DOWNSTREAM PROCESSING

Dept. of Chemical Eng., N I T, Tiruchirappalli

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Recovery of intracellular products

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In the biotechnology industry, there is quite a

challenge to the biochemists and chemical

engineers in the downstream processing. The diverse purification methods of the research laboratory at the bench scale are to be eventually scaled up to the production floor. The methods are used in complementary fashion to develop costeffective methods in quick time and enable the

companies to bring the products.

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Chromatography Methods
Therapeutic proteins in this industry are recovered from complex sources and have to be very pure and efficacious to be approved for use as medicinal products. Use of chromatographic techniques helps to purify the biotechnology proteins to a state of very high purity. Each chromatographic technique has its advantages and disadvantages. One single chromatography step is seldom capable of giving a product of the desired quality in terms of homogeneity and purity. The different types of chromatography procedures that are used in downstream processing

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Chromatography technique
Gel Chromatography Ion exchange Chromatography Affinity Chromatography Hydrophobic interaction Chromatography

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Ion Exchange Chromatography

Ion Exchange Chromatography relies on charge-charge interactions between the protein of interest and charges on a resin (bead). Ion exchange chromatography can be subdivided into cation exchange chromatography, in which a positively charged protein of interest binds to a negatively charged resin; and anion exchange chromatography, in which a negatively charged protein of interest binds to a positively charged resin. One can manipulate the charges on the protein by knowing the pI of the protein and using buffers of different pHs to alter the charge on the protein. Once the protein of interest is bound, the column is washed with equilibration buffer to remove unattached entities.

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Then the bound protein of interest is eluted off using an elution buffer of increasing ionic strength or of a different pH. Either weakens the attachment of the protein of interest to

the bead and the protein of interest is

bumped off and eluted from the resin. Ion exchange resins are the cheapest

of the chromatography media available

and are therefore almost always used as a step in biopharmaceutical protein production purification.
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Hydrophobic Interaction Chromatography (HIC)

HIC is finding dramatically increased use in production chromatography. Antibodies are quite hydrophobic and therapeutic antibodies are the most important proteins in the biopharmaceutical pipeline. Since the molecular mechanism of HIC relies on unique structural features, it serves as a nonredundant option to ion exchange, affinity, and gel filtration chromatography. It is very generic, yet capable of powerful resolution. Usually HIC media have high capacity and are economical and stable. Adsorption takes place in high salt and elution in low salt concentrations.

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Pervaporation
Pervaporation is a membrane process, in which a liquid mixture is brought in contact with a membrane at the upstream side and the permeate is removed as a vapor at the permeate side. The driving force for the process is established by reducing the relative pressure at the downstream side by either the use of an inert carrier gas or an applied vacuum. The vapor is usually obtained as a liquid in a condenser.
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Fig. : Schematic representation of pervaporation processes

feed retentate membrane module vapor condenser vacuum pump condensed permeate feed non condensables

vacuum pervaporation
retentate

membrane module condenser blower condensed permeate

carrier gas recycle

carrier gas pervaporation

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Reference books
1. Fundamentals of Biochemical Engineering, Rajiv Dutta, Ane Books India, [ chapter 10] 2. Bioseparations, Principles and Techniques, 2nd edition, B. Sivasankar, PHI 2006 3. Biochemical Engineering A Textbook for Engineers, Chemists and Biologists, Shigeo Katoh and Fumitake Yoshida, WILEYVCH Verlag GmbH

4. P. Stanbury, A. Whitaker and S. J. Hall "Principles of Fermentation Technology" 2nd Ed., Aditya Books (P) Ltd, N. Delhi.1997.