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 cost-

effective methods in quick time and enable the

companies to bring the products.

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 24

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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 non-

redundant 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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vapor

non condensables

feed retentate

membrane module

vacuum pump

condensed permeate

condenser

feed retentate

membrane module

condensed permeate

condenser

blower

carrier gas recycle

vacuum pervaporation

carrier gas pervaporation

Fig. : Schematic representation of pervaporation processes

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, WILEY-

VCH Verlag GmbH

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