Beruflich Dokumente
Kultur Dokumente
4/4/2007
Characteristics of Bioseparations
Starting Materials
Fermentation broth (bacteria and yeasts, mycelial fungi and streptomycetes, mammalian or insect cell cultures) or defined media and complex media Biological materials (blood, plant and animal tissues or organs) Product concentration is usually dilute
Quality of products
activity, purity, contaminants For biologics, consistency of products
The structure or composition of the product is not very well defined, e.g. virus, glycoprotein. The sugar of protein has a lot of heterogeneity Prof. Wei-Shou Hu
Concentrations of product in the effluent streams from fermentors Molecular weight Ethanol Amino Acids Vitamin B12 Cellulase Penicillin Monoclonal Antibody Recombinant Interferon tPA Polysaccharide (xanthan gum) 180K 46 110 (Avg) 1355 ~300K Concentration 70-100 g/l 20-120 g/l 2-5 g/l 15 g/l 30 g/l 50-150 mg/l 25 g/l 50 mg/l 10 g/l $400/mg $50/kg $2000/g Price 40/lb $2/kg
Fermentation Broth of MSG Production Monosodium Glutamate Molasses (45% solid) 50% (w/w) NH4OH After Fermentation Residual unfermented molasses Cell mass Glutamic acid NH4OH
The product stream from complex medium fermentation can be very complex.
Prof. Wei-Shou Hu
4/4/2007
Molecular components of an E. coli cell Percent total weight Water Proteins Nucleic acids DNA RNA Carbohydrates Lipids Building block molecules and intermediates Inorganic ions 1 6 3 2 2 1 ~1000 ~50 ~40 ~500 70 15 ~3000 Number of each kind
Recombinant interferon in E. coli as inclusion bodies Glucose 45 g/l Medium NH4 Other salts Dry Cell mass Product Interferon Other salts 20 g/l 0.1 g/g cell mass 10 g/l
1
Some rDNA proteins are produced inside E coli cells (intracellular).
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Purification
Adsorption, elution chromatography, ultrafiltration, electrophoresis, precipitation, crystallization
Polishing
Crystallization, drying, auxiliary process, solvent recovery, water preparation
Prof. Wei-Shou Hu
4/4/2007
Example of a recombinant protein purification (see flow chart of recombinant antibody production)
Step Volume of output stream (liter) Antibody in output stream (g) Total Protein in output stream (g) Specific antibody (g antibody/g total protein) Total DNA (ng) Specific DNA (ng/mg protein)
Cell Culture Broth Centrifugation Isolation/Solid Removal Microfiltration Ultrafiltration Affinity Chromatography Diafiltration Polishing Gel permeation chromatography Ion exchange Diafiltration Purification