Production of Monoclonal Antibodies
From Hybridoma to Mammalian Cell Culture
Ana Azevedo
([email protected])
1
Seminar Outline
•
Introduction
•
Antibodies :: Definition, Structure
•
Hybridoma Technology 
•
Applications (Therapeutic antibodies)
•
Antibody Engineering
•
Antibody Manufacturing
•
Cell Culture
•
Cell Line Development
•
Process Development
•
Bioreactor Operation Mode 
2
1973
Recombinant DNA Technology
by Stanley Cohen and Herbert Boyer
Stanley Cohen and Herbert Boyer combined their
efforts to create “recombinant DNA”.
This
technology would radically change molecular biology
and have a huge impact in the development of the
biotechnology industry.
3
Recombinant DNA Technology
 Isolation of plasmid DNA from
bacteria (E.coli)
 Isolation of DNA containing gene of
interest
 Gene inserted into plasmid
 Plasmid put into bacterial cell
 Cells cloned with gene of
interested are replicated
and the coded protein
synthesised
4
1982
Humulin
(recombinant human insulin, E. coli)
The first biopharmaceutical product launched into the market,
marked the arrival of the biopharmaceutical industry.
5
What are Antibodies?
Antibodies belong to a family of large
molecules known as immunoglobulins (Ig):
 Glycoproteins
 4 polypeptide chains:
2 heavy chains
2 light chain
 Y shaped
 Hinge region
Flexible
Disulphide briges
6
Immunoglobulins Structure
The primary structure (amino acid sequence) reveals:
Variable region
The sections that make up the tips of
the Y's arms vary greatly from one
antibody to another, creating a pocket
uniquely shaped to enfold a specific
antigen.
Constant region
The stem of the Y serves to link the
antibody to other participants in the
immune defenses. This region is
identical in all antibodies of the same
class.
Antigen
binding sites
Antigen
binding sites
variable
constant
Fab
Light chain
Light chain
Fc
Heavy
chain
Heavy
chain
7
www.biology.arizona.edu
Immunoglobulins Structure
The constant region of the heavy chains define the antibody class. There are
five immunoglobulins (Ig) classes: IgG, IgA, IgM, IgD and IgE.




Size
Charge
Amino acid composition
Carbohydrate content
Dimer
Pentamer
Monomers
IgG
IgD
IgE
80%
1%
< 1%
IgM
IgA
6%
13%
Kindt el al., Kuby Immunology, 6 ed, Macmillan Education, 2006
8
Immunoglobulins Structure
IgG subclasses
IgG subclasses differ in the number and arrangement of the interchain
disulphide bonds (thick black lines) linking the heavy chains.
IgG1
IgG2
Disulphide
bridge
IgG3
IgG4
Hinge
region
10
Kindt el al., Kuby Immunology, 6 ed, Macmillan Education, 2006
Immunoglobulins Structure
Immunoglobulin fragments :: Proteolytic digestion (papain and pepsin)
Papain
Fab – antigen binding Fragment
(monovalent)
Pepsin
Fc - crystalline Fragment
F(ab')2 –antigen binding Fragment
(bivalent)
en.wikipedia.org/wiki/Fragment_antigen-binding
11
Monoclonal vs. Polyclonal Antibodies
When an antigen enters the body, some
of the immune system's B-cells will
produce antibodies that bind to that
antigen.
Each B-cell produces only one kind of
antibody, but different B-cells will
produce
structurally
different
antibodies that bind to different
epitopes of the antigen.
Antibodies in this natural mixture are known as polyclonal antibodies.
12
Monoclonal vs. Polyclonal Antibodies
Monoclonal Antibodies
Recognise only one epitope at the
surface of the antigen
Polyclonal Antibodies
Recognise several epitopes at
the surface of the antigen
Monoclonal and polyclonal have very distinct production process.
13
Polyclonal Antibodies
Immunising
antigen bound
to carrier
protein
Antibodycontaining
serum is
collected
and purifier
Animal generates antibodies
14
Polyclonal Antibodies
Mouse
Rabbit
Goat
Test bleed
0.2 mL
2.5 – 5 mL
5 - 10 mL
Production
bleed
-
10 mL
200 mL
2.5 mL
50-75 mL
0.5 – 1 L
Terminal
bleed
Animal Selection: Based on the amount of antibodies needed.
15
Polyclonal Antibodies
Polyclonal antibodies are one of the most widely used research reagents.
