Animal Cell Biotechnology

ANIMAL CELL BIOTECHNOLOGY

Human health and well-being is threatened by numerous diseases. These may be infectious diseases or 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.

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 (Epo) and Folliclestimulating 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. Our research focuses on the medium, process and reactor design addressing the above mentioned problems.

Erythropoietin (Epo) 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 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 a defect in sperm production, or castration.

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)

 History Animal cell culture

Why, media, applications, comparison microbial cell culture

Regulations

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

1900 Harrison

characteristics for in vitro cell growth:

Cells require an anchor like the lymph clots and the cover slip. Cells require nutrients provided by the lymph. Cells grow very slow; 20 hours doubling time compared to 20 minutes for bacteria This means cell cultures are vulnerable to contamination

History
Carrel (surgeon, 1923) Aseptic techniques
Carrel Flask

1912-1946 Culture Chicken Embryo Fibroblast

Plasma+tissue homogenate

Polio
1940/1950s Major Polio epidemics

Polio product Polio vaccine first

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)

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 1960s RIVM

Monkey Kidney cells

Polio

http://www.rivm.nl/en/

Hybridoma
1975 Kohler and Milstein B-cell antibody Mortal Myeloma single chain immortal

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 Oxygen CO2 Gas outlet Viable-cell concentration (109 cells.dm-3)
1.4

Probes for: pH Dissolved Oxygen Temperature Heating

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 50 100 150 200

Culture time (hours)

An example of the use of animal-cell culture in the study of toxicology is shown. Toxaphene is a pesticide used in the US until the eighties in cotton farming and in fish industry to remove all fish out of lakes with the intention to culture one specific type of fish. It is a mixture of 180 types of polychlorinated cyclic hydrocarbons and very persistent. Here a culture of hepatocytes on microcarriers is exposed to different toxaphene concentrations of 0 and 10 microgram per liter. In the 10 microgram/l culture no growth occurred and no oxygen was consumed, while glucose was rapidly converted to lactate. Probably toxaphene inhibits processes in the mitochondria thus inhibiting respiration. The only way to generate ATP is through glycolysis. Apparently this is only just enough to maintain cells and not for growth.

Cartilage

http://www.isotis.com/

EPO

http://www.amgen.com/

Erythropoeitin (EPO)
Erythropoeitin is a glycoprotein hormone produced in the kidneys that stimulates redblood-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.

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 1.0
Cx (g/l)

Stationary decline exponential

Yeast Hybridoma

0.1 0.0 0.0

Minimum density 0.0

0.0 0

Lag phase
50 100

Time (h)

Desinfection step

Tissue isolation

Incubation&growth

Primary cells

Passage number
Ln(total cells)

70

generations

Transformation
Characteristics
Infinite life span High growth potential Low growth factor dependence Suspension growth aneuploid

Methods
Mutagens Viruses Oncogens Spontaneous Tumors

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
Glucose Amino acids Glutamine HCO3 H2PO4 Salts Vitamins/Spore pH Osmolarity Mammalian 4 g/l 0.01-0.15 g/l 1 g/l 3.5 g/l 0.1 g/l 4.5 g/l NaCl More 7.2 300 mOsm Insect 2.5 g/l 0.1-1.5 g/l 1 g/l 0.35 g/l 1 g/l 1g/l MgSO4,KCl Less 6.4 350 mOsm

Media
Insect cells
Fumaric & alfaketo-glutaric acid, Malic & succinic acid Maltose & Sucrose &Threhalose

Mammalian cells
Pyruvate Hypoxanthine, thymidine Linoleic acid Hepes buffer Phenol red

Serum
0-20% Serum
Growth factors Transferrin (Fe) Lipids Insulin Shear protection Detoxification

Problems
Infectious agents (viruses, prions) Variable composition Expensive High protein content problems in DSP

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)

Comparison with Yeast/Bacteria System

Mammalian Insect Size (m) Dry cell weight
(10-10 g/cell)

Yeast
5 10

Bacteria
1 1

15 300

20 600

Doubling time (h)

Biomass (g/dm3)

12-48
0.5 1

28-48
2

0.3
20

0.3
20 0.1

Productivity (g/g/day)

Cont
Mammalian Insect Nutritional Media cost
($/dm3)

Yeast <1 variable +/-

Bacteria <1 no --

Complex 20 yes ++ simple difficult 20 Lytic +

simple

secretion Post-transl. Mod. DSP Scale-up

complex
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 CHO PER-C6 Fibroblast Epithelial Epithelial Epithelial Fibroblast Fibroblast Fibroblast Baby hamster kidney Chinese Hamster Ovary Human Keratonocyt Canine Kidney Monkey Kidney Mouse tumor fibroblast Mouse fibroblast

MDCK
Vero L 3T3

Common cell lines

HeLa Namalwa MRC 5 Epithelial Human cervical carcinoma

Lymphoblast Human lymphoblastoma cell

Fibroblast Human embryonic lung fibroblast Human embryonic lung
Spodoptera Frugiperda
trichopluisa ni

WI-38 Sf21,Sf9 T.ni 5

Fibroblast

Insect
Insect
High Five

Products/Proteins
TPA: Tissue Plasmogenic Activator FSH: Follicle stimulating Hormone EPO: Erythropoin Interferon Factor VIII blood clotting Monoclonal antibodies Remicade Reopro Contract production Genentech Diosynth Amgen Biogen Novo Centocor

DSM Biologics

Vaccines
Polio Rabies Flue Foot and mouth disease Classical swine fever Other RIVM Pasteur Merieux Duphar ID-Lelystad ID-Lelystad/Bayer 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 Generic

New Drug Application

Abbreviated NDA

Biologicals
Viruses Therapeutic sera Toxins & antitoxin Vaccines Blood products Cell & gene therapeutics Therapeutic proteins Oligonucleotides (antisense) Peptides/hormones

Approval Processes
Basic safety: dosages Preclinical Cells, Guinea pigs Investigational new drug application (IND) Phase 1 20-50 humans 50-200 patients Hundreds patients

Pharmacological actions, Product safety & side effects

Effectiveness: optimal dosage, application scheme etc. Final safety and effectiveness

Phase 2
Phase 3

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 Basic research IND application Phase 1 1-3 0.5-2 0.5-3 1-5 1-3 3-100 5-100 10 100-1000 Dollars 106 0.5-5 Prod. Scale dm3 1

Phase 2
Phase 3 PLA 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)