & Polyclonal (natural) Antibodies
• Antibiotics, Plant & Animal Extracts, Allergens
• Vaccines, Cell & Gene Therapy
• Human & Xenogenic Cells & Tissues
• Blood & Blood Derivatives
¾ Early recombinant proteins were simple proteins, usually
replacement products for natural products
• Insulin
• Human Growth Hormone
• Alpha Interferon
¾ Today’s recombinant proteins and monoclonal antibodies
are more complex
Cost of Manufacturing of Biopharmaceuticals
¾ Processes are fixed-cost driven
¾ Manufacturing costs typically 15 – 25% of COGs
¾ Basic cGMP background identical to chemical drugs
¾ Complexity of products results in demanding technical
processes and high capital investments
¾ Factors influencing manufacturing costs
• Process design and plant capacity
• Operating Strategy
• Equipment and Facilities Costs
• Materials Costs
• Labor Costs
• Overhead Structure
Price and scale are inversely related…
Price, $/g

Production Scale, kg/yr

Ref: U. Gottschalk. bioLOGICS USA, 2004

Production Hosts Influence Manufacturing Costs
Bacteria Æ Yeast Æ Insect cells Æ Mammalian cells

Simple proteins Complex Proteins

No post-translational modifications Post-translational modifications
Relatively simple fermentation More complex fermentation
Generally lower COGs Relatively high COGs

¾ Production host will determine…

•
Quantity of product produced
•
Quantity and type of contaminants
•
Post-translational modifications, i.e., glycosylation
•
Economics & regulatory issues
¾ Compared to microbial fermentation, cell culture systems
are 50 – 60 fold less productive per liter
Effect of Expression Level on Manufacturing Costs

¾ Most products in development,

such as monoclonal antibodies 1.0
(MAb), require mammalian cell
culture
0.8
¾ Cell culture costs contribute

Relative Cost
approximately 50% of total
0.5
manufacturing costs
¾ Expression level is most
0.3
important parameter in
determining cell culture cost
0.0
¾ Expression level drives required
0 4 8 12
bioreactor capacity and overall
Relative Titer
facility size
 World-wide Manufacturing Capacity:
Today and Future
3,500
Reactor Volume ('000 L)

3,000

2,500

2,000

1,500

1,000

500

0
2003 2004 2005 2006 2007 2008
Year
Forecast Industry-wide Capacity Top 8 Potential Volume Drivers Fail
Capacity utilization remains <60% through 2007
Most Probable Case Top 8 Potential Volume Drivers Succeed
Capacity utilization increases to approx. 85% by 2007 Capacity fully utilized by 2007
Estimating Capacity Requirements for MAbs

¾ Total MAb requirements 100 Kg/yr

¾ Crude MAb (60% overall yield) 167 Kg/yr
¾ Total cell culture (0.5 g/L expression) 0.33 M L/yr
¾ Estimated batch size (25 batches/yr) 13,000 L

¾ Each MAb requires approximately one 15,000 L

bioreactor to meet market demand

¾ Increase in expression level to 2 g/L lowers requirement

to 3,500 L bioreactor per product or 7 batches per year
 Cost Implications of Process Optimisation

200 100%

160 80%

Re la tive Fe rm e nta tio n

N um be r o f B a tc he s

120 60% Assumptions:

R e quire d

Co s t %
¾ Product Requirement:
35 kg/yr
80 40%
¾ 65% overall yield

40 20%

0 0%
0.1 0.3 0.6 1.9 2.8
Product Titre (g/L)

2000L sc ale 5000L Sc ale Cost

Ref: Lonza GS brochure, 2004

 Historical and Projected Expression Levels

15,000

12,500
Expression Level, mg/L

10,000
10,000

7,500

5,000

2,500
10 100
0
1975 1985 1995 2005 2015
Year
Can Expression Levels be Increased?
¾ MAb expression levels have increased significantly over time
from less than 100 mg/L in 1975 to greater than 3 g/L today
¾ Predicted yields of >10 g/L within the next decade

Ref: G. Slaff. bioLOGICS USA, 2004

Cell Line Development and Production
Expression Vector Construction
and Transfection of Host Cell

Selection of individual clones

Screening for high expression

Preparation of cell banks for production

Expansion through growth and dilution

to produce inoculum for final reactor volume

Growth to high density and production

Each step in the process can be

optimized to increase expression
Mammalian Protein Expression Vector Features
¾ Promoter and enhancer drive transcription of desired protein
• CMV promoter commonly used today
¾ Selectable marker
• Dihydrofolate Reductase (DHFR)
− Native DHFR is duplicated in response to increasing Methotrexate (MTX)
− Technology developed in early 80’s uses amplification of DHFR on an
expression vector to co-amplify gene encoding biopharmaceutical product
• Glutamine Synthetase (GS)
¾ Improved promoters and enhancers can increase protein expression
• CHEF-1a promoter currently used for some products in clinic
• Ubiquitous Chromatin-opening Elements (UCOE)
− Allows prolonged expression in transfected pools to rapidly generate pre-
clinical material
− Enables selection of production clone by screening fewer initial clones
Improving Clone Selection
¾ Speed to identification of final high expressing production clone is critical to
rapid product development
¾ New strategies for clone selection allow more clones to be analyzed,
increasing odds of finding the rare high expressing clone
• Certain selectable markers increase percentage of high expressors
• High throughput screening methods can increase the likelihood that a
high expressing clone will be found
Titer µg/ml

