This document provides an overview of downstream processing in biotechnology. It discusses the key steps involved which include: 1) cell disruption to release intracellular products, 2) clarification to remove cells and debris, 3) concentration of the product stream, 4) purification which may involve multiple steps such as extraction and crystallization, and 5) final product formulation. Specific techniques are described at each step, for example centrifugation and filtration for clarification, evaporation and ultrafiltration for concentration, and liquid-liquid extraction, crystallization, and various types of chromatography for purification. The goal of downstream processing is to produce a highly purified final product from the complex fermentation broth in an efficient manner.

1. [1]
Background To The Downstream Processing
After making the product in a fermenter, one might assume that the work is done. But this is
not so; we have just begun. The production of biomolecules is governed by the aqueous
environment needed for microorganisms. The
product can be the micro-organism itself, or a
metabolite excreted in the solution or contained in
inclusion bodies, but the fermenter may contain up
to 95% water and much effort has to be put in
concentrating the product. There is a correlation
between the concentration of a product in the broth
and its price in the market place. The more dilute a
product, the higher the cost prize. Removal of
water is one thing, there are many additional
problems in downstream processing (DSP). The
product may be intracellular and the cells have to
be disrupted to release the product. The fermenter
fluid may be complex containing compounds
resembling the product, which makes it difficult to
purify the product. Even so, a high purity may be
needed: in pharmaceutical products up to 99.999%
purity is required. These problems govern the
approach used to separate the product. Usually the
following steps are required:
1. Cell disruption (only when dealing with an
intracellular product),
2. Clarification (separation of the cells and
cell debris from the liquid),
3. Concentration of the product stream,
4. Purification (often in multiple steps),
5. Product formulation (giving the product a
suitable form).
1… Cell disruption
Ideally, products are released by cells into the fermentation fluid to allow direct recovery. If
the microorganism does not excrete the product, genetic engineering may modify the cells such
that the product is excreted. But this is not always possible, and the excreted product may be
unstable. So some products have to be released by disruption of cells. Cell walls can be
disrupted in several ways. These methods form two main groups: mechanical and non-
mechanical. The non-mechanical methods are used on a small scale. The important ones are:
 Drying (freeze drying, vacuum drying),
 Osmotic shock (a change of ionic strength of the solution causing the cells to swell and
burst),
 Temperature shock,
 Chemo-lysis (addition of surface active chemicals, solvents, antibiotics or enzymes to
degrade the cell walls).

2. [2]
For large applications mechanical methods are used:
 Ultrasonic disrupters,
 Bead mills
 Homogenisers.
Ultrasonic disrupters are frequently used in the laboratory but are too expensive on a plant
scale. Bead mills consist of a cylindrical vessel containing rotating discs and beads. The cells
in the process fluid are disrupted by shear forces between the beads. The process is
accompanied by heat production and cooling is required. This pressures the size of the
equipment. On a production scale the homogeniser is most often used. This is a valve that can
withstand pressures of 40 to 100 MPa. The fluid is forced through the valve slit with a high
velocity to disrupt the cell walls. Behind the slit, cavitation causes further disruption. The
homogeniser is a simple piece of equipment but has some disadvantages. It is noisy, it can only
process large volumes, has a high wear rate, and needs expensive pumps to operate. The
effectiveness of the homogeniser depends on the microorganism. Bacterial cells are easily
disrupted but yeast cells are not.
2… Clarification
Before concentration and purification of the product, cells or cell debris are removed. This
clarification yields a clear liquid containing the dissolved product. Two major techniques are
available: centrifugation and filtration. Large particles can be removed from the liquid by
sedimentation but in biotechnology this is only used for large agglomerates.
2.1… Centrifugation
If the gravity is not strong enough to separate the particles in a reasonable time, we can apply
a centrifugal field. The suspension is put into two identical tubes that are rotated. If separation
is apparent after 10 minutes of centrifugation at 3000 g, then this separation method can be
used on plant scale. In larger centrifuges the residence time is lower but so is the sedimentation
distance.
In plant operations, two types of centrifuges are frequently used:
 The tubular centrifuge
 The disc stack centrifuge.
A tubular centrifuge can be seen as a sedimentation vessel turned on its side and with an
increased acceleration force.
Disc stack centrifuges can be compared to a tilted sedimentation vessel with a large area and
increased acceleration force. Disc stack centrifuges contain conical plates (discs) at a short
distance from each other
2.2… Filtration
Another way to separate particles from suspensions is filtration. The particles are retained by a
filter medium, which is a porous fabric or felt. On the surface of the medium the particles form
bridges over the pores and create a filter cake: this acts as the medium for the next particles.
The liquid is forced through the filter by a pressure difference.

