Bio-separations Copyright 2003 Genentech, Inc.

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 -4 -3 -2 -1 0 1 2 3 4 5 6 7 Log10 Annual Production (ton/year)

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 IgG eluted Membrane concentration Unbound material

The Problem Overall Material Balance for IgG Production (kg/batch) Component Inlet Outlet Product Ammonium sulfate 64.69 64.69 Bio mass 0.00 0.87 Glycerol 1.85 1.85 IgG 0.00 0.22 0.14 Growth media 21.76 8.41 Na3 citrate 0.80 0.80 Phosphoric acid 1,041 1,041 Sodium hydrophosphate 6.83 6.81 Sodium chloride 55.18 55.19 Sodium hydroxide 6.83 6.81 Tris-HCL 0.69 0.69 Water 11,459 11,459 Water for Injection 18,269 18.269 Total 30,928 30,928 0.14

IgG Economics – Commercial Plant Direct Fixed Cost $ 15.3 million Total capital investment $ 16.3 million Plant throughput 6.2 kg of IgG per year Manufacturing cost $ 5.64 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 47.4 % Net present value ( 7 % ) $ 32.5 million

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

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

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

Crystallization / Precipitation

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

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

K (PEG phase / Dextran phase) PEG 6000 - 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 0 1 2 3 4 5 6 7 8 Protein Molecular Weight (X 10-4 Daltons)

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