bio pharmaceutical production cost

8/6/2019 Bio Pharmaceutical Production Cost

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Recent Advances in Cell Culture Technology:Improvements in Biopharmaceutical

Production and Cost

International Knowledge Millennium ConferenceOctober 31 - November 2, 2004

Hyderabad, India

Howard L. Levine, Ph.D.

BioProcess Technology Consultants, Inc.

Acton, MA


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Biopharmaceutical Products

Products made by or composed of viable organisms andbiopolymer 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 Early recombinant proteins were simple proteins, usually

replacement products for natural products

Insulin Human Growth Hormone Alpha Interferon

Todays recombinant proteins and monoclonal antibodiesare more complex


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Cost of Manufacturing of Biopharmaceuticals

Processes are fixed-cost driven

Manufacturing costs typically 15 25% of COGs

Basic cGMP background identical to chemical drugs

Complexity of products results in demanding technicalprocesses and high capital investments

Factors influencing manufacturing costs

Process design and plant capacity Operating Strategy

Equipment and Facilities Costs

Materials Costs Labor Costs

Overhead Structure


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Price and scale are inversely related

Pric

e,

$/g

Production Scale, kg/yr

Ref: U. Gottschalk. bioLOGICS USA, 2004


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Production Hosts Influence Manufacturing Costs

Bacteria Yeast Insect cells Mammalian cells

Complex Proteins

Post-translational modificationsMore complex fermentation

Relatively high COGs

Simple proteins

No post-translational modificationsRelatively simple fermentation

Generally lower COGs

Production host will determine

Quantity of product produced

Quantity and type of contaminants Post-translational modifications, i.e., glycosylation

Economics & regulatory issues Compared to microbial fermentation, cell culture systems

are 50 60 fold less productive per liter


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Effect of Expression Level on Manufacturing Costs

Most products in development,

such as monoclonal antibodies

(MAb), require mammalian cell

culture

Cell culture costs contribute

approximately 50% of total

manufacturing costs Expression level is most

important parameter in

determining cell culture cost

Expression level drives required

bioreactor capacity and overall

facility size

0.0

0.3

0.5

0.8

1.0

0 4 8 12

Relative Titer

RelativeCost


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World-wide Manufacturing Capacity:

Today and Future

0

500

1,000

1,500

2,000

2,500

3,000

3,500

2003 2004 2005 2006 2007 2008

Year

ReactorVolume('000L)

Forecast Industry-wide Capacity Top 8 Potential Volume Drivers FailCapacity utilization remains


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Estimating Capacity Requirements for MAbs

Total MAb requirements 100 Kg/yr

Crude MAb (60% overall yield) 167 Kg/yr

Total cell culture (0.5 g/L expression) 0.33 M L/yr

Estimated batch size (25 batches/yr) 13,000 L

Each MAb requires approximately one 15,000 Lbioreactor to meet market demand

Increase in expression level to 2 g/L lowers requirement

to 3,500 L bioreactor per product or 7 batches per year


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Cost Implications of Process Optimisation

0

40

80

120

160

200

0.1 0.3 0.6 1.9 2.8

Product Titre (g/L)

Num

berofBatches

Required

0%

20%

40%

60%

80%

100%

RelativeFermentatio

n

Cost%

2000L scale 5000L Scale Cost

Assumptions:

Product Requirement:35 kg/yr

65% overall yield

Ref: Lonza GS brochure, 2004


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Historical and Projected Expression Levels

0

2,500

5,000

7,500

10,000

12,500

15,000

1975 1985 1995 2005 2015

Year

Expressio

nLevel,mg/L

10 100

10,000


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Can Expression Levels be Increased?

