Final Sample

DOWNSTREAM
PROCESSING
INTRODUCTION
The extraction and purification of a biotechnological product
from fermentation is referred to as Downstream Processing
(DPS) or Product Recovery

Downstream processing mainly refers to the recovery and
purification of biosynthetic products, particularly
pharmaceuticals, from natural sources such as animal or plant
tissue or fermentation broth, including the recycling of
salvageable components and the proper treatment and
disposal of waste.

It is an essential steps in the manufacture of pharmaceuticals
such as antibiotic, hormones (e.g. insulin and human growth
hormone), antibodies (e.g. infliximab and abciximab) and
vaccines; antibodies and enzyme used in diagnostics;
industrial enzymes; and natural fragrance and flavor
compounds.

STAGES IN DOWNSTREAM PROCESSING
SOLID-LIQUID SEPARATION
The first step in product recovery is the separation
of whole cells (biomass) and other insoluble
ingradients from the culture broth.

Several methods for solid-liquid separation are:
Flotation
Flocculation
Filtration
Centrifugation

FLOTATION
When a gas is introduced into the liquid broth it
forms bubbles. The cells an other solid particles get
adsorbed on the gas bubbles. These gas bubbles
rise to the foam layer which can be collected and
removed.

FLOCCULATION
In the flocculation the cells for cell debrise from
large aggregates to settle down for easy removal.

The process of flocculation depends on the nature
of cells and the ionic constituents of the medium.

Addition of flocculating agents (inorganic salt
organic polyelectrolyte, mineral hydrocolloid is often
necessary to achieve appropriate flocculation.

FILTRATION
Filtration is the most commonly used technique for
separating the biomass and culture filtrate.

The efficiency of filtration depends on many factors-
the size of organism, presence of other organism,
viscosity of the medium and temperature. Several
filters such as:
Depth filters
Absolute filters
Rotary drum vacuum filters

CENTRIFUGATION
Use of the centrifugal force foe the separation of
mixtures

More-dense components migrate away from the
axis of the centrifuge

Less-dense components migrate toward the axis
CENTRIFUGATION
DECANTER CENTRIFUGE
RELEASE OF INTRACELLULAR PRODUCT
Cell Disruption: Cell disruption is a method or
process for releasing biological molecules from the
inside a cell. The main aim is that the disruption
must be effective and the method should not be too
harsh so that the product recovered remains in its
active form.

Cell disrupted by three methods:
Physical method
Chemical method
Enzymatic method
PHYSICAL METHOD
Physical method such as:
Mechanical:
Ultrasonication
High-pressure homogenisation
Impigment
Grinding with glass beads

Non-mechanical:
Osmotic Shock
Heat shock
ULTRASONICATION
It is common laboratory-scale method for cell
disruption applies ultrasound (typically 20-50 kHz)
to the sample (sonication). In principle, the high
frequency is generated electronically and the
mechanical energy is transmitted to the sample via
a metal probe that oscillates with high frequency.
The probe is placed into the cell-containing sample
and the high-frequency oscillation causes a
localized low pressure region resulting in cavitation
and impaction, ultimately breaking open the cells.
HIGH-PRESSURE HOMOGENISATION
The high-pressure homogeniger consists of a
positive displacement piston pump with one or
more plungers. The cell suspension is drawn
through a check vale into the pump cylinder and, on
the pressure stroke, is forced through an adjustable
discharge valve with restricted orifice.

IMPINGEMENT
In this procedure, a stream of suspended cells at
high velocity and pressure are forced to hit either a
stationary surface or a second stream of
suspended cells (impinge literally mean to stroke or
hit). The cell are disrupted by the forces created at
the point of contact Microfluidizer is a device
developed based on the principle of impingement.
GRINDING WITH GLASS BEADS
The cells mixed with glass beads are subjected to
a very high speed in a reaction vessel. The cells
break as they are forced against the wall of the
vessel by the beads.

OSMOTIC SHOCK
This method involves the suspension of cells (free
from growth medium) in 20% buffered sucrose. The
cell are then transferred to water at about 4 C.
Osmotic shock is used for the release of hydrolytic
enzymes and binding proteins gram negative
bacteria.

HEAT SHOCK
Breakage of cells by subjecting them to heat is
relatively easy and cheap. But this tech. can be
used only for a very few heat stable intracellular
products.
CHEMICAL METHOD
Treatment with alkalies, organic solvents and
detergents can lyse the cells to release the
contents.
ENZYMATIC METHOD
Cells desruption by enzymatic method has certain
advantages i.e., lysis of cells occurs under mild
conditions in selective manner.
CONCENTRATION
The filtrate that is free from suspended particles (cells,
cell debris etc.) usually 80-90% of water. The desired
product is a very minor constituent. The water has to be
removed to achieved the product concentration.

The methods for concentrating biological products by:
Evaporation
Liquid-liquid extraction
Membrane filtration
Precipitation and adsorption

The actual procedure adopted depends on the nature of
the desired product and cost of factor.
PURIFICATION
The biological products of fermentation are very
effectively purified by chromatography. It is basically
an analytical technique dealing with the separation
of closely related compounds from a mixture.

FORMULATION
Formulation broadly refers to the maintenance of
activity and stability of biotechnolical product during
storage and distribution. The formulation of low
molecular weight products (solvents and organic
products) can be achieved by concentrating them
with removal of most of the water. For certain small
molecules, (antibiotic, critic acid), formulation can
be done by crystallization by adding salts.
REFERENCES
U. Satyanarayana, Biotechnology; Downstream
Processing, pg no: 270-280.

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