Animal Cell Biotechnology
ANIMAL CELL BIOTECHNOLOGY
Human health and well-being is threatened by
numerous diseases.
•infectious diseases
•diseases due to some malfunction in the body
Finding remedies for and, if possible, preventing
human disease is a major area of scientific research.
Biotechnology research focuses on optimizing the
production process for bacterial and virus vaccines
and pharmaceutical proteins. For this we make use of
detailed metabolic models in mammals, which in turn
are also used to study the effect of food components
on mammalian cell physiology.
•virus vaccines (Add-on) (papers from us)
•pharmaceutical proteins
Pharmaceutical Proteins
Animal cell culture increasingly attracts interest due
to the ability of animal cells to produce qualitative
excellent pharmaceutical proteins. Examples of such
proteins are Erythropoietin (Epogen, Amgen) and
Follicle-stimulating hormone (FSH). However,
compared to microbial production systems, animal
cell cultures are characterized by low biomass
concentrations, low productivities, slow growth rates,
and a high shear sensitivity.
Research focuses on the medium, process and
reactor design addressing the above mentioned
problems.
Erythropoietin (Epogen, Amgen)
The principal factor responsible for the regulation of red
blood cell production during steady-state conditions and
for accelerating recovery of red blood cell mass
following hemorrhage
Erythropoeitin (EPO)
Erythropoeitin is a glycoprotein hormone
produced in the kidneys that stimulates red-
blood-cell production in the bone marrow.
Patients with kidney failure produce less
EPO and consequently have lower levels of
red blood cells (up to 1/3 of a normal
person) and have severe anemia. Epogen
solves this problem. Because the active
protein is modified after translation it can
only be produced in animal cells in the
correct form.
EPO http://www.amgen.com/
Follicle-stimulating hormone (FSH)
•Produced by the Pituitary gland, causes the follicle to
develop in females, then stimulates the follicle to
produce estrogen
•In males: spermatogonia stimulating (with the aid of
testosterone) the production of sperm (sperm
production)
–History
–Animal cell culture
•Why, media, applications, comparison
between microbial cell culture
–Regulations
History
History
1880 Roux Embryonic chicken in saline
frog embryo
1900 Harrison
• Anchorage dependent
• Nutrients
• Relative slow growth rate
• Doubling 1 day vs 20 minutes bacteria
• Contamination
characteristics for in vitro cell growth:
1. Cells require an anchor like the lymph clots (the
cover slip)
2. Cells require nutrients provided by the lymph.
3. Cells grow very slow; 20 hours doubling time
compared to 20 minutes for bacteria This means
cell cultures are vulnerable to contamination
1900 Harrison
Carrel (surgeon, 1923)
Aseptic techniques
Carrel Flask
1912-1946 Culture Chicken Embryo Fibroblast
Plasma+tissue homogenate
Polio
1940/1950’s Major Polio epidemics
Polio
Polio vaccine
primary monkey kidney cells*
human diploid lung fibroblast
Antibiotics: Penicillin and streptomycin
1950s
Hela cell line: human carcinoma
Chemically defined media (Eagle, Earle)
Consistency
Sterilization
Reduced chance of contamination
Insect cell lines (Grace)
Why Animal
cell?