They can be used for a range of research applications, including:
•
•
•
•
•
•
•
western blotting
immunohistochemistry
immunocytochemistry
flow cytometry
immunoprecipitation
neutralization assays
ELISAs
Immunoglobulin preparations were first used
therapeutically in the 1950s as
immunoglobulin replacement therapy for
primary immunodeficiency disorders.
16
1975
Hybridoma Technology
by César Milstein and Georges Kohler
Arrangement for continuous growth of myeloma
cells used by Milstein in 1972.
The discovery of hybridoma technology by Kohler
and Milstein heralded a new era in antibody
research and clinical development. Mouse
hybridomas were the first reliable source of
monoclonal antibodies and were developed for a
number of in vivo therapeutic applications.
17
Hybridoma Technology
B-Cells
 The mouse is immunised
with the target antigen.
 The spleen is removed
and antibody-producing
cells (B-cells) are isolated.
Myeloma
 B-cells are fused with
myeloma cells and the
resulting hybridomas are
tested for antibody
production.
Hybridoma
Cell Culture
 Screening for antibody production.
 Producing clones are selected and
grown.
18
Myelomas cells
Myeloma cells are derived from tumours of plasma
cells (immortal cells). All mouse myeloma cells
commonly used for hybridoma production are of
BALB/c mouse origin.
The myeloma cells are selected beforehand to ensure that:
• Do not secrete antibody themselves
• Lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene,
making them sensitive to the HAT medium.
Most commonly used cell lines (descendants of MOPC-21):
• Sp2/0 (Sp 2/0-Ag-14)
• NS0
• P3-X63Ag8.653
• NS1 (P3-NS1-Ag 4-1) :: loss of endogenous immunoglobulin heavy chains
19
Hybridoma Technology
B-cells from
mouse spleen
Myeloma cells
Cell Fusion
(fusogen :: PEG)

Hypoxanthine
Aminopterin
Thymidine




B-cells die within few days
Myeloma cells die as they can
not grown in HAT medium
Only the hybrid cells
(hybridomas) survive
20
HAT Selection
Cell type:
B-cell
Hybridoma
Myeloma
Genotype:
HGPRT+
HGPRT+/HGPRT-
HGPRT-
HAT fate:
Dies
Survives
Dies
Immortal with functional DNA synthesis:
Unable to synthesise
DNA:
1) Immortality from
myeloma cell
2) Full HGPRT gene
allows DNA synthesis
from hypoxanthine and
thymidine
1) Aminopterin blocks
de novo pathway
2) Lack of the HGPRT
gene causes a loss-offunction of the salvage
pathway
Explanation: Mortal:
1) Functional DNA
synthesis, but
2) Dies eventually due
to limited number of
replication cycles
21
Hybridoma Technology
22
What are they used for?
 Purification
Immunoprecipitation
Affinity Chromatography
 Therapeutic use
 Detection
ELISA
Flow cytometry
Pregnancy tests
 Diagnosis
Imagiology
23
Therapeutic Antibodies
Up to date, the FDA has approved 42 MAbs to treat different types of
diseases, but many more are under development. In deed, monoclonal
antibodies represent a third of all biopharmaceuticals under development.
24
PhRMA Report, Medicines in Development, 2013
Therapeutic Antibodies :: Market Share
Oncology
Autoimmune & Inflammatory
Cardiovascular diseases
Transplant rejection
Infectious diseases
Others
Total revenues in 2007 :: 12,612 million dollars1
Revenues forcast to 2015 :: 62,658 million dollars2
25
1Frost&Sullivan, 2008
2Elvin
et al., Int. J. Pharm., 440 (2013) 83–98
Therapeutic Antibodies :: Market Share
Number of products approved 20141
Revenues in 20102
20
12
3
3
2
2
Oncology
Autoimmune & Inflammatory
Cardiovascular
Organ Transplantation
Infectious diseases
Other Indication
26
1Janice
Reichert, 2014 (www.antibodysociety.org)
2Howard
L Levine, mAbs 7 (2015) 9-14
1984
Muromonab-CD3
(trade name Orthoclone OKT3)
It was the first monoclonal antibody to be approved for clinical use in humans.
It is an immunosuppressant drug given to reduce acute rejection in patients
with organ transplants.
