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Bio-separations Copyright 2003 Genentech, Inc..

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Presentation on theme: "Bio-separations Copyright 2003 Genentech, Inc.."— Presentation transcript:

1 Bio-separations Copyright 2003 Genentech, Inc.

2 Production vs Cost Log10 Price ($ / kg)
9 8 7 6 5 4 3 2 1 Erythropoietin a-Interferon Taxol Log10 Price ($ / kg) Digitalis Penicillin Ethanol Log10 Annual Production (ton/year)

3 Molecules of Interest Proteins

4 Molecules of Interest Antibiotics (Tetracycline)

5 Molecules of Interest a helix b sheet
tPA – Tissue Plasminogen Activator

6 Amino Acids

7 Amino Acids Methionine Aspartic Acid (pK = 3.9) Lysine (pK = 10.5)

8 The “Chemical Plants” Bacteria

9 The “Chemical Plants” Plants

10 The “Chemical Plants” Yeast

11 The “Chemical Plants” Fungi

12 The “Chemical Plants” Transgenic Host

13 Recombinant DNA Technology
James Watson Francis Crick Rosalind Franklin Maurice Wilkins

14 Eukaryotic (animal / plant) Cell

15 Prokaryotic (bacteria) Cell

16 Cell Disruption Cell Homogenizer Bead Mill

17 The “Chemical Reactor”
Fermentation Vessels

18 The Production Process

19 Monoclonal Antibody Production
Bioreactor fluid with cells Sterile filtration Cells Continuous centrifugation IgG product Formulation 100,000 MWCO membrane Affinity chromatography IgG eluted Membrane concentration Unbound material

20 The Problem Overall Material Balance for IgG Production (kg/batch)
Component Inlet Outlet Product Ammonium sulfate Bio mass Glycerol IgG Growth media Na3 citrate Phosphoric acid 1,041 1,041 Sodium hydrophosphate Sodium chloride Sodium hydroxide Tris-HCL Water 11, ,459 Water for Injection 18, Total 30, ,

21 IgG Economics – Commercial Plant
Direct Fixed Cost $ 15.3 million Total capital investment $ 16.3 million Plant throughput kg of IgG per year Manufacturing cost $ million / year Unit production cost $ 908 / g of IgG Selling price $ 2,500 / g of IgG Revenues $ 15.5 million / year Gross profit $ 9.9 million / year Taxes (40 %) $ 4.0 million / year Net profit $ 7.4 million / year Internal Rate of Return % Net present value ( 7 % ) $ 32.5 million

22 Human Insulin Production
Precipitation Chromatography Bioreactor fluid with cells Product Dialysis Cell disruption Cell debris Sulfonation Centrifugal extraction Dialysis Cleaning pro insulin Cyanogen bromide

23 Penicillin Production
Solvent Isopropanol Filtration Bioreactor fluid with cells Washing Solvent Filtration Filtration Drying Extraction Amyl acetate Precipitation Product Extract Filtration Activated carbon treatment Carbon and impurities

24 Bio-separation Technologies
Crystallization / Precipitation Liquid-liquid extraction Membrane filtration Chromatography Field based separations

25 Crystallization / Precipitation

26 Crystallization / Precipitation
Movement toward liquid film Diffusion toward crystal surface Surface adsorption Surface diffusion Reaction Well-mixed bulk liquid Solid State Crystal Phase Liquid Film

27 Liquid-liquid Extraction
Aqueous two-phase extraction Phase 1 - 4% polyethylene glycol in water Phase 2 - 4% dextran in water Dr = g / cc s = 1.2 dyne / cm PEG Dextran

28 K (PEG phase / Dextran phase)
PEG Dextran 500 Distribution Coefficient 3.0 2.0 1.0 0.1 0.05 Trypsin a Chymotrypsin Ovalbumin Lysozyme Insulin Transferrin K (PEG phase / Dextran phase) a - Amylase BSA Protein Molecular Weight (X 10-4 Daltons)

29 Centrifugal Extractor
Light phase out Light phase in Heavy phase in Heavy phase out Podbielniak Centrifugal Extractor

30 Membrane Filtration Permeate Retentate Feed Porous membrane
= mm Microfiltration = mm Ultrafiltration

31 Membrane Filtration Copyright 2003 Genentech, Inc.

32 Ultrafiltration Copyright 2003 Genentech, Inc.

33 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.

34 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.

35 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.

36 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

37 Field Based Separations


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