proteomics 6 lecture8 - Production of Protein Pharmaceuticals

소개

1

목차

Review

Six Step Process

Cell Growth Needs

Prototrophs vs. Auxotrophs

Shake Flask Incubator

Fermentors & Bioreactors

Fermentor Scale Up

Cell Isolation/Harvesting

Protein Isolation & Purification

Cell Disruption Methods

Protein Isolation Methods

Electrophoresis

Isoelectric Point (pI)

IEF Principles

Isoelectric Focusing

Column Chromatography

Affinity Chromatography

Size Exclusion Chrom

Size Exclusion Chromatography (SEC)

Sephadex Structure

Ion Exchange Chromatography (IEC)

Ion Exchange Chromatography

IEC Nomenclature

IEC Groups

IEC Techniques

IEC Rules of Thumb

Conclusion

본문내용

Review
Escherichia coli
Other bacteria
Pichia pastoris
Other yeast
Baculovirus
Animal cell culture
Plants
Sheep/cows/humans
Cell free
Polyhedra
Six Step Process
Isolation of gene of interest
Introduction of gene to expression vector
Transformation into host cells
Growth of cells through fermentation
Isolation & purification of protein
Formulation of protein product
Cell Growth Needs
A sterile carbon, nitrogen, hydrogen and oxygen source (air and H2O) + trace metals (Zn, Fe, Cu, Ca, Mg, Mn)
A sterile energy source (light, sugar, acetate, methanol, ethanol)
A constant (or near constant) temperature above 20 oC
A growth regulating chemical (antibiotic)
Prototrophs vs. Auxotrophs
Protrophic cells (bacteria, plants) can produce all essential amino acids, nucleic acids, carbohydrates and lipids from simple nutrients (water, oxygen, nitrogen or ammonia, CO2)
Auxotrophic cells (yeast, insect cells, mammalian) need vitamins, essential amino acids (His, Cys), sugars, lipids, etc. to grow because they have lost this ability through evolution (bacterial symbiosis)
106 cells/mL
109-1010 cells/mL
Growth density
105 cells/mL
1 cell
Seeding density
10000-100000 nm
100-2000 nm
Size
Very susceptible
Less affected
Environmental FX
Key for buffering
Sometimes
CO2 Requirement
Complex
Usually simple
Nutritional Rqmt
Low
High
O2 Requirement
1-5% per hour
10-50% per hour
Growth Rate
Present
Present
Cell membrane
Generally absent
Generally present

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