Cell Culture Technology for

Pharmaceutical and Cellular

Therapies

Sadettin S. Ozturk, Ph.D.

Centocor Inc.
200 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

Sadettin S Ozturk

1
 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.
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2
 The antibody – a.k.a
“Immunoglobulin”

-The Variable region is different for each

Antibodies are produced by B-cells as part antibody and determines its specificity.
of immune response. Each antibody is -The Constant region is identical for each type
specific to a specific antigen of antibody and allows recognition by your
Sadettin S Ozturk immune cells.

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
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Sadettin S Ozturk

Engineering of Antibodies

Fab

Fc

Murine Chimeric
Humanized IgG Fully Human IgG
IgG IgG
Current
Products in
“Humanization” of antibody minimizes/eliminates Development
immune reaction when injected to the patients

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4
 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

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

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 Production of Monoclonal Antibodies

Seed Bioreactor
50L

Production Bioreactors
300L, 1500L, 5000L, 20,000L

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Monoclonal Antibody Production Process

Bioreactor and Product Capture

Inoculum Production
Bioreactor Bioreactor

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6
 Monoclonal Antibody Production
Process

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Monoclonal Antibody Production

Process

Shipment to
Fill and
Finish Site

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7
 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)

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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)

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 Purification of Monoclonal Antibodies
using Column Chromatography

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Concentration and Diafiltration of

Monoclonal Antibodies

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9
 Centocor : An Antibody
Company

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 Centocor Timelines
Merger with
REMICADE®
Centocor Leiden Mfg. Launched Launched in
Founded 1982 Plant Opens 1993 ReoPro® 1998 1999 ERA & AS 2005

IPO Raised Marketing RETAVASE® REMICADE® REMICADE®

1979 1987 1995 1999 2004 launches in
$21 Million; Alliance with Approved; Launched in
First Diag. Lilly for REMICADE® Rheumatoid psoriasis
Product ReoPro® Launched in Arthritis (OUS),
Approved by Crohn’s ulcerative
FDA colitis (U.S.)
and PsA
(U.S.)

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

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

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 REMICADE®
Rheumatoid
Arthritis – signs &
symptoms of RA,

Approvals inhibiting x-ray

diseases progression,
and improving physical

U.S.
Rheumatoid functioning in patients
Arthritis – Rheumatoid Arthritis – not previously treated
signs and with methotrexate
physical function in
symptoms failed methotrexate Ankylosing
patients Spondylitis
Rheumatoid
Arthritis – Psoriatic
structural Crohn’s Crohn’s Arthritis
Crohn’s
damage Disease – Disease –
Disease
luminal CD fistulizing Ulcerative
Colitis

1998 1999 2000 2001 2002 2003 2004 2005 2006

Rheumatoid Psoriasis
Crohn’s Crohn’s
Crohn’s Arthritis – (OUS)
Disease – Disease –
Disease – structural luminal CD fistulizing
fistulizing damage
EU

Ankylosing Psoriatic
Rheumatoid Arthritis – Spondylitis Arthritis
physical function in
failed methotrexate
patients
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Number of Patients Treated

Worldwide With REMICADE®

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 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 arthritis
Crohn’s Disease
Psoriasis
Multiple 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

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 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

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Autoimmune disorders: Psoriasis

• 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

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 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

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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

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 The use of antibody based
treatment for Psoriasis

Psoriasis activity before and after

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

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

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Development of Antibodies for

Pharmaceutical Therapies

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 Antibody Development and
Commercialization Process

Clinical Development
Early Development Late Development Submit BLA
Target Reg. Review
Research
Preclinical Studies Phase I / II Clinical Trials Phase III Clinical Trials Filing Approval

Drug Development
Cell Line Selection Process Launch
Clinical Manufacturing
and Purif./Form. Dev. Validation Preparation

Process Development Tech. Transfer

Manufacturing Investment Assay Validation

Decision
Year Year Year Year Year Year
-3 1 2 3 4 5

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Steps in Drug Development

Pharmaceutical Development

Discovery Research Cell Line

Cell Line Development
Development

Identify Create New Media Development

Media Development
Molecular
Target Molecular Entity

Bioreactor Process
Bioreactor Process
Development
Development
Clinical
Develop
Initiate Clinical Trials Purification
Process

Make Clinical Supplies Develop Formulation

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 Cell Line Development
-Gene
Develop -Promoter Insert into
expression -Enhancer expression
Clone product
vector -Selective plasmids
gene cDNA marker