Recombinant DNA 1970
• All proteins in E. coli
– No post-translational modifications
• Glycosylation
• Folding
– Inclusion / excretion
• Large complex proteins require
animal cells
Microcarriers
Van Wezel 1960’s RIVM
Monkey Kidney cells
Polio
http://www.rivm.nl/en/
Media, applications
(Methodology)
Kohler and Milstein Nature 256, 495 - 497 (07 August 1975);
Hybridoma
1975 Kohler and Milstein
Myeloma
B-cell
single chain
antibody
immortal
Mortal
Hybridoma
monoclonal antibody
immortal
Nowadays
• Serum-free media
• Genetic modification cells
• Bioreactors
• Large-scale (20 m3)
• Physiological studies
– Understand function of cells (Toxicology)
• Tissue engineering (Skin, Cartilage, Liver)
• Biologicals (pharmaceutical proteins)
– Monoclonal Antibodies
– Hormones (EPO, FSH)
– Enzymes (-glucosidase)
– Vaccines (Polio)
Toxaphene
Gas inlet
Nitrogen Gas outlet Viable-cell concentration (109 cells.dm-3)
Oxygen
CO2
1.4
Probes for:
1.2
pH
Dissolved Oxygen 1.0
Temperature
0.8
Heating
0.6
0.4
0.2
0.0
0 50 100 150 200
Culture time (hours)
Animal cell culture
• Stem cells&tissue engineering
–Skin, Liver
–Bone and Cartilage
• Pharmaceuticals
–MKZ (ID-Lelystad)
–Classical swine fever (Bayer)
–Small pox
–Remicade (Centocor)
–-Glucosidase (Pharming)
Cartilage
http://www.isotis.com/isotis/isotiswebv3.nsf/wwwVwContent/l2annualreport2004.htm
Break
Multicellular environment
• Complex nutritional requirements
– 20 amino acids, minerals, vitamins, glucose,
serum/growth factors
• Fragile/ shear sensitive
– Oxygenation
• Limited life-span
– Transformed cells, cancerous cells
• Bad growth
– Slow growth rates, low biomass, cell death
Growth curve
10.0
Stationary
1.0
0.1 decline
Cx (g/l)
exponential Yeast
0.0
Hybridoma
0.0
Minimum
density 0.0 Lag phase
0.0
0 50 100
Time (h)
Desinfection step
Tissue isolation
Incubation&growth
Primary cells
Passage number
Ln(total cells)
70 generations
Transformation
• Characteristics • Methods
– Infinite life span – Mutagens
– High growth – Viruses
potential – Oncogens
– Low growth factor – Spontaneous
dependence
– Tumors
– Suspension growth
– aneuploid
Not all transformed cell lines can form tumors, but
all tumors contain transformed cells
Normal cells?
• Diploid
• Anchorage
dependent
• Finite life span
• Non-malignant
Basic Media
Mammalian Insect
Glucose 4 g/l 2.5 g/l
Amino acids 0.01-0.15 g/l 0.1-1.5 g/l
Glutamine 1 g/l 1 g/l
HCO3 3.5 g/l 0.35 g/l
H2PO4 0.1 g/l 1 g/l
Salts 4.5 g/l NaCl 1g/l MgSO4,KCl
Vitamins/Spore More Less
pH 7.2 6.4
Osmolarity 300 mOsm 350 mOsm
Media
• Insect cells • Mammalian cells
– Fumaric & alfa- – Pyruvate
keto-glutaric – Hypoxanthine,
acid, Malic & thymidine
succinic acid – Linoleic acid
– Maltose & – Hepes buffer
Sucrose
&Threhalose – Phenol red
Serum
• 0-20% Serum • Problems
– Growth factors – Infectious agents
– Transferrin (Fe) (viruses, prions)
– Lipids – Variable
composition
– Insulin
– Expensive
– Shear protection
– High protein
– Detoxification
content
problems in DSP
Serum
• Serum/protein free media
–Transferrin / ferrous citrate
– Lipid concentrate
– Extracts Yeast extract
– Insulin
– Bovine Serum Albumin (f.a.
transport/detoxification)
– Pluronic (shear protectant)
• Completely mammalian origin free
(MOF)
Comparison between
microbial cell culture
Comparison with Yeast/Bacteria
System
Mammalian Insect Yeast Bacteria
Size (m) 15 20 5 1
Dry cell weight 300 600 10 1
(10-10 g/cell)
Doubling time (h) 12-48 28-48 0.3 0.3
Biomass (g/dm3) 0.5 2 20 20
Productivity (g/g/day) 1 0.1
Cont’
Mammalian Insect Yeast Bacteria
Nutritional Complex simple
Media cost ($/dm3) 20 20 <1 <1
secretion yes Lytic variable no
Post-transl.
++ + +/- --
Mod.
