cell culture technology for pharmaceutical and...

1

Sadettin S Ozturk

Cell Culture Technology forPharmaceutical and Cellular

Therapies

Sadettin S. Ozturk, Ph.D.Sadettin S. Ozturk, Ph.D.Centocor Centocor Inc.Inc.

200 Great Valley Parkway, Malvern, PA 19355200 Great Valley Parkway, Malvern, PA 19355

Sadettin S Ozturk

Outline

1. Introduction to Monoclonal Antibodies

2. Production of Monoclonal Antibodies

3. Introduction of Centocor

4. Autoimmune disorders

5. Development of Monoclonal Antibody based Pharmaceutical Therapies

6. Case study: Fed-batch Process development for Monoclonal Antibodies

2

Sadettin S Ozturk

Immune Response as First Line of Defense

When a pathogen (bacteria, foreign proteins, virus,.) enters the blood stream it is recognized, attacked, and eliminated by a sophisticated defense mechanism: Body’s Immune Response

Sadettin S Ozturk

Immune System: The Body's First Line of Defense

Lymphatic vessels form a circulatory system that operates in close partnership with blood circulation.

Organs and tissues of the immune system dot the body in a protective network of barriers to infection.

3

Sadettin S Ozturk

The antibody – a.k.a “Immunoglobulin”

-The Variable region is different for each antibody and determines its specificity.

-The Constant region is identical for each type of antibody and allows recognition by your immune cells.

Antibodies are produced by B-cells as part of immune response. Each antibody is specific to a specific antigen

Sadettin S Ozturk

Evolution of Antibody Technology:Development of Antibody Based Medicines

• Utilization of antibodies as medicines took some time:– B-cells cannot be expanded in vitro for practical purposes – It is difficult to find out which B-cell makes a specific antibody

• Kohler and Milstein discovered Hybridoma Technology and cloning in 1975: A revolution in antibody technology– Hybridoma cells (a fusion of B-cell and myeloma cell) can be expanded

indefinitely– Utilization of cloning techniques allows to isolate cells that make a

specific antibody: These antibodies are called Monoclonal Antibodies (Mab)

• Expansion of hybridoma cells in vivo (mouse) or in vitro (bioreactors) allowed the development of first MAbs

• Over the last 30 years antibody technology was further developed– Engineering of antibodies to make them more “human”– To use cell lines other than hybridoma cells (CHO, NS0, etc)– The use of antibody fragments and fusion proteins– The use of antibodies for targeted drug delivery

4

Sadettin S Ozturk

Sadettin S Ozturk

Murine IgG

ChimericIgG

Fab

Fc

Humanized IgG Fully Human IgG

Current

Products in

Development

Engineering of Antibodies

“Humanization” of antibody minimizes/eliminates immune reaction when injected to the patients

5

Sadettin S Ozturk

Monoclonal Antibodies as Medicines

• There are 18 approved antibody treatments in the market for:• Autoimmune disorders• Cancer• Asthma• Organ rejection

• Sales of antibodies is expected to be $13 Billion in 2005• There are 500 new antibody products in development• There are 75 new antibodies in clinical trials• Sales of antibodies is expected to be $26 Billion in 2010• Some of the indications require as high as 2,000 kg/year product• These antibodies are produced in large (20,000L) bioreactors

Sadettin S Ozturk

Therapeutic Antibodies Approved to Date

* Nature Biotechnology, Sept. 2005, 23(9), p.1075

6

Sadettin S Ozturk

Production of Monoclonal Antibodies

Seed Bioreactor

50L

Production Bioreactors

300L, 1500L, 5000L, 20,000L

Sadettin S Ozturk

InoculumBioreactor

ProductionBioreactor

Monoclonal Antibody Production ProcessBioreactor and Product Capture

Monoclonal Antibody Production ProcessBioreactor and Product Capture

7

Sadettin S Ozturk

Monoclonal Antibody Production Process

Monoclonal Antibody Production Process

Sadettin S Ozturk

Shipment toFill and

Finish Site

Monoclonal Antibody ProductionProcess

Monoclonal Antibody ProductionProcess

8

Sadettin S Ozturk

Production Bioreactors: Continuous Perfusion Operation

• Cells are retained in the bioreactor by physical means

• Cells grow, stay in the bioreactor, and produce proteins

• Media is added and harvest is collected continuously

• Can be operated for months• Usually compact bioreactors

(1000L)

Sadettin S Ozturk

Production Bioreactors: Batch Operation

• Inoculate with media and cells

• Cells grow and produce proteins

• All of the contents are harvested after typically 2 weeks

• Usually very large bioreactors (20,000L)

9

Sadettin S Ozturk

Purification of Monoclonal Antibodies using Column Chromatography

Sadettin S Ozturk

Concentration and Diafiltration of Monoclonal Antibodies

10

Sadettin S Ozturk

Centocor : An Antibody Company

Sadettin S Ozturk

11

Sadettin S Ozturk

Centocor Timelines

1999

1998Centocor Founded

IPO Raised $21 Million; First Diag.