Host Cell

Transfection

Evaluate in
Bioreactors Development
Cell bank (DCB)

Master Clone selection

Amplify, Amplify, for high
Cell bank (MCB)
Clone, producing cells
Clone,
Select, Select,
Master working
Cell bank (MWCB)
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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)
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 Bioreactor Process Development
• 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
• 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
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Process Development Options

Process/Media
Perfusion
Commercial
Batch Fed-batch
Proprietary

Therapeutic
Selection Monoclonal Antibody
System Host cell
Sp2/0
gpt/MHX NS0
DHFR/MTX CHO-dhfr-
GS/MSX CHOK1SV
Neo/G418
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 Case Study: Development of a Fed-
batch Process for Monoclonal
Antibody Production

— 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
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Cell Line Development:

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

ATP ADP + Pi

GS
Glutamate + NH3 L-glutamine

MSX
MSX = L-Methionine Sulfoximine

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

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 Cell Line Development: Schematic
Overview of Plasmid Construction
- Start with a research cell line (expressing < 20 mg/L)
- Isolate RNA, reverse transcribe to generate cDNA
mRNA 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’ plasmid
HC LC
SV40 poly A
Amplification
CNTO X LC hCMV-IE
by PCR
Kozak Not I promoter
sequence and intron
Kozak
hCMV-IE
sequence
promoter
and intron GS CNTO X
Clone CNTO X HC
Double Gene
VH VL 11479 bp
Ck
CH1
hCMV SV40 poly A
CH2 hCMV
GS cDNA
Sal I
CH3 β-lactamase
SV40 promoter Pvu I
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(Amp resistance)
GS

Cell Line Development: Process of Developing

High Producing GS-CHO Cell Lines
STATIC CULTURE Transfections
3-6 weeks
200-300 transfectants
1 week Rank order clones by
96w plates 96w single-point ELISA
60-100 transfectants
Rank order clones by
24w plates 2 weeks
24w Neph. (overgrow)
3-10 parental cell lines

SUSPENSION SUBCLONES
(shake flask culture)
8 weeks Adapt to CD-CHO 3 weeks Subclone

3-10 parental cell lines 30-100 subclone cell lines

3 weeks Perform shake flask
3 weeks Rank 24w (Neph.)
growth profiles
1-3 parental cell lines 3-10 subclone cell lines
6 weeks Prepare and test DCBs 8 weeks
Adapt to CD-CHO
1-3 parental cell lines 3-10 subclone cell lines
3 weeks Perform shake flask
12-16 weeks Bioreactor
growth profiles
process development

~ 4.5 months ~ 6 months

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21
 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 interior
HEPA filtration CCD camera 1µm encoders

Wash
and
sterilise

Stacker for microplates Holder for 5 Culture dishes

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 Cell Line Development: Transfection
and Colony Screening

Rank Order Clones: 96w ELISA / 24w Nephelometry

Clone # 96w ELISA Titer 24w Neph Titer

3 21 26.6 mg/L 76.0 mg/L
4 23 20.3 mg/L 52.9 mg/L
6 36 18.7 mg/L 61.8 mg/L
16 113 37.1 mg/L 67.5 mg/L
20 123 25.3 mg/L 93.6 mg/L
21 127 4.1 mg/L 59.1 mg/L
22 134 25.3 mg/L 79.7 mg/L

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Cell Line Development: RESULTS

- 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
VCD vs. Days
VCD (x 10^6 cells/mL)

5
Specific Productivity 4
3
Antibody Titer (mg/L)

2
400 1
y = 18.055x 0
300 2
0 1 2 3 4 5 6 7 8 9 10 11 12 13
R = 0.9474 Days
200
y = 17.049x Titer Accumulation vs. Days
100 2
R = 0.9445
Antibody Titer (mg/L)

400
0 300
200
0 5 10 15 20 25
100

Integral of Viable Cells (E6/mL * days) 0

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

Days
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 Fed-batch Process Development:
Temperature and pH Optimization in Batch
VCC
C1180A Batch
Cultures
Temp and pH DOE