Y
It binds to the T cell receptor-CD3-complex
on the surface of circulating T cells, initially
leading to an activation, but subsequently
inducing blockage and apoptosis of the T
cells. This protects the transplant against
the T cells.
OKT3
CD3
Activated
T-cells
Apoptosis
27
Human Anti-Mouse Antibodies
Initial treatments using monoclonal antibodies produced using the
hybridoma technology were not as effective as doctors had hoped...
The problem: HAMA response
"Patients receiving murine antibodies, particularly in high amounts, may form
human antibodies against these foreign proteins or human anti-mouse
antibodies (HAMA), which usually occur within 2–3 wk after the first mAb
administration and within hours or days after a repeated administration"
The solution: Recombinant Antibodies
Antibody engineering
28
Antibody Engineering
From Mouse to Men
100% Murine
33% Murine
66% Human
10% Murine
90% Human
100% Human
29
Complementary Determining Regions (CDR)
30
Brekke & Sandlie, Nature Rev. Drug. Disc. 2 (2003) 52
Antibody Engineering
Mouse hybridoma technology
Antibody libraries
Transgenic mouse
Human hybridomas
Genetic Engineering
V gene cloning / CDR grafting
Mammalian Expression
32
Therapeutic Antibodies :: Market Share
Number of mAbs approved per category
18
12
7
5
Human
Humanized
Chimeric
Murine
38
Janice Reichert, 2014 (www.antibodysociety.org)
Antibody Engineering
There are two main classes of recombinant antibodies.
 The first is based on the intact immunoglobulin molecule and is designed to
reduce the immunogenicity of the murine molecule;
 The second class of molecules consists of fragments of antibody molecules.
Fab2
39
Hollinger & Hudson, Nature Biotechnol. 23 (2005) 1126
Antibody Fragments
40
Hollinger & Hudson, Nature Biotechnol. 23 (2005) 1126
How are mAbs produced?
Genetic engineering
Cell line development
Upstream processing
Downstream processing
Formulation
Quality control
Regulation and
registration
Marketing
41
Antibodies :: Cell culture
All monoclonal antibodies approved for therapeutic use are produced by animal cell culture,
mainly using CHO (Chinese hamster ovary) or myeloma (NS0 or Sp2/0) cells.
42
Antibodies :: Cell culture
J.M. Reichert, mAbs 4, 1-3 (2012)
All monoclonal antibodies approved for therapeutic use are produced by animal cell culture,
mainly using CHO (Chinese hamster ovary) or myeloma (NS0 or Sp2/0) cells.
43
Antibodies :: Cell culture
All monoclonal antibodies approved for therapeutic use are produced by animal cell culture,
mainly using CHO (Chinese hamster ovary) or myeloma (NS0 or Sp2/0) cells.
Disadvantages:
• Lower growth rate: Lower productivity
• Complex nutrition and higher raw material cost
• More difficult in manufacturing
Advantages:
• Product secreted: Active biologically
• Capable of post-translational modification
• Glycosylation provides:
• Higher biological activity
• Greater solubility
• Longer half-life
CHO cells
44
Antibodies :: Cell culture
IgG-Fc Glycosylation
-Gln-Tyr-Asn297-Ser-Thr-Tyr-Arg|
Fuc -- GlcNAc
α1-6
|
β1-4
GlcNAc
β1-4
|
Man
α1-6
|
Man GlcNAc Man
α1-3
β1-4
β1-2
β1-2
|
|
β1-4
β1-4
GlcNAc
GlcNAc
|
|
α2-6
α2-6
Gal
Gal
|
|
Neu5Ac
Neu5Ac
45
Source: Nature Reviews Drug Discovery 8, 226-234 (2009)
Antibodies :: Cell culture
Host
Glycan Type
Bacteria
None
Yeast
Glycan Structure
High mannose
Insect
Fucosylated core
structure
Plant
Xylosylated and
fucosylated core
structure
Mammalian
Complex biantennary
Symbols: N-acetyl-glucosamine (); mannose (); fucose (); xylose (); galactose (); sialic acid ().
