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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
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+
+
+
+
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+
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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