Number of Clones Screened

Ref: B. Adamson, Manupharma, 2004
Cell Line Development – DHFR

Steps required for DHFR amplification

¾ Transfect cells and select in 0.01 – 0.2 nM MTX
¾ Identify clone with highest level of transgene expression
(generally 1 – 3 pg/cell/day)
¾ Dilute isolated clone and reselect in higher MTX level
¾ Identify clone with highest level of transgene expression (up to
12 pg/cell/day)
¾ Repeat until sufficient transgene expression is obtained

Amplification of DHFR gene requires 12 weeks per cycle

and can take up to 5 cycles to obtain a clone with high
enough expression for today’s biopharmaceutical
products
Cell Line Development – GS
Glutamine synthetase can be used to more rapidly
select high expressing cell lines without amplification

Ref: Lonza GS brochure, 2004

Process Optimization with the GS System

Cell Line Process Antibody (mg/L)

A 1 139

A 2 334

A 3 585

B 3 1917

B 4 2829

Ref: Lonza GS brochure, 2004

CHEF-1 Vector for Improved Expression
¾ Expression vector uses the homologous Chinese hamster EF1-α promoter
¾ CHEF1 promoter permits rapid isolation of highly productive cell lines
• Intrinsic expression in CHO cells significantly higher than CMV promoter
•
Time required for cell line development reduced by 6 - 12 months
compared to methods that utilize gene amplification
¾ Low number of integrated plasmid copies (10-20) increases genetic stability

80
70
Titer from pooled CHO
transfectants (mg/L)

60
50 CMVp
40
30 CHEF1p
20
10
0
Ig

Ig

Ig

-2
1
1

b-
E

1-

2-

3-

ab
A
M
P

M
M

Ref: Icos CHEF-1 brochure, 2004

Host Cell Line Improvements

¾ CHO cell is standard host for most biopharmaceutical products

today
¾ Change of cell line not likely for biochemical and regulatory
reasons
¾ Develop CHO host that meets production criteria
• Adapted to growth in suspension
• Grows in animal-product-free-media
¾ Addition of genes encoding rate-limiting transcription and
translation factors (ie, RNA polymerase subunits, specific
tRNAs) may increase expression levels
¾ Improvement in carbohydrate structure and uniform distribution
of sugars can improve product function and yields from
downstream processing
Characteristics of a Desirable Process
[Product] = qp x [cells] x ∆time
¾ Maximum specific productivity (qp)
¾ Maximum cell concentration
¾ Optimum time
¾ Robust, reproducible, and scalable
1600

14 1400

12 1200
VCD (x 10E6) / viability (%)

10 1000

Titer (mg/l)
Harvest
8 800

6 600

4 400

2 200

0 0
0 2 4 6 8 10 12 14
Days
Media Optimization Strategies
¾ Optimal media for growth and for production must do the
following:
• Provide necessary nutrients at appropriate levels and times
• Keep energy source high so energy production is not rate
limiting
• Buffer to maintain pH within defined process parameters
¾ Regulations now require that media be free of animal-derived
components
• Soy media and other plant sources are under evaluation
• Synthetic media have been developed
¾ Feed rates for glucose and other nutrients can be optimized
¾ Optimization of media and feed strategies can lead to 4 – 10
fold increase in protein expression levels from same cell line
and bioreactor
Media Development and Process Optimization

Ref: B. Adamson. Manupharma, 2004

Erbitux production history: Effect of Feed Changes

¾ Process 1
• 300 mg/L
• Used for initial clinical trials through Phase 2
¾ Process 2
• Change feed strategy and optimize media
• 600 mg/L
• Used for Phase 3 and commercial launch
¾ Process 3
• Currently introducing new feed strategy that enables
production at level of >1 g/L from same initial cell line
• To be implemented as post-approval changes

Ref: G. Pendse, BioProcess International, 2004

Conclusions

¾ Increasing expression level decreases manufacturing

cost and facility requirements
¾ There are multiple approaches to increasing expression
levels
• Intrinsic methods such as vector design and host cell
line development
• Extrinsic methods such as media selection and
process optimization
¾ Expression levels for monoclonal antibodies today are
typically in the 1 – 3 g/L range
¾ Expression levels of >10 g/L are predicted in the next
decade
Thank you!
BioProcess Technology Consultants, Inc.
289 Great Road, Suite 303
Acton, MA 01720
978.266.9153 (phone)
978.266.9152 (fax)
hlevine@bioprocessconsultants.com
www.bioprocessconsultants.com