BKB4493

Chapter 1 Introduction to Bioseparations Engineering

Whats there at the end of the road?

Explain the nature of bioseparation Outline the stages involve in downstream processing, objective of each stage, and typical unit operations for each stage Calculate purity, specific activity, and yield as quality indicators in purification

Introduction
Bioseparation engineering systematic study of the scientific and engineering principles utilized for the large-scale purification of biological products Downstream processing separation and purification segment of a bioprocess which followed biological reaction [purification of an antibiotic following microbial fermentation Purification major expenses in the production of most fermentation products [>50% of total manufacturing costs]
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What is separated?
Categories Solvents Organic acids Vitamins Amino acids Antibiotics Sugars and carbohydrates Semi-purified proteins Examples Ethanol, acetone, butanol Citric acid, lactic acid, butyric acid Ascorbic acid, B12 Lysine, phenylalanine, glycine Penicillin, streptomycin, gentamycin Glucose, fructose, starch, dextran, xanthan Industrial enzymes, egg proteins, milk proteins

Purified proteins Therapeutic proteins, monoclonal antibodies, hormones, vaccines Cells Bakers yeast, lactobacillus
Source: Ghosh (2006) 4

Nature of Bioseparation
Low [biological products]
Monoclonal antibodie 0.1 mg/ml in mammalian cell culture supernanant Large volume are needed from the very beginning

Target product and impurities/byproducts have almost similar chemical and physical properties
Challenging techniques have to be selective

Nature of Bioseparation
Stringent quality requirement for consumable products
Therapeutic proteins should be free from endotoxins and pyrogens

Biological products are susceptible to denaturation (pH, ionic strength, shear rates)
Techniques have to be gentle

Many biological products are thermolabile

Carried out at sub-ambient temperature
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Basis of Separation

Source: Shuler and Kargi (2002)

Bioseparation Techniques

RIPP Scheme
Liquid-solids separations (dewatering, concentration, particle removing) @ Recovery Solute-solute separations (Isolation, Purification) Solute-liquid separations (Polishing)

Bioseparation Techniques
Stage Recovery (separation of insolubles) Isolation Objective(s) Remove or collect cells, cell debris Reduce volume Remove materials having properties widely different from those of target product Reduce volume Typical Unit Operations Filtration, sedimentation, extraction, adsorption, centrifugation Extraction, adsorption, ultrafiltration, precipitation

Purification

Remove remaining impurities, which typically are similar to those of target product
Remove liquids Convert product to crystalline form (not always possible)

Chromatography, affinity methods, precipitation

Polishing

Drying, crystallization

Source: Harrison et al. (2002)

Example of bioseparation
Separation and purification of intracellular enzymes
fermentation lyophilization Cell removal and concentration Cell disruption Removal of cell debris Protein precipitation or aqueous twophase extraction

dialysis
Solvent precipitation Chromatographic purification

ultrafiltration
Source: Shuler and Kargi (2002) 10

Basic Principles of Engineering Analysis

Frequently asked question How much? How fast? Based on three criteria material balance, equilibria, flux
equilibria A+B C, Keq=

C A B
y x

flux = coefficient x driving force Fick's Law, JD =-D Darcy's Law, JW dc dx = Lp P

In extraction, K=

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Process and Product Quality

product purity = product + impurities purityn purification fold = purity0 units of biological activity specific activity = mass product formed yield = product in feed

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Assignment 1 (due: Monday, 07/01/2008)

Calculation for the purification of a recombinant protein The purification of a recombinant protein is carried out starting with 100 liters of a clarified cell lysate (i.e., the cells have been lysed, and the cell debris has been removed to give a clarified solution), which has a total protein concentration of 0.36 mg/ml and a recombinant protein concentration of 2.2 U/ml, where U denotes units of biological activity of the recombinant proteins. It is known that the completely pure recombinant protein has a specific activity of 40.0 U/mg. Purification is continued until a chromatography step that yields 2.0 liters of a fraction containing the protein, with a total protein concentration of 1.11 mg/ml and a recombinant protein concentration of 43.2 U/ml. For the recombinant protein, calculate the starting and ending purity, the starting and ending specific activity, and the percentage yield and fold purification through the chromatography step.

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References
Ghosh, R. Principles of Bioseparations Engineering. World Scientific, Singapore (2006) Harrison, R. G., Todd, P., Rudge, S. R. and Petrides, D. P. Bioseparations Science and Engineering. Oxford University Press. Oxford (2002) Shuler, M. L. and Kargi, F. Bioprocess Engineering: Basic Concepts, 2nd edition. Prentice Hall, Upper Saddle River (2002)

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