Product Approved by

FDA

Leiden Mfg. Plant Opens

Marketing Alliance with

Lilly for ReoPro®

Launched ReoPro®

RETAVASE®

Approved; REMICADE®

Launched in Crohn’s

In the past five years, sales have grown from $500 million to over $3 billion.

1979 1987 1995

1982 1993

Merger with

REMICADE®

Launched in Rheumatoid

Arthritis

1999

2004

REMICADE®

Launched in ERA & AS 2005

REMICADE®

launches in psoriasis

(OUS), ulcerative

colitis (U.S.) and PsA

(U.S.)

Sadettin S Ozturk

Centocor Products

• Launched 1998– Approved in over 80 countries

World Wide– 2004 WW sales $2.63 Billion– Indications: RA / CD / AS /

PsA/UC

• Launched 1995– Approved in over 50

countries World Wide– 2004 WW sales $363Million – Indications: PCI

12

Sadettin S Ozturk

REMICADE® Approvals

1999 2000 2001 2002 2003 2004 2005 2006

Rheumatoid Arthritis –physical function in failed methotrexate

patientsRheumatoid Arthritis –structural damage

Psoriatic ArthritisCrohn’s

Disease –luminal CD

Rheumatoid Arthritis – signs &

symptoms of RA, inhibiting x-ray

diseases progression, and improving physical functioning in patients not previously treated

with methotrexate

Rheumatoid Arthritis –signs and symptoms

Crohn’s Disease

Ulcerative Colitis

1998

U.S

.E

U

Crohn’s Disease –fistulizing

Ankylosing Spondylitis


Rheumatoid Arthritis –physical function in failed methotrexate

patients

Rheumatoid Arthritis –structural damage

Crohn’s Disease –

luminal CD

Ankylosing Spondylitis


Psoriatic Arthritis

Psoriasis (OUS)

Sadettin S Ozturk

Number of Patients TreatedWorldwide With REMICADE®

13

Sadettin S Ozturk

Autoimmune disorders:What Are They?

Disorders caused by an immune response against the body's own tissues.

Immune system disorders occur when the immune response is inappropriate, excessive, or lacking.

Rheumatoid arthritisCrohn’s DiseasePsoriasisMultiple sclerosis (MS)Systemic lupus erythematosus

Sadettin S Ozturk

Autoimmune disorders: Rheumatoid Arthritis

inflammation begins in the tissue lining your joints and then spreads to the whole joint (hand joints are the most common site, but it can affect most joints in the body)

• muscle pain• deformed joints• Weakness• Fatigue• loss of appetite• weight loss• becoming confined to bed in severe cases

14

Sadettin S Ozturk

Autoimmune disorders:Crohn's Disease

• Chronic autoimmune disease where immune cells attack any part of the gastrointestinal tract

• The lining of the intestine may ulcerate and form channels of infection, called fistulas

• Ulcerative colitis is a similar inflammation of the colon, or large intestine

Sadettin S Ozturk

• Immune-mediated, genetic disease manifesting in the skin and/or the joints

• Psoriasis and psoriatic arthritis affect more than 4.5 million people in the United States

• A person's quality of life—including emotional health—can be seriously jeopardized

Autoimmune disorders: Psoriasis

15

Sadettin S Ozturk

Autoimmune disorders:Multiple sclerosis (MS)

– weakness and trouble with coordination, balance, speaking, and walking

– paralysis– Tremors– numbness and tingling feeling in arms, legs, hands, and feet

Lupus– swelling and damage to the joints, skin, kidneys, heart,

lungs, blood vessels, and brain– “butterfly” rash across the nose and cheeks– rashes on other parts of the body– painful and swollen joints– sensitivity to the sun

Sadettin S Ozturk

Autoimmune disorders:A lot of things can go wrong in the immune

system to result in autoimmune disorders• T cell proliferation and interferon production • Differentiation of T-cells• Cytokine production• Cytokine, receptor binding• B-cell differentiation• Antibody production• Migration of cells to the tissue

Antibodies can be used to intercept or block these events

16

Sadettin S Ozturk

The use of antibody based treatment for Psoriasis

J Invest Dermatol. 2004 Dec;123(6):1037-44.