MAb expression levels have increased significantly over timefrom less than 100 mg/L in 1975 to greater than 3 g/L today

Predicted yields of >10 g/L within the next decade

Ref: G. Slaff. bioLOGICS USA, 2004


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Cell Line Development and Production

Expression Vector Constructionand 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 beoptimized to increase expression


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Mammalian Protein Expression Vector Features

Promoter and enhancer drive transcription of desired protein

CMV promoter commonly used today

Selectable marker

Dihydrofolate Reductase (DHFR)

Native DHFR is duplicated in response to increasing Methotrexate (MTX)

Technology developed in early 80s uses amplification of DHFR on an

expression vector to co-amplify gene encoding biopharmaceutical product

Glutamine Synthetase (GS)

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


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Improving Clone Selection

Speed to identification of final high expressing production clone is critical torapid product development

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

Number of Clones Screened

Titerg/ml

Ref: B. Adamson, Manupharma, 2004


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Cell Line Development DHFR

Steps required for DHFR amplification

Transfect cells and select in 0.01 0.2 nM MTX

Identify clone with highest level of transgene expression

(generally 1 3 pg/cell/day) Dilute isolated clone and reselect in higher MTX level

Identify clone with highest level of transgene expression (up to12 pg/cell/day)

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 highenough expression for todays biopharmaceuticalproducts


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Cell Line Development GS

Glutamine synthetase can be used to more rapidly

select high expressing cell lines without amplification



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Process Optimization with the GS System

28294B

19173B

5853A

3342A

1391A

Antibody (mg/L)ProcessCell Line



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CHEF-1 Vector for Improved Expression

Expression vector uses the homologous Chinese hamster EF1- promoter 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 monthscompared to methods that utilize gene amplification

Low number of integrated plasmid copies (10-20) increases genetic stability

0

10

2030

40

50

60

70

80

E1

E2

P1-Ig

P2-Ig

P3-Ig

MDC

MAb-1

Mab-2

Titerfrom

pooledCHO

transfectants

(mg/L)

CMVp

CHEF1p

Ref: Icos CHEF-1 brochure, 2004


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Host Cell Line Improvements

CHO cell is standard host for most biopharmaceutical productstoday

Change of cell line not likely for biochemical and regulatory

reasons Develop CHO host that meets production criteria

Adapted to growth in suspension Grows in animal-product-free-media

Addition of genes encoding rate-limiting transcription andtranslation factors (ie, RNA polymerase subunits, specifictRNAs) may increase expression levels

Improvement in carbohydrate structure and uniform distributionof sugars can improve product function and yields fromdownstream processing


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Characteristics of a Desirable Process

[Product] = qp x [cells] x time Maximum specific productivity (qp)

Maximum cell concentration

Optimum time

Robust, reproducible, and scalable

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14Days

VCD(x

10E6)

VCD(x

10E6)/viability(%)

viability(%)

0

200

400

600

800

1000

1200

1400

1600

Harvest

Titer(mg/l)


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Media Optimization Strategies

Optimal media for growth and for production must do thefollowing:

Provide necessary nutrients at appropriate levels and times

Keep energy source high so energy production is not ratelimiting Buffer to maintain pH within defined process parameters

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

Feed rates for glucose and other nutrients can be optimized Optimization of media and feed strategies can lead to 4 10

fold increase in protein expression levels from same cell line

and bioreactor


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Media Development and Process Optimization

Ref: B. Adamson. Manupharma, 2004

bi d i hi ff f d h


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Erbitux production history: Effect of Feed Changes

Process 1

300 mg/L

Used for initial clinical trials through Phase 2

Process 2

Change feed strategy and optimize media

600 mg/L

Used for Phase 3 and commercial launch

Process 3

Currently introducing new feed strategy that enablesproduction at level of >1 g/L from same initial cell line

To be implemented as post-approval changes

Ref: G. Pendse, BioProcess International, 2004

C l i


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Conclusions

Increasing expression level decreases manufacturing

cost and facility requirements

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 andprocess optimization

Expression levels for monoclonal antibodies today are

typically in the 1 3 g/L range

Expression levels of >10 g/L are predicted in the next

decade

Th k !


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Thank you!

BioProcess Technology Consultants, Inc.

289 Great Road, Suite 303

Acton, MA 01720

978.266.9153 (phone)

978.266.9152 (fax)[email protected]

www.bioprocessconsultants.com

bio pharmaceutical production cost

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