3. [3]
In practice two modes of filtration are used:
 Batch filtration with plate filters
 Continuous filtration with rotating drum vacuum filters.
The plate filters have a large number of hollow frames. These are covered with the filter
medium. The liquid flows from outside to inside and the solids are retained on the filter
medium. After a certain time the space between the frames is filled with filter cake and the
pressure drop of the equipment increases. The frames are disassembled and the cake removed.
This can be done automatically.
The rotating vacuum drum filter is a large drum that rotates around a horizontal axis. The filter
medium covers the outside of the drum. The drum rotates slowly through a trough containing
the feed. The vacuum in the drum sucks in the liquid, leaving the solids as a cake on the outside.
Only the lower part of the drum is submerged in the feed, the rest of the surface can be used to
wash and dry the filter cake. At the end of the cycle, the cake is scraped off the drum by a knife
and collected for further processing.
3… Concentration
After clarification, the feed is still dilute and has to be concentrated before extracting and
purifying the product. This usually means removing water. Concentration can also purify the
product stream: sometimes this is enough to obtain the desired end-product purity.
3.1… Evaporation
The oldest and simplest method of concentration is to evaporate water or solvent from the
mixture. Biological products tend not to be very stable, so temperature and exposure time
should be kept low. The temperature can be kept low by working under reduced pressure.
3.2… Precipitation
Precipitation can be used in several parts in the process. Early in the process it can remove
water or salt and give as table intermediate. During the recovery process it can be used for
concentration, for example, before a chromatography step. In the final stage it can be applied
to obtain a solid end-product. During precipitation, the solubility of the desired product is
lowered until the solution is oversaturated and the product precipitates. The solid precipitate is
separated from the solution by a settler, centrifuge or filter. The precipitation can also be
performed in a fractionating manner (the precipitates obtained contain different fractions of the
different products). The solubility is mostly decreased by adding salts, typically (NH4)2SO4,
or organic solvents such as acetone or ethanol.
3.3…Ultrafiltration
Concentrating can also be performed using membranes. Two types are frequently used in
biotechnology:
 Microfiltration membranes (MF)
 Ultrafiltration membranes (UF).
MF membranes have pores of 0.2--0.5 µm diameter and thus retain cells. MF resembles
ordinary filtration. Ultrafiltration membranes have pores of approximately 10 nm diameter and
thus retain proteins, but not water and salts. Membranes in the form of porous tubes of 1--5

4. [4]
mm diameter are most common. A thin coating on the surface of the tube is the actual
membrane. It can consist of a porous (sponge-like) polymer, porous carbon or porous ceramic.
A UF membrane can be used in two ways: to concentrate or to wash out salts from the product
(diafiltration). A major difference between ultrafiltration and ordinary (dead-end) filtration is
the type of flow along the membrane. In UF the liquid containing the solids flows along the
membrane (hence the term cross-flow filtration). A pressure difference of several hundred kPa
is applied across the membrane. This is the driving force for transport of water and salt through
the membrane. Increasing the pressure across the membrane initially increases the flux, but
only to a limited extent. The water transports protein towards the membrane where it
accumulates. At a certain pressure difference the protein precipitates on the membrane.
4… Purification
After releasing the product from the cell and subsequent clarification and concentration of the
process stream, a contaminated product stream is available for further processing. In the
downstream processing of bio-products two major groups of purification processes can be
distinguished: those for bulk products such as citric acid, enzymes and penicillin and those for
small-scale pharmaceutical processes.
During bulk purification extreme purity is not required. A few per cent of impurities are often
allowed. Two important techniques can be identified: liquid-liquid extraction and
crystallization. Both make use of an auxiliary phase that selectively extracts a certain
component from the mixture.
4.1… Extraction
Single-stage extraction is often not sufficient and several steps may be needed. For small
molecules like citric acid and penicillin, organic solvents such as butanol, butyl acetate or larger
amines are used. For larger and more complex biomolecules, aqueous two-phase extraction can
be applied. These systems are composed of an aqueous mixture of a salt and a polymer or two
polymers. In certain concentration ranges these separate into two phases. The dense bottom
phase contains most of the salt, whereas the top phase mainly contains the polymer. Both
systems are relatively gentle to sensitive proteins.
Four factors govern the extraction:
 Mass balances (overall, phase, local)
 Phase and reaction equilibria (K)
 Hydrodynamics
 Mass transfer and reaction rates (k).
4.2… Crystallisation
When a solution is super-saturated the solvent contains more product than at equilibrium and
as a result the product crystallizes. The crystals are separated by filtration or centrifugation.
This yields a fairly pure product. A single crystallization step can often produce the desired
purity, but large quantities of the product may be left in the solution.
Two ways to obtain super-saturation are cooling (lowering the solubility of the product) and
evaporation (increasing the concentration of the product). Crystallization resembles
precipitation but it differs in the way that super-saturation is obtained. Crystallization makes