Psoriasis activity before and after treatment with a specific antibody: 0.1 mg/kg dose (1 week post-treatment [baseline not available] and 16 weekspost-treatment); 1.0 mg/kg dose (baseline and 16 weeks post-treatment)

Sadettin S Ozturk

Development of Antibodies for Pharmaceutical Therapies

17

Sadettin S Ozturk

Process Development Tech. Transfer

Review Approval

Reg. FilingPhase III Clinical Trials

Submit BLA

Preclinical Studies

Early Development

Phase I / II Clinical Trials

Drug Development

Late Development

Clinical Development

Assay Validation

Cell Line Selectionand Purif./Form. Dev. Clinical Manufacturing Process

ValidationLaunch

Preparation

Year-3

Year1

Year2

Year3

Year4

Year5

TargetResearch

Manufacturing InvestmentDecision

Antibody Development and Commercialization Process

Sadettin S Ozturk

Pharmaceutical Development

Clinical

Identify Molecular

Target

Create New

Discovery Research

Molecular Entity

Initiate Clinical Trials

Cell Line Development

Media Development

Bioreactor Process Development

Cell Line Development

Media Development


Develop Purification

Process

Make Clinical Supplies Develop Formulation

Steps in Drug Development

18

Sadettin S Ozturk

Clone productgene cDNA

Insert intoexpressionplasmids

Cell Line Development-Gene-Promoter-Enhancer-Selectivemarker

Transfection

Clone selectionfor highproducing cells

Amplify,Clone,Select,

Amplify,Clone,Select,

Host Cell

DevelopmentCell bank (DCB)

Evaluate inBioreactors

MasterCell bank (MCB)

Master workingCell bank (MWCB)

Develop expression vector

Sadettin S Ozturk

Media Development

• Cell culture medium contains:

Salts, trace elements, glucose, amino acids,other nutrients, vitamins, buffers, etc.

• Early media formulations used serum or other animal derived proteins (albumin)

• Issues related to safety (BSE), availability, and cost became driving force to eliminate serum and to develop animal product free (APF) (safer and economical)

• Today chemically defined medium (CDM) is a reality for many cell culture based processes (consistent and traceable)

• Most of the companies use specially formulated in-house proprietary media formulations for their processes (independence)

19

Sadettin S Ozturk


• Batch, fed-batch, and perfusion process options are in use for commercial production

• It is possible to get 5 g/L titers in fed-batch and about 50 MM cells/mL in perfusion

• Bioreactor process development involves– Optimization of culture environment

(pH, temperature, DO, CO2)– Optimization of media exchange rates– Development of feeding solutions and

feeding strategies

• Advances in biochemical engineering made it possible to grow animal cells in conventional bioreactors (no need for specialized systems)• Today stirred-tank based bioreactors are in operation at sizes up to 20,000L

Sadettin S Ozturk

Process/Media

SelectionSystem Host cell

gpt/MHX

GS/MSXDHFR/MTX

Sp2/0

CHO-dhfr-NS0

CHOK1SV

Perfusion

Fed-batch

Neo/G418

Monoclonal Antibody

BatchCommercial

Proprietary

Therapeutic

Process Development Options

20

Sadettin S Ozturk

� Cell line: CHOK1SV

� Glutamine Synthetase selection system

� Animal product free medium

� Fed-batch process in stirred tank bioreactors

� Optimized pH, temperature

� Optimized feeding schedule

� Process Scale-up

� Consistency

Case Study: Development of a Fed-batch Process for Monoclonal

Antibody Production

Sadettin S Ozturk

Glutamine Synthetase (GS) catalyzes the biosynthesis of glutamine from glutamate and ammonia, providing the only pathway for L-glutamine formation in the cell

Glutamate + NH3 L-glutamine

ATP ADP + Pi

MSX

GS

MSX = L-Methionine Sulfoximine

In the absence of glutamine, the GS enzyme is essential for cell survival

Cell Line Development: Introduction/Selection System

21

Sadettin S Ozturk

VH

GS

hCMV

VL

hCMV

CH3

CH1

CH2

Ck

Clone

by PCRAmplification

mRNA cDNA

- Start with a research cell line (expressing < 20 mg/L)