46
Antibodies :: Cell culture
Cell lines:
• Hybridoma (mostly for development :: source of gene sequences)
• Chinese Hamster Ovary (CHO)
• Mouse Myeloma (NS0; Sp2/0)
• Human Embrionic Kidney (HEK)
• Human embryonic retina (PER.C6)
HEK cells
NS0 cells
BHK cells
PER.C6 cells
47
Chinese Hamster Ovary cell line
1919
• Typing pneumococci in research labs, in stead of mice;
• Vectors for transmitting certain diseases – epidemiology;
1948
• The Chinese hamster arrived at the USA (smuggle);
• Concern about a possible used as biological warfare;
• Two scientists arrested and accused of war crimes;
1958
• Dr. Theodore Puck isolated an ovary from a female hamster and establish a new cell line
that was able to grown in culture plates: The CHO cell line;
• Cells were found to be resilient and easily cultivated in vitro with a fast generation time;
• CHO cells were used in numerous biomedical studies;
1987
• First biopharmaceutical produced in animal cells was approved: Activase
(tissue plasminogen activator) to treat acute myocardial infraction;
Mammalian equivalent of the model bacterium Escherichia coli
48
Antibodies :: Cell culture
Specifications/Requirements:
• Regarded as safe by regulatory agencies (FDA, EMEA)
• Relatively stable genetic profile
• Efficient and simple transfection of rDNA
• Capacity to grow in suspension
• High cell growth rate
• High antibody production
Specific productivity: 20 pg/cell/day
Green: Expression of
exogenous GFP in animal cells;
Blue: DAPI staining of cell
nucleus
High expression
Lower expression
49
Antibodies :: Cell culture
@ Lab scale (T-flasks, Roller bottles and spinner flasks)
50
Antibodies :: Cell culture
Scale-up :: Roller bottles
51
Antibodies :: Cell culture
Scale-up :: Roller bottles
52
Antibodies :: Cell culture
1 L Bioreactor
150 L Bioreactor
53
Antibodies :: Cell culture
10 000 L Bioreactor at Bohering Ingelheim
54
Antibodies :: Cell culture
Disposable bioreactors
Wave Bioreactor (500 L)
55
Antibodies :: Cell culture
Disposable bioreactors
Cube Bioreactor (1200 L)
56
Antibodies :: Cell culture
Disposable bioreactors
Bag Bioreactor (2000 L)
57
Process Optimization
Past versus Present
60
Wurum, Nature Biotechnol., 22 (2004) 1393
Process Optimization
The steady increase in cell culture productivity is the result of intensive efforts
to understand and optimize all aspects of the cell culture production process.
Improvements have been made in:
Evolution of Mammalian Cell Culture Media
• Cell line construction and
selection
• Media development
• Bioreactor design
• Operating parameters
• Feed strategies
Animal-Free components
• Molecular biology
Animal-Free
Chemically
Defined Media
Animal-Free
Media
Serum-Free
Media
Chemically
Protein-Free
Defined Media
Media
Serum
+
Basal Media
Defined Formulation
61
Process Optimization :: Bioreactors
Bioreactors
Important improvements have been made in the design of traditional bioreactors, and new
types of bioreactor are also being developed. Work is also progressing on techniques to
improve the performance of bioreactors, including perfusion culture, the use of
microcarriers, and methods of suppressing apoptosis and of monitoring cell growth in real
time.
• Stirred-tank bioreactor
• Airlift bioreactor
• Hollow-fibre bioreactor
• Fluidized and packed bed
bioreactor
• Membrane bioreactor
• Disposable bioreactors
• Rotary Cell Culture System
(developed by NASA for tissue culture)
66
Process Optimization :: Feed Strategies
Bioreactor Culture Mode
The main carbon, energy and nitrogen sources used in cell culture media are
glucose and glutamine. However, their rapid metabolism leads to a very
inefficient use, causing their rapid depletion from the culture medium and
eventually the accumulation of lactate and ammonium ions in the medium,
that have inhibiting effects for the cells.
