Downstream Processing

Downstream processing (DSP) refers to the recovery and purification

of biosynthetic products from

• Natural sources such as animal or plant tissue.

• Fermentation of microorganisms in native or genetically modified
form.

The products ranges from antibiotics, hormones (e.g. insulin and

human growth hormone), antibodies and enzymes used in diagnostics,
vaccines, industrial enzymes to more recently recombinant proteins.

In a collective manner DSP can be used as a term for the processes that
follows fermentation i.e.
• Cell harvesting.
• Cell disruption for intracellular proteins.
• Product purification from cell extracts or the growth medium
(culture supernatant)
• Confirmation of target protein recovery.
 Fermentation
In context to industrial biotechnology, The industrial
microorganisms are grown under controlled conditions with an
aim of optimizing the growth of the organism or production of a
target microbial product.

Fermentation is carried out in vessels known as Fermentors.

The types of fermentor ranges from

simple 3-10Lt vessels

complex integrated system of automated control.

Type of fermentation process
Fermentation in liquid media is of three types depending
upon the mode of operation:
• Batch Fermentation
• Fed-batch Fermentation
• Continuous Fermentation

Batch Fermentation

• Simplest mode of operation

• Reactor is filled with medium, fermentation allowed to
proceed. No feed after inoculation.
• After finishing of fermentation, reactor is emptied for
DSP and refilled again for new batch of fermentation.
 Fed-Batch fermentation
• Modification of Batch type fermentation
• Reactor is filled with medium, fermentation allowed to
proceed. More feed periodically added after inoculation with

exhaustion of feed added at start.

• Product for DSP is obtained periodically and after finishing of
fermentation process as well.
• Most widely
Phases used
of cell process
growth in industries.
during Batch and Fed-Batch

• Lag phase
• Exponential phase
• Stationary phase
• Death phase
 Continuous Fermentation
• Fresh media supplied continuously
• Bioreactor fluid continuously removed, Cells receive fresh
medium continuously.
• Products and cells continuously removed for processing.
• Can be operated for longer periods.

• growth rate of the cells can be optimized by controlling the

flow rate of the feed entering the reactor thus resulting in high
productivity
Cell harvesting

Fermentation process results in Biomass containing cells

which must be separated for further processing.
The separation of microbial cell normally involves :
• Filtration
• Centrifugation.
Filtration
• Used in case of very small sized microbial cells.
• Useful for industries being cheapest option.
• Slurry can be fed directly to filteration unit attached with
the output of the bioreactor.
 Centrifugation

Used when filtration is not satisfactory, although expensive but

essential in cases when :
• Filtration is slow and difficult.
• Cells must be obtained free of filter aids.
• Continuous separation to a high standard of hygiene is required.

Types of centrifuges

Table top centrifuges Ultra Centrifuge

Low speed 1000 – 15,000 r.p.m High speed 10,000 – 70,000 r.p.m
 Cell disruption
In case of intracellular expression, cellular contents have to be
released. Cell free extract is needed for further Processing. Various
methods have been devised for cell disintegration such as :
Physio-mechanical methods :
• Liquid shear
• Freeze thawing
• Ultra sonication
Chemical methods :
• Detergent
• Osmotic shock
• Alkali treatment
• Enzyme treatment
 Recovery and Purification of fermentation products

Recovery of protein involves following stages :

Centrifugation and filtration of culture supernatant in case of
extracellular expression and cell extract in case of intracellular
expression
Concentration of centrifuged cell free extract on membranes.
Concentration involves
passing of supernatant
through membranes of
different cutoff values
i.e. pore sizes. For
smaller volumes
centricons and
ultrafilteration unit is
used, for larger
volumes tangential
Ultra filtration unit Tangential flow cartridge
flow cartridge is
preferred
Purification