- Isolate RNA, reverse transcribe to generate cDNA

- Use sequence information from genomic constructs to

design PCR primers to isolate specific HC and LC cDNAs

- Clone cDNAs into Lonza GS vectors, pEE 6.4 and pEE 12.4

- Construct a GS ‘double-gene’ plasmidHC LC

11479 bp

CNTO X LC

SV40 poly A

SV40 poly A

Pvu I

Not I

Sal I

GS CNTO XDouble Gene

hCMV-IE promoter and intron

Kozak sequence

hCMV-IE promoter and intron

Kozak sequence

CNTO X HC

β-lactamase (Amp resistance)

GS cDNA

SV40 promoter

Cell Line Development: Schematic Overview of Plasmid Construction

Sadettin S Ozturk

STATIC CULTURE Transfections3-6 weeks

200-300 transfectants

60-100 transfectants

1 week

2 weeks

96w plates

24w plates

Rank order clones by96w single-point ELISA

Rank order clones by24w Neph. (overgrow)

3-10 parental cell lines

SUSPENSION(shake flask culture)

SUBCLONES

3 weeks

3 weeks

8 weeks

3 weeks

6 weeks

Adapt to CD-CHO

Perform shake flask

growth profiles

Prepare and test DCBs

Bioreactorprocess development




Subclone

Rank 24w (Neph.)

Adapt to CD-CHO8 weeks

3 weeks Perform shake flaskgrowth profiles

30-100 subclone cell lines



12-16 weeks

Cell Line Development: Process of Developing High Producing GS-CHO Cell Lines

~ 4.5 months ~ 6 months

22

Sadettin S Ozturk

Cell Line Development: Clone Selection

Immuno-precipation method for rapid selection of high expression/ secretion clones:

Patent: WO 2005 / 020924 A2 (Publication date 10 March 2005)

Sadettin S Ozturk

Automated Halo-Colony Picking

ClonePix interiorClonePix interior

HEPA filtration

Holder for 5 Culture dishes

Wash and

sterilise

Stacker for microplates

1µm encodersCCD camera

23

Sadettin S Ozturk

Clone # 96w ELISA Titer 24w Neph Titer3 21 26.6 mg/L 76.0 mg/L4 23 20.3 mg/L 52.9 mg/L6 36 18.7 mg/L 61.8 mg/L

16 113 37.1 mg/L 67.5 mg/L20 123 25.3 mg/L 93.6 mg/L21 127 4.1 mg/L 59.1 mg/L22 134 25.3 mg/L 79.7 mg/L

Rank Order Clones: 96w ELISA / 24w Nephelometry

Cell Line Development: Transfection and Colony Screening

Sadettin S Ozturk

- Expand in 24w, T-flasks, and cryopreserve- Adapt highest expressing cell lines to APF medium (CD-CHO)- Adapt to suspension culture in shake flasks- Perform 10-passage stability study- Perform growth profiles in shake flasks in APF medium- Transfer cell line(s) to bioreactor process development group

Specific Productivity

y = 18.055xR2 = 0.9474

y = 17.049xR2 = 0.9445

0

100

200

300

400

0 5 10 15 20 25

Integral of Viable Cells (E6/mL * days)

An

tib

od

y T

iter

(m

g/L

)

VCD vs. Days

0

1

23

4

5

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Days

VC

D (

x 10

^6 c

ells

/mL

)

Titer Accumulation vs. Days

0

100

200

300

400

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Days

Ant

ibod

y Ti

ter

(mg/

L)

Cell Line Development: RESULTS

24

Sadettin S Ozturk

VCC C1180A Batch

Temp and pH DOE

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

4.500

0 2 4 6 8 10 12 14 16 18 20

Day

VC

C

H005H006H007H008H009H010J003J004J005J006

H00635 C7.2

H01036.5 C7.0

H00738 C6.8

H00938 C7.2

H00535 C6.8

J00338 C7.2

H00835 C7.2

J00435 C6.8

J00538 C6.8

J00636.5 C7.0

Fed-batch Process Development: Temperature and pH Optimization in Batch

Cultures

Sadettin S Ozturk

% Viability C1180A Batch

Temp and pH DOE

0

20

40

60

80

100

120

0 5 10 15 20 25

Day

% V

iab

le

H005H006H007H008H009H010J003J004J005J006

H00635 C7.2

H00738 C6.8

H00938 C7.2

H01036.5 C7.0

H00535 C6.8

J00338 C7.2

J00435 C6.8

J00538 C6.8

J00636.5 C7.0

H00835 C7.2

Fed-batch Process Development: Temperature and pH Optimization in Batch Cultures

25

Sadettin S Ozturk

C1180A Temp and pH DOE Titer Comparison

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0 2 4 6 8 10 12 14 16 18 20

Day

Titer H005Titer H006Titer H007Titer H008Titer H009Titer H010Titer J003Titer J004Titer J005Titer J006

