ChEn 5751 - Spring 2007

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

ChEn 5751 Bioseparation Overview

Properties utilized in bioseparations

size, density, solubility, partitioning, mobility, charge, hydrophobic interactions, biological molecular interactions, etc.

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

10% (w/w) 10 g/l 120 g/l

The product stream from complex medium fermentation can be very complex.

In general concentration is low, product value varies widely.

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

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).

12

Typical Operations of Bioseparation

Removal of solids
Filtration, centrifugation, microfiltration

Common Stages of Bioseparation

Removal of solids Isolation (volume reduction) Purification Polishing

Isolation of product (volume reduction)

Cell disruption, extraction, adsorption, ultrafiltration, precipitation

Purification
Adsorption, elution chromatography, ultrafiltration, electrophoresis, precipitation, crystallization

Polishing
Crystallization, drying, auxiliary process, solvent recovery, water preparation

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

4/4/2007

Cell Culture Process for Production of Factor VIII

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

1000 950 1200 60 18 20 80 100 20

1500 1450 1420 1420 1380 1380 1200 1150 1150

1600 1500 1470 1430 1390 1390 1200 1150 1150

0.93 0.97 0.97 0.99 0.99 0.99 >0.99 >0.99 >0.99

286 278 240 230 11.5

ND ND ND 0.2 0.2 ND 0.2 0.2 0.01

Example of Bioseparation: Penicillin G Production

Lyophilized Spores Medium: Corn steep liquor hydrolyzed starch, oil, urea, phenyl acetic acid Agar Slant Culture Shaker Flasks (1 ) 100 m3 5000 kg Pen G Seed Culture (50 ) Washing Water 40 m3 Secondary Seed Culture (1 m3)

Example of Bioseparation: Monoclonal Antibody (IgG) Production

Production
T-flask Frozen Ampule 1

Fermentor
Centrifugation

10

100

m3
10 m3 20 Kg Antibody

10m3

Production fermentor 100 m3 Cooling Water

Isolation
Waste Treatment cooling
Holding Tank

Defined medium glucose, amino acids, lipids, salts, 10 mg/ proteins

Buffer Filtrate Holding Tank

Retentate Microfiltration 1 m Buffer Filtrate

Removal of Solids Isolation

Rotary Drum Filter

Biomass (3000kg) Pen G (50 kg) Sulfuric Acid 1000 kg

Cells, 10 kg Waste Disposal

Holding Tank

Holding Tank 4C Aqueous Solvent 130 m3 Centrifugal Extractor

Refrigerated Cooling Water Butyl acetate, 10,000 kg (Solvent) Spent Carbon Carbon Recovery Carbon Recycle Solvent + Impurity Solvent Recovery Impurity Waste Disposal

Elution Buffer

Washing Buffer, Regeneration Buffer

Ultrafiltration MWCO 100K

Holding Tank Retentate

Purification
Protein A Affinity Chromatography Elution Buffer
Holding Tank

Butylacetate phase Carbon Treatment 60% w/w potassium acetate acetone, 1m3 11m3 Stirred Tank Extractor Filter Potassium salt of Pen G

Viral Inactivation Tank Buffer Diafiltration

Ceramic Viral Filter

Flow Through

DEAE ion exchange chromatography

Holding Tank

Purification

Eluted Adsorbed DNA

Polishing

Waste Treatment Formulation Buffer

Polishing
Vacuum Dryer Penicillin G K Salt 5000 Kg
+

sterile, filtration 0.2 m

Diafiltration, exchange to water for injection

Filling

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

4/4/2007

Cohn Fractionation of Blood Proteins

Example: Fractional Precipitation

Ethanol fractionation of human plasma protein factors: pH, temperature, ionic strength & protein concentration change Cohns method 6:
primary means of preparing albumin, -globulin, fibrinogen
Effectively combine pH, temperature, solvent and salt concentrations that affect the solubility of different components of plasma to separate them.