fresh medium
nutrient
feeding
Batch
Fed-Batch
cells
spent medium
Continuous / Perfusion
67
Process Optimization :: Feed Strategies
Product
Culture system
Bioreactor Train Scale
ReoPro
Continuous/Perfusion (spinfilter)
10 – 500 L
Zenapax
Fed-batch (stirred tank)
Not disclosed
Simulect
Continuous/Perfusion
(membrane)
Not disclosed
Synagis
Fed-batch (stirred tank)
400 – 10000 L
Remicade
Continuous/Perfusion (spinfilter)
10 – 500 L
Herceptin
Fed-batch (stirred tank)
80 – 12000 L
MyoScint
Continuous/Perfusion (spinfilter)
10 – 500 L
Humaspect
Continuous/Perfusion (hollowfibre)
Not disclosed
68
Process Optimization :: Feed Strategies
Feeding Regime :: Batch versus Fed-batch
Number of Viable Cells (PER.C6)
Antibody Titre
Fed-batch :: 3-fold increase in MAb titre from 0.4 to 1.2 g/L
69
Marco Boorsma, DSM Biologics, 3rd. European. Biotechnology Workshop, Ittingen, Switzerland, 2004
Process Optimization :: Feed Strategies
Feeding Regime :: Batch/ Fed-batch versus Perfusion
Simulation of a comparison between batch / fed-batch / perfusion
(cell specific productivity :: 5 pg/cell/day)
Number of Viable Cells
Antibody Titre
700
600
20
Ab conc. (mg/l)
Viable cells (x106 cells/ml)
25
15
10
Batch
5
Fed-batch
500
Batch
400
Fed-batch
300
Perfusion
200
100
Perfusion
0
0
0
2
4
6
8
10
Culture Time (d)
12
14
16
0
2
4
6
8
10
12
14
16
Culture Time (d)
70
Veronique Chotteau, Workshop on Industrial scale cultivation of cells in pharmaceutical or antibody production systems, Sweden, 2009
Process Optimization :: Feed Strategies
Feeding Regime :: Batch/ Fed-batch versus Perfusion
Simulation of a comparison between batch / fed-batch / perfusion
(cell specific productivity :: 5 pg/cell/day)
Product Mass
3000
Fed-batch (Final vol 1 L)
2500
Ab mass (mg)
Perfusion (1 L/day)
2000
Perfusion :: 2600 mg
Fed-Batch :: 620 mg
Batch :: 180 mg
Batch (1 L)
1500
1000
500
0
0
3
6
9
12
15
18
21
24
27
30
Culture Time (d)
71
Veronique Chotteau, Workshop on Industrial scale cultivation of cells in pharmaceutical or antibody production systems, Sweden, 2009
Process Optimization :: Feed Strategies
Feeding Regime :: Batch/ Fed-batch versus Perfusion
Fed-batch
Perfusion
 Feed concentrate
 Build up of Metabolites
 Osmotic Increase
 Changing Environment
 Reduced Cell Viability
+ Concentrated Harvest
+ Batch Identification
+ Medium Feed
+ Wash up of Metabolites
+ No Osmotic Increase
+ Constant Environment
+ High Cell Viability
 Dilute Harvest
 Large Harvest
72
Rolf Duwenga, DSM Biologics, BioProcess International™ Conference & Exhibition, Raleigh, 2009
XD® Process:: Extreme Density Cell-culture
Fresh Medium
Spent Medium
Alternating Tangential
Flow (ATF) System
Cells & MAb
73
Rolf Duwenga, DSM Biologics, BioProcess International™ Conference & Exhibition, Raleigh, 2009
XD® Process:: Extreme Density Cell-culture
74
Rolf Duwenga, DSM Biologics, BioProcess International™ Conference & Exhibition, Raleigh, 2009
Feeding Regime :: Fed-batch versus Perfusion versus XD®
Fed-batch
Perfusion
+ Medium Feed
 Feed concentrate
+ Wash up of Metabolites
 Build up of Metabolites
+ No Osmo Increase
 Osmo Increase
+ Constant Environment
 Changing Environment
XD®
+ High Cell Vialbility
 Reduced Cell Vialbility
+ Concentrated Harvest
 Dilute Harvest
+ Batch Identification+ Medium Feed
 Large Harvest
+ Wash up of Metabolites
+ No Osmo Increase
+ Constant Environment
+ High Cell Vialbility
+ Concentrated Harvest
+ Batch Identification
75
Rolf Duwenga, DSM Biologics, BioProcess International™ Conference & Exhibition, Raleigh, 2009
How mAbs act?
Complement Activation and
consequente cell lysis
Antibodies promote cell
mediated cytotoxicity
(activation of macrophages or
natural killer cells)
Radioactive isotopes, toxins or
enzyme can be conjugated to
the anti-tumour antibody,
which then specifically targets
the radiation, toxin or drug to
the tumour cell.
+
76
Peter Wood, Understanding Immunology, 2nd edition, Pearso Education imited, 2006
Herceptin (Trastuzumab)
Célula Normal
Célula Cancerígena
77
Rituxan
(Rituximab)
78
Thank you for your attention!
Questions?