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Animal Cell Biotechnology
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ANIMAL CELL BIOTECHNOLOGY
Human health and well-being is threatened bynumerous diseases. These may be infectious diseasesor 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 theproduction process for bacterial and virus vaccines andpharmaceutical proteins. For this we make use of
detailed metabolic models in mammals, which in turnare also used to study the effect of food components onmammalian cell physiology.
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Pharmaceutical Proteins
Animal cell culture increasingly attracts interest dueto the ability of animal cells to produce qualitativeexcellent pharmaceutical proteins. Examples of such
proteins are Erythropoietin (Epo) and Follicle-stimulating hormone (FSH). However, compared tomicrobial production systems, animal cell culturesare characterized by low biomass concentrations,
low productivities, slow growth rates, and a highshear sensitivity. Our research focuses on themedium, process and reactor design addressing theabove mentioned problems.
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Erythropoietin (Epo)
the principal factor responsible for the regulationof red blood cell production during steady-stateconditions and for accelerating recovery of red bloodcell mass following hemorrhage
Follicle-stimulating hormone (FSH)
produced by the Pituitary gland,causes the follicle to develop in females,
then stimulates the follicle to produce estrogen,and helps to stimulate sperm production in males ---When FSH is elevated, in males, it generally reflects adefect in sperm production, or castration.
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Stem cells&tissue engineering Skin, Liver
Bone and Cartilage Pharmaceuticals MKZ (ID-Lelystad)
Classical swine fever (Bayer) Small pox
Remicade (Centocor)
-Glucosidase (Pharming)
Animal cell culture
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History
Animal cell culture
Why, media, applications, comparison
microbial cell culture
Regulations
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History
1880 Roux Embryonic chicken in saline
1900 Harrison
Anchorage dependent Nutrients Relative slow growth rate
Doubling 1 day vs 20 minutes bacteria Contamination
frog embryo
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characteristics for in vitrocell growth:
Cells require an anchor like the lymph clots and thecover slip.Cells require nutrients provided by the lymph.
Cells grow very slow; 20 hours doubling timecompared to 20 minutes for bacteria This means cellcultures are vulnerable to contamination
1900 Harrison
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History
Carrel (surgeon, 1923)
Aseptic techniques
Carrel Flask
1912-1946 Culture Chicken Embryo Fibroblast
Plasma+tissue homogenate
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Polio
1940/1950s Major Polio epidemics
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Polio
Antibiotics: Penicillin and streptomycin
Polio vaccine first product
primary monkey kidney cellshuman diploid lung fibroblast
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1950s
Hela cell line: human carcinoma
Chemically defined media (Eagle, Earle)ConsistencySterilizationReduced chance of contamination
Insect cell lines (Grace)
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Recombinant DNA 1970
All proteins in E-coli
No post-translational modifications
Glycosylation Folding
Inclusionexcretion
Large complex proteins requireanimal cells
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MicrocarriersVan Wezel 1960s RIVM
Monkey Kidney cells
Polio
http://www.rivm.nl/en/
http://www.rivm.nl/en/http://www.rivm.nl/en/ -
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Hybridoma
1975 Kohler and Milstein
B-cellantibody
Mortal
Myelomasingle chain
immortal
Hybridoma
monoclonal antibodyimmortal
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Serum-free media
Genetic modification cells
Bioreactors Large-scale (20 m3)
Nowadays
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Physiological studies Understand function of cells (Toxicology)
Tissue engineering (Skin, Cartilage, Liver)
Biologicals (pharmaceutical proteins)
Monoclonal Antibodies
Hormones (EPO, FSH) Enzymes (-glucosidase)
Vaccines (Polio)
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Toxaphene
Probes for:
pH
Dissolved Oxygen
Temperature
Gas inlet
Nitrogen
Oxygen
CO2
Gas outlet
Heating
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 50 100 150 200
Viable-cell concentration (109 cells.dm-3)
Culture time (hours)
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An example of the use of animal-cell culture in thestudy of toxicology is shown. Toxaphene is a pesticide
used in the US until the eighties in cotton farming andin fish industry to remove all fish out of lakes with theintention to culture one specific type of fish. It is amixture of 180 types of polychlorinated cyclichydrocarbons and very persistent. Here a culture of
hepatocytes on microcarriers is exposed to differenttoxaphene concentrations of 0 and 10 microgram perliter. In the 10 microgram/l culture no growth occurredand no oxygen was consumed, while glucose was
rapidly converted to lactate. Probably toxapheneinhibits processes in the mitochondria thus inhibitingrespiration. The only way to generate ATP is throughglycolysis. Apparently this is only just enough tomaintain cells and not for growth.
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Cartilage
http://www.isotis.com/
http://www.isotis.com/isotis/isotiswebv3.nsf/lookupDefaultHomepages/$first -
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EPO http://www.amgen.com/
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Erythropoeitin is a glycoprotein hormoneproduced in the kidneys that stimulates red-blood-cell production in the bone marrow.Patients with kidney failure produce lessEPO and consequently have lower levels ofred blood cells (up to 1/3 of a normalperson) and have severe anemia. Epogen
solves this problem. Because the activeprotein is modified after translation it canonly be produced in animal cells in thecorrect form.