Chromatography techniques are used to isolate and purify

fermentation products
Chromatography is concerned with the passage and
separation of different solutes as liquid is passed through
a column i.e. liquid chromatography.
Depending on the mechanism by which the solutes may
be differentially held in a column ,the techniques can be
grouped as follows:
• Adsorption chromatography
• Ion –exchange chromatography
• Gel-permeation chromatography
• Affinity chromatography
• Reverse phase chromatography
• High performance liquid chromatography
 Gel-Filtration Chromatography
Gel-filtration chromatography separates molecules on the basis


of size.
It involves passing a solution through a column (long tube) that


is packed with beads of a hydrated insoluble material such as

dextran, agarose or polyacrylamide

The smaller molecules in the solution

spend more time interacting with the
beads while larger molecules pass by
Consequently, the larger molecules
leave the column first
 Ion-Exchange Chromatography
•
Ion-exchange chromatography separates molecules based on
differences in net charge
•
Proteins with a net positive charge will be retained on
negatively-charged columns such as carboxymethyl-cellulose
•
Proteins with a net negative charge will be retained on
positively- charged columns such as diethylaminoethyl-cellulose
 Affinity Chromatography
• Affinity chromatography can be used to purify proteins that
have a high specific affinity to some chemical group
• For example, concanavalin A, shown above, is a sugar-binding
protein with a high affinity for glucose. A column with glucose
residues attached will retain concanavalin A, which can then be
later released by adding a solution of free glucose

• This technique is not always be applicable

because the specific high-affinity groups may
not always be obtainable
• However, for certain categories of proteins
such as transcription factors, the high
affinity group can be a specific sequence of
DNA, which is easily prepared
 HPLC (High Pressure Liquid Chromatography)

HPLC is a very powerful technique for separation and

purification of biomolecules.
• HPLC is a high resolution column chromatographic technique.
• Improvements in the nature of column packing materials for a
range of chromatographic tech-niques (e.g. gel permeation and
ion-exchange)
• yields smaller, more rigid and more uniform beads. This allows
packing in columns with minimum spaces between the beads
thus minimizing peak broadening of eluted species.
• Distinguished from liquid chromatography because of
improved media (in terms of their selectivity and physical
properties) for the solid (stationary) phase through which the
mobile (fluid) phase passes.
 Confirmation of purified target protein

Analysis of crude as well as purified product for presence of

target protein is an essential step in downstream processing.
There are techniques available for efficient separation of
proteins, some of the frequently used techniques for the
aforesaid purpose are :

• SDS-PAGE
• 2D-PAGE
• Isoelectrofocussing
 SDS-PAGE
SDS-PAGE, Sodium dodecyl polyacrylamide gel electrophoresis,
is a technique widely used in biochemistry, forensics, genetics
and molecular biology to separate proteins according to their
electrophoretic mobility

Electrophoretic mobility depends on :

• Molecular weight
• Higher order protein folding
• Posttranslational modifications
The SDS gel electrophoresis of
samples having identical
charge to mass ratios results in
fractionation by size.
 Isoelectric focussing

Separation is based on the charge a protein possesses.

Every protein has a unique

isoelectric point, a pH on
which the net charge on
molecule becomes Zero.
In presence of ampholytes and a charged field, a protein
molecules migrates till the net charge on it becomes nil, thus
resulting in efficient sepration.

Better resolution even at larger sample volumes.

Does not requiure denaturation of proteins thus maintaining
their biological activity.
 2D Gel electrophoresis

• It is a form of gel electrophoresis commonly used to analyze

proteins
• Begins with 1-D electrophoresis i.e. PAGE and seprates
proteins on the size basis
• Second step of separation based on second property i.e.
isoelectric point in a direction 90˚ from the first.
• Results in molecules spread
across the 2-D gel, molecules are
more effectively separated in 2-D
electrophoresis than in 1-D
electrophoresis.
Compared to alternative methods (e.g. SDS-PAGE), IEF
offers the following advantages
• efficient
• expressive
• economic (no sophisticated equipment required)
• easy (clear, one-dimensional separation principle)
• fast
• sensitive

IEF of maize seeds

Left-right
Female, male, F1 generation seeds