H00938 C7.2

H00635 C7.2 H008

35 C7.2

H01036.5 C7.0

H00738 C6.8

H00535 C6.8

J00338 C7.2

J00538 C6.8

J00636.5C7.0

J00435 C6.8

Fed-batch Process Development: Temperature and pH Optimization in Batch Cultures

Sadettin S Ozturk

• GS-CHO Cell line producing a fully human antibody

• Animal Product Free Medium• pH, DO, and temperature set-points from batch

optimization study• Feeding solutions include

– Glucose, plant hydrolysate, MEM, NEAM, Vitamins, Specially formulated cocktails

• Feeding strategies include daily additions of pre-determined amounts to the bioreactor

Fed-Batch Process Development: Feeding Strategies


26

Sadettin S Ozturk


CD CHO Fed Batch Viable Cell Density Comparison

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Day

Via

ble

Cel

l Den

sity

(x1

0E6

cells

/mL

)

M04E001 M04E002 M04E003 M04E004 MO4E007 M04E008 M04D014 M04D015M04F038 M04F039 M04E031 M04F032

Glu, BRX, Nucleosides, PHyd

Glu, PHyd BioGro

BRX=MEM+NEM+Vitamins PHyd=Plant Hydrosylate

Glu, PHyd MEM, NEAM, GS

BatchGlu, PHyd

Glu, PHyd MEM

Sadettin S Ozturk


CD CHO Fed Batch Antibody Comparison

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Day

An

tib

od

y (m

g/L

)

E001 E002 E003 E004 E007 E008 D014 D015

M04F038 M04F039 M04E031 M04F032

Glu, BRX, Nucleosides, PHyd

Glu, Phyd, BioGro

Glu, Phyd, MEM, NEAM, GS

Batch

Glu, PHyd

Glu, Phyd, MEM

BRX=MEM+NEM+VitaminsPHyd=Plant Hydrosylate

27

Sadettin S Ozturk

Fed-Batch Process Development: Results

0

1

2

3

4

5

6

7

8

Maxim

um

Via

ble

cell

s, M

M/

mL

Bat

ch

Glu

, so

y

Glu

,soy

,MEM

Glu

, so

y,Bio

Gro

Glu

, so

y, M

EM

,N

EM

, G

S S

upp

Glu

, BRX,

soy,

nucl

eosi

des

Sadettin S Ozturk

Fed-Batch Process Development: Results

0

200

400

600

800

1000

1200

1400

1600

IgG

, m

g/

L

Bat

ch

Glu

, so

y

Glu

,soy,

MEM

Glu

, so

y,Bio

Gro

Glu

, so

y, M

EM

,N

EM

, G

S S

upp

Glu

, BRX,

soy,

nucl

eosi

des

28

Sadettin S Ozturk

Non-reduced Reduced

In-process Testing : Agilent 2100 Bioanalyzer

Sadettin S Ozturk

Process Scale-up and Commercialization

29

Sadettin S Ozturk

Process Scale-up and Consistency

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10 12 14

Days

VC

C (

x10E

6 ce

lls/m

L)

Sadettin S Ozturk

Process Scale-up and Consistency

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12 14

Days

Tit

er (

mg

/L)

30

Sadettin S Ozturk

Process Consistency: SDS-PAGE

ReducedReduced

Non-ReducedNon-Reduced

Sadettin S Ozturk

Process Consistency: IEF

8.258.107.897.74

8.258.107.897.74

31

Sadettin S Ozturk

Process Consistency: Tryptic Peptide Maps

Ref StdRef Std

Batch-1Batch-1

Batch-2Batch-2

Batch-3Batch-3

Sadettin S Ozturk

Conclusions1. Monoclonal Antibodies evolved over the years to

become an essential part of biotechnology

2. Monoclonal Antibodies can be used as an effective therapy for immune disorders

3. There are several processing options for the manufacture of Monoclonal Antibodies. The final choice may depend on a variety of reasons

4. Development and manufacturing of Monoclonal Antibodies require extensive optimization, consistency, and comparability studies

5. It can be tedious, frustrating, costly, and very risky, but making a drug that can help people’s life is worth it.

cell culture technology for pharmaceutical and...

Documents