Erythropoeitin (EPO)
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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
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0.0
0.0
0.0
0.0
0.1
1.0
10.0
0 50 100Time (h)
Cx(g
/l)
YeastHybridoma
Growth curve
Minimum
densityLag phase
exponential
Stationary
decline
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Desinfection step
Tissue isolation
Incubation&growth
Primary cells
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Passage number
70 generations
Ln(total cells)
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Transformation
Characteristics Infinite life span
High growthpotential
Low growth factordependence
Suspension growth
aneuploid
Methods Mutagens
Viruses
Oncogens Spontaneous
Tumors
Not all transformed cell lines can form tumors, butall tumors contain transformed cells
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Normal cells?
Diploid
Anchoragedependent
Finite life span Non-malignant
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Basic Media
Glucose
Amino acids
HCO3
Mammalian Insect4 g/l 2.5 g/l
0.01-0.15 g/l 0.1-1.5 g/l
Glutamine 1 g/l 1 g/l3.5 g/l 0.35 g/l
H2PO4 0.1 g/l 1 g/l
pH 7.2 6.4Osmolarity 300 mOsm 350 mOsm
Salts
Vitamins/Spore4.5 g/l NaCl 1g/l MgSO4,KCl
More Less
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Media
Insect cells
Fumaric & alfa-
keto-glutaric acid,Malic & succinicacid
Maltose & Sucrose&Threhalose
Mammalian cells
Pyruvate
Hypoxanthine,thymidine
Linoleic acid
Hepes buffer Phenol red
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Serum
0-20% Serum Growth factors
Transferrin (Fe) Lipids
Insulin
Shear protection Detoxification
Problems Infectious agents
(viruses, prions)
Variablecomposition
Expensive
High proteincontent problems inDSP
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Serum
Serum/protein free media Transferrine / ferrous citrate
Lipid concentrate Extracts Yeast extract
Insulin
Bovine Serum Albumin (f.a. transport/detoxification) Pluronic (shear protectant)
Completely mammalian origin free (MOF)
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Comparison with Yeast/Bacteria Syste
Mammalian Insect Yeast Bacteria
Size (m)
Dry cell weight(10-10 g/cell)
Productivity (g/g/day)
15 20 5 1
300 600 10 1
1 0.1
Biomass (g/dm3) 0.5 2 2020
Doubling time (h) 12-48 28-48 0.3 0.3
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Cont
Mammalian Insect Yeast Bacteria
secretion
DSP
Scale-up
yes Lytic variable no
difficult simple
simple complex
Post-transl.Mod.
++ + +/- --
Nutritional Complex simple
Media cost ($/dm3) 20 20
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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 forothers)
In-vivo half life
Excretion vs inclusion bodies
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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
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Cells vs whole animals
Pharmaceutical proteins Economic
Product concentration/down-streamprocessing
Scale-up
Proven technology
Time to market
Reproducibility, robustness
Validation/safety
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Canine KidneyEpithelial
Common cell lines
BHK
CHO
PER-C6
MDCK
Vero
L
3T3
Baby hamster kidneyFibroblast
Chinese Hamster OvaryEpithelial
Mouse fibroblastFibroblast
Mouse tumor fibroblastFibroblast
Monkey KidneyFibroblast
Human KeratonocytEpithelial
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Common cell lines
HeLa
Namalwa
MRC 5
WI-38
Human cervical carcinomaEpithelial
Human lymphoblastoma cellLymphoblast
Human embryonic lungfibroblast
Fibroblast
Human embryonic lungFibroblast
Sf21,Sf9 Insect Spodoptera Frugiperda
T.ni 5 Insect trichopluisa niHigh Five
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Products/Proteins
TPA: Tissue Plasmogenic Activator GenentechFSH: Follicle stimulating Hormone DiosynthEPO: Erythropoin Amgen
Interferon BiogenFactor VIII blood clotting NovoMonoclonal antibodies Centocor
Remicade
ReoproContract production DSM Biologics
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Vaccines
Polio RIVMRabies Pasteur MerieuxFlue Duphar
Foot and mouth disease ID-LelystadClassical swine fever ID-Lelystad/Bayer
Other Intervet
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Dangers
1955 Bad Polio vaccinebatch
250 people ill
11 dead
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Food and Drug Administration
(FDA)
Food and Drug Act
Biological and non-biological
Public Health Act
Biological, pre-market approval
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Over the counter drugs (aspirin)
Prescription (most biologicals)
Pioneer
Generic
Products
New Drug Application
Abbreviated NDA
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Biologicals
Viruses Therapeutic sera
Toxins & antitoxin
Vaccines
Blood products
Cell & gene therapeutics
Therapeutic proteins
Oligonucleotides (antisense)
Peptides/hormones
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Approval Processes
Preclinical
Phase 1
Phase 2
Phase 3
Basic safety: dosagesCells,Guinea pigs
Pharmacological actions,Product safety & side effects
20-50humans
Effectiveness: optimal dosage,
application scheme etc.
50-200
patientsFinal safety and effectivenessHundreds
patients
Investigational new drug application (IND)
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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)
Applications
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Time, finances, scale
Basic research
IND application
Phase 1
Phase 2
Phase 3PLA 1-3 3-100
1-3 0.5-5 1
0.5-2
0.5-35-100 10
1-5 100-1000
10 250
Years Dollars106
Prod. Scaledm3
Total
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Fast track
Clear public health advantage
Orphan drugs
Live threatening diseases Clear therapeutic advances
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Product in trials = product on market
Production process fixed
Each change requires FDA approval
Demonstrate product not changed
CO2 Problem
Notes
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Documentation
Extensive
Integrity must be validated
Electronic documents
Field compliance staff
Unannounced inspections
Criminal prosecution
Imprisonment (board of directors)
Other