DSP simple complex
Scale-up difficult simple
animal cell Culture?
• Tissue engineering/physiology
– Obvious
• Pharmaceutical protein & vaccine
production
– Product quality / post-translational
modifications
• Glycosylation, Sulfonation and folding
• Activity
• Immunogenity (wanted for vaccines, not wanted
for others)
• In-vivo half life
– Excretion vs inclusion bodies
Cells vs whole organs/animals
• Physiology studies: Guinea Pigs
– Isolating effects on cellular level
– Reproducibility
– Ethics
• Tissue engineering:
– Genetically modified organisms as organ
donor
• Non-ethical
• Risks
– Patients own material: stem cells
Cells vs whole animals
• Pharmaceutical proteins
– Economic
– Product concentration/down-stream processing
– Scale-up
– Proven technology
– Time to market
– Reproducibility, robustness
– Validation/safety
Common cell lines
BHK Fibroblast Baby hamster kidney
CHO Epithelial Chinese Hamster Ovary
PER-C6 Epithelial Human Keratonocyt
MDCK Epithelial Canine Kidney
Vero Fibroblast Monkey Kidney
L929 Fibroblast Mouse tumor fibroblast
3T3 Fibroblast Mouse fibroblast
Common cell lines
HeLa Epithelial Human cervical carcinoma
Namalwa Lymphoblast Human lymphoblastoma cell
MRC 5
Fibroblast Human embryonic lung fibroblast
WI-38 Fibroblast Human embryonic lung
Sf21,Sf9 Insect Spodoptera Frugiperda
T.ni 5 Insect trichopluisa ni
High Five
Products/Proteins
TPA: Tissue Plasmogenic Activator Genentech
FSH: Follicle stimulating Hormone Diosynth
EPO: Erythropoin Amgen
Interferon Biogen
Factor VIII blood clotting Novo
Monoclonal antibodies Centocor
Remicade
Reopro
Contract production DSM Biologics
Vaccines
Polio RIVM
Rabies Pasteur Merieux
Flue Duphar
Foot and mouth disease ID-Lelystad
Classical swine fever ID-Lelystad/Bayer
Other Intervet
Dangers
• 1955 Bad Polio vaccine batch
– 250 people ill
– 11 dead
Food and Drug
Administration (FDA)
• Food and Drug Act
– Biological and non-biological
• Public Health Act
– Biological, pre-market approval
Products
•Over The Counter drugs (aspirin)
• Prescription (most biologicals)
– Pioneer
New Drug Application, NDA
– Generic
Biologicals
• Viruses
• Therapeutic sera
• Toxins & antitoxin
• Vaccines
• Blood products
• Cell & gene therapeutics
• Therapeutic proteins
• Oligonucleotides (antisense)
• Peptides/hormones
Approval Processes
Preclinical Cells, Basic safety: dosages
Guinea pigs
Investigational new drug application (IND)
Phase 1 20-50 Pharmacological actions,
humans Product safety & side effects
Phase 2 50-200 Effectiveness: optimal dosage,
patients application scheme etc.
Phase 3 Hundreds Final safety and effectiveness
patients
Applications
• Product License Application
• Establishment License Application
– Only for biologicals
• All changes must be FDA approved
• Product must be in phase 3
• Good Manufacturing Practice (GMP)
Time, finances, scale
Years Dollars Prod. Scale
106 dm3
Basic research
1-3 0.5-5 1
IND application
Phase 1 0.5-2
5-100 10
Phase 2 0.5-3
Phase 3 1-5 100-1000
PLA 1-3 3-100
Total 10 250
Fast track
• Clear public health advantage
–Orphan drugs
–Live threatening diseases
–Clear therapeutic advances
Notes
• Product in trials = product on market
– Production process fixed
– Each change requires FDA approval
• Demonstrate product not changed
• CO2 Problem
Other
• Documentation
– Extensive
– Integrity must be validated
• Electronic documents
• Field compliance staff
– Unannounced inspections
– Criminal prosecution
– Imprisonment (board of directors)