Plasma
Ethanol 8% -3C Ethanol 25% pH 6.9 -5C Add ice water Ethanol 18% pH 5.2 Ethanol 40% pH 5.8

Fraction I 61% fibrinogen

ppt

Fraction II, III 37% r-globulin 48% -globulin All 1-lipoprotein plasminogen prothrombin

ppt

Fraction IV-I 1-lipoprotein ceruloplasmin

Fraction IV-4 transferin haptoglobulin

pH 4.8 (with sodium acetate/acetic and acid buffer) Fraction V

Albumin (ppt)

MSG Isolation & Crystallization

HCl

Fermentation Broth Evaporator Concentrate Neutralizer Cooling Water

MSG Isolation & Crystallization (cont)

The only discharge is mother liquor 1. All the others are recycled to increase the recovery yield
Cooling Water

Steam NaOH

Neutralizer Filter Neutralizer Filter

Active carbon

MSG Solution Carbon, other solids

Crystallizer GA (-crystal) Continuous Centrifuge GA Mixing Tank Continuous Centrifuge GA

discard solids

Mother Liquor 1--discard Removal of solids (cells and insoluble unused media components) Mother Liquor 2
(to form -crystal of GA)

HCl NaOH

Ion Exchange

decolorization

Eluent Pure MSG Solute

Discard

NaOH, Steam

Crystal Transformation

Steam
Crystallizer Cooling Water

Continuous Centrifuge GA

Mother Liquor 3

Cooling Water

Vacuum Crystallizer 60-70C Centrifuge

MSG

Mother Liquor

H2O

Mixing Tank Continuous Centrifuge GA Mixing Tank

Mother Liquor 4

Dryer
GA: Glutamic acid

Sieve

Big chunks Products

Re-dissolve

Prof. Wei-Shou Hu

ChEn 5751 - Spring 2007

4/4/2007

Unit Operation

Properties Used in Separation

Application
Solid removal Solid removal Solid Removal Isolation Isolation Isolation, Purification Purification, Isolation Purification, Isolation Purification, Isolation

Unit Operation
Filtration Centrifugation Microfiltration Extraction
Solvent Extraction Aqueous Two-Phase Extraction

Properties Used in Separation

Size Size, density Size

Application
Solid removal Solid removal Solid Removal

Major Characteristics
Produces relatively dry cake Produces paste or cell concentration Retentate solid content, not very high

Filtration Size Centrifugation Size, density Microfiltration Size Extraction Solvent Extraction Partition Aqueous Two-Phase Extraction Partition Adsorption Ion Exchange Chromatography Charge Affinity Chromatography Molecular interaction Hydrophobic Interaction Protein-ligand interaction Chromatography (Transition) Metal Ion Sequence-specific tag-metal interaction Chromatography Elution Chromatography, (Liquid Chromatography, HPCL) Gel Permeation Size and shape of molecules Chromatography Reverse Phase Size, molecular interaction Chromatography Chromatofocusing Charge, Mobility Displacement Chromatography Molecular interactions Electrophoresis Charge, Mobility Ultrafiltration Size Reverse Osmosis Size, Molecular diffusivity Precipitation Solubility Crystalization Solubility, Molecular interactions

Partition Partition Charge Molecular interaction Protein-ligand interaction Sequence-specific tag-metal interaction Size and shape of molecules Size, molecular interaction Charge, Mobility Molecular interactions Charge, Mobility Size Size, Molecular diffusivity Solubility Solubility, Molecular interactions

Isolation Isolation Isolation, Purification Purification, Isolation Purification, Isolation Purification, Isolation Purification Purification Purification Purification Purification Isolation, Purification Isolation, Purification Isolation Purification, Polishing

Adsorption
Ion Exchange Chromatography Affinity Chromatography Hydrophobic Interaction Chromatography (Transition) Metal Ion Chromatography Gel Permeation Chromatography

Purification Purification Purification Purification Purification Isolation, Purification Isolation, Purification Isolation Purification, Polishing

Elution Chromatography (Liquid Chromatography, HPLC)

Reverse Phase Chromatography Chromatofocusing Displacement Chromatography

Electrophoresis Ultrafiltration Reverse Osmosis Precipitation Crystalization

Prof. Wei-Shou Hu