Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Bio-separations Copyright 2003 Genentech, Inc. Production vs Cost 10 9 Erythropoietin a-Interferon Log10 Price ($ / kg) 8 7 6 Taxol 5 4 Digitalis 3 2 Penicillin 1 Ethanol 0 -4 -3 -2 -1 0 1 2 3 4 5 Log10 Annual Production (ton/year) 6 7 Molecules of Interest Proteins Molecules of Interest Antibiotics (Tetracycline) Molecules of Interest a helix b sheet tPA – Tissue Plasminogen Activator Amino Acids Amino Acids Methionine Aspartic Acid (pK = 3.9) Lysine (pK = 10.5) The “Chemical Plants” Bacteria The “Chemical Plants” Plants The “Chemical Plants” Yeast The “Chemical Plants” Fungi The “Chemical Plants” Transgenic Host Recombinant DNA Technology James Watson Francis Crick Rosalind Franklin Maurice Wilkins Eukaryotic (animal / plant) Cell Prokaryotic (bacteria) Cell Cell Disruption Cell Homogenizer Bead Mill The “Chemical Reactor” Fermentation Vessels The Production Process Monoclonal Antibody Production Bioreactor fluid with cells Sterile filtration Cells Continuous centrifugation IgG product Formulation 100,000 MWCO membrane Affinity chromatography Unbound material IgG eluted Membrane concentration The Problem Overall Material Balance for IgG Production (kg/batch) Component Inlet Outlet Ammonium sulfate Bio mass Glycerol IgG Growth media Na3 citrate Phosphoric acid Sodium hydrophosphate Sodium chloride Sodium hydroxide Tris-HCL 0.69 Water Water for Injection 64.69 0.00 1.85 0.00 21.76 0.80 1,041 6.83 55.18 6.83 64.69 0.87 1.85 0.22 8.41 0.80 1,041 6.81 55.19 6.81 11,459 18,269 11,459 18.269 Total 30,928 30,928 Product 0.14 0.69 0.14 IgG Economics – Commercial Plant Direct Fixed Cost Total capital investment Plant throughput Manufacturing cost Unit production cost Selling price Revenues Gross profit Taxes (40 %) Net profit Internal Rate of Return Net present value ( 7 % ) $ 15.3 million $ 16.3 million 6.2 kg of IgG per year $ 5.64 million / year $ 908 / g of IgG $ 2,500 / g of IgG $ 15.5 million / year $ 9.9 million / year $ 4.0 million / year $ 7.4 million / year 47.4 % $ 32.5 million Human Insulin Production Bioreactor fluid with cells Precipitation Chromatography Product Dialysis Cell disruption Cell debris Sulfonation Centrifugal extraction Dialysis Cleaning pro insulin Cyanogen bromide Penicillin Production Solvent Isopropanol Bioreactor fluid with cells Filtration Washing Solvent Filtration Filtration Drying Extraction Amyl acetate Product Precipitation Extract Activated carbon treatment Filtration Carbon and impurities Bio-separation Technologies Crystallization / Precipitation Liquid-liquid extraction Membrane filtration Chromatography Field based separations Crystallization / Precipitation Crystallization / Precipitation Well-mixed bulk liquid 1) 2) 3) 4) 5) Movement toward liquid film Diffusion toward crystal surface Surface adsorption Surface diffusion Reaction Solid State Crystal Phase Liquid Film Liquid-liquid Extraction Aqueous two-phase extraction Phase 1 - 4% polyethylene glycol in water Phase 2 - 4% dextran in water Dr = 0.2 g / cc s = 1.2 dyne / cm PEG Dextran PEG 6000 - Dextran 500 Distribution Coefficient K (PEG phase / Dextran phase) 3.0 Trypsin 2.0 a Chymotrypsin Ovalbumin 1.0 Insulin Lysozyme Transferrin a - Amylase BSA 0.1 0.05 0 1 2 3 4 5 6 7 Protein Molecular Weight (X 10-4 Daltons) 8 Centrifugal Extractor Light phase out Light phase in Heavy phase in Heavy phase out Podbielniak Centrifugal Extractor Membrane Filtration Permeate Retentate Feed Porous membrane = 0.02-10 mm Microfiltration = 0.001-0.2 mm Ultrafiltration Membrane Filtration Copyright 2003 Genentech, Inc. Ultrafiltration Copyright 2003 Genentech, Inc. Ion-exchange Chromatography + + + + + - + +- + +- + + Ion-exchange columns are packed with small beads that carry positive or negative charges that retard proteins of opposite charge. The association between a protein and the matrix depends on the pH and ionic strength of the solution passing down the column. These can be varied in a controlled way to achieve an effective separation. Gel-filtration Chromatography Gel filtration columns separate proteins according to size. The matrix consists of tiny porous beads. Protein molecules that are small enough to enter the holes in the beads are delayed and travel more slowly through the column. Proteins that cannot enter the beads are washed out of the column first. Such columns also allow an estimate of the protein size. Affinity Chromatography Affinity columns contain a matrix covalently coupled to a molecule that interacts specifically with the protein of interest. (e.g. an antibody ,or an enzyme substrate). Proteins that bind specifically to such a column can be finally released by a pH change or concentrated salts solution addition. The final product is highly purified. Affinity Chromatography AFpak ACB-894(an affinity column) with a cibacron blue ligand is recommended for the analysis of albumin and NAD-dependent enzymes. Sample Bovine serum albumin Column :Shodex AFpak ACB-894 Eluent :(A);0.1M Potassium phosphate (pH5.0) (B);0.1M Potassium phosphate (pH7.5) + 1.5M KCl Step gradient:(A) to (B) Flow rate :1.0mL/min Detector :Shodex UV (280nm) Column temp.:Ambient BSA Field Based Separations