4.500
J006
H010 H006 H008
36.5 C
36.5 C 35 C 35 C
4.000 7.0
7.0 7.2 7.2

3.500

H005
3.000 H006
H007
2.500 H005 H008
35 C
VCC

J004 H009
6.8 H010
35 C
2.000 J003
6.8
J004
1.500 J005
H007 J006
38 C
1.000 J005
6.8
H009 38 C
38 C 6.8
0.500 J003
7.2 38 C
7.2
0.000
0 2 4 6 8 10 12 14 16 18 20
Day
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Fed-batch Process Development:

% Viability
C1180A Batch
Temperature and pH
OptimizationTemp
in and
Batch
pH DOE Cultures

120
H005
35 C J004
6.8 35 C
100 6.8
H009
38 C
7.2 J003 H005
80 38 C H006
7.2 H007
H007 H008
% Viable

38 C J005 H009
60 6.8
38 C H010
6.8
J003
H006 J004
40 35 C J005
H008
7.2 J006
35 C
7.2
H010
20 36.5 C
J006
7.0
36.5 C
7.0

0
0 5 10 15 20 25
Day
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 Fed-batch Process Development:
C1180A
Temp and pH DOE
Temperature and pH
OptimizationTiter
in Comparison
Batch Cultures

600.00

H010 J006
36.5 C 36.5C
500.00
7.0 7.0

H005
35 C Titer H005
6.8 J004 Titer H006
400.00
35 C Titer H007
6.8 Titer H008
H007
38 C Titer H009
300.00 6.8 J005 Titer H010
38 C
Titer J003
6.8
H006 Titer J004
35 C Titer J005
200.00 7.2 H008 Titer J006
35 C
7.2
H009
100.00 38 C
7.2 J003
38 C
7.2

0.00
0 2 4 6 8 10 12 14 16 18 20
Day
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Fed-Batch Process Development:

Feeding Strategies

• 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

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25
 Fed-Batch Process Development: Feeding
Strategies
CD CHO Fed Batch Viable Cell Density Comparison
9.00

8.00 Glu, BRX, BRX=MEM+NEM+Vitamins

Nucleosides, PHyd=Plant Hydrosylate
PHyd
7.00
Viable Cell Density (x10E6 cells/mL)

6.00 Glu, PHyd

MEM, Glu, PHyd
NEAM, GS MEM
5.00

4.00 Glu,
PHyd
BioGro
3.00

2.00

Batch
1.00 Glu, PHyd

0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Day
M04E001
Sadettin S Ozturk M04E002 M04E003 M04E004 MO4E007 M04E008 M04D014 M04D015
M04F038 M04F039 M04E031 M04F032

Fed-Batch Process Development: Feeding

Strategies
CD CHO Fed Batch Antibody Comparison
1600

Glu, BRX,
1400 Nucleosides,
PHyd

Glu, Phyd,
1200 MEM
Glu, Phyd,
MEM,
1000 NEAM, GS
Antibody (mg/L)

800
Glu, Phyd,
BioGro

600

Glu, PHyd
400

Batch
200 BRX=MEM+NEM+Vitamins
PHyd=Plant Hydrosylate

0
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
E001 E002 E003 E004 E007 E008 D014 D015
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M04F038 M04F039 M04E031 M04F032

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 IgG, mg/L

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Maximum Viable cells, MM/mL

200
400
600
800
1000
1200
1400
1600

0
0
1
2
3
4
5
6
7
8

Batch

Batch

Glu, soy

Glu, soy

Glu,soy,MEM

Glu,soy,MEM

Glu, soy,
BioGro
Results

Glu, soy,
BioGro

Results
Glu, soy, MEM,
NEM, GS Supp
Glu, soy, MEM,
NEM, GS Supp

Glu, BRX, soy,

nucleosides
Glu, BRX, soy,
nucleosides
Fed-Batch Process Development:

Fed-Batch Process Development:

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 In-process Testing : Agilent 2100
Bioanalyzer
Non-reduced Reduced

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Process Scale-up and

Commercialization

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 Process Scale-up and Consistency

10

8
VCC (x10E6 cells/mL)

0
0 2 4 6 8 10 12 14

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Days

Process Scale-up and Consistency

2500

2000
Titer (mg/L)

1500

1000

500

0
0 2 4 6 8 10 12 14
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Days

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 Process Consistency: SDS-PAGE

Reduced

Non-Reduced

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Process Consistency: IEF

8.25
8.10
7.89
7.74

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 Process Consistency: Tryptic
Peptide Maps
Ref Std

Batch-1

Batch-2

Batch-3

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Conclusions
1. 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
Sadettin S Ozturk worth it.

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