5. [5]
no use of additives and is the older and more common technique. Inorganic salts such as sodium
and ammonium sulphates, and certain organic compounds such as sucrose and glucose, are
produced in quantities exceeding 100 million tons per year. Most bulk pharmaceuticals and
organic, fine chemicals are marketed as crystalline products.
Crystallization is important for three reasons:
 The crystals are often very pure -- this is important in the finishing step in ultra-
purification
 The production of uniform crystals facilitates subsequent finishing steps like filtration
and drying
 It improves product appearance -- which is important for consumer acceptance.
5… Ultra-purification
Many pharmaceutical products, especially those introduced intravenously into the human body,
have to be extremely pure. Purity requirements of 99.999% are not rare. Sorption processes
that use a solid auxiliary phase are commonly used and the separation of large molecules like
proteins.
In bioprocess technology the matrix consists of spherical beads with diameters of 10--100 µm,
placed in a column. The beads are made into a gel, appearing as molecular cross-linked fibers
with water-filled pores of about 10 nm diameter, which is large enough for proteins to penetrate.
Different proteins have a different interaction with the matrix. A protein that interacts strongly
will be retarded more than a non-bonding protein. The properties controlling the interactions
are:
 Pore size
 Polarity or hydrophilic character of the pore surface
 Charge of the surface
 Shape of the molecules in the surface.
All properties may play a role in a separation, but one property is normally overruling and
determines the name of the sorption process. These processes are:
 Gel filtration (separation on size)
 Adsorption (hydrophobic interaction, separation on polarity)
 Ion exchange (separation on charge)
 Affinity chromatography (separation on shape: some molecules perfectly fit on the
surface).
 Electrophoresis
Interaction of the protein with the matrix also depends on the protein and its concentration; on
the concentration of other components in solution; on the characteristics of the solvent; on the
pH and I of the solvent and on the temperature. The sorption material has to be:
 Insoluble
 Macro-porous
 mechanically stable
 hydrophilic
 properly shaped
 Biochemically stable.

6. [6]
There are several types: organic polymers, inorganic pellets and composite materials. The
inorganic sorbents are applied in color removal and consist of activated carbon, silica or
alumina. There are two ways to operate a sorption column: as a chromatography column and
as an adsorption/regeneration system.
5.1… Chromatography
In elementary chromatography there is a constant flow of eluent (a suitable fluid) through the
column. At a certain moment a pulse of feed is injected at the entrance of the column. This may
consist of components A and B, with B travelling faster than A. After some time the peaks of
both components are collected as separate fractions at the outlet of the column. The separation
suffers from peak broadening and can be improved by using a longer column or a lower rate of
elution.
Only a limited amount of feed can be processed in this way. Increasing the throughput can be
done by injecting a band of feed into the column. At given eluent velocity more column length
will be needed but, on average, better use is made of the column material. Another method is
to increase the feed concentration and to enter the area of non-linear chromatography. The
bands will form triangular peaks: with a sharp front but a diffuse tail. Again the column length
needs to be increased but, on average, the column material is used more efficiently. A gradient
in the feed (eluent) composition can also be applied, using variation in pH, I or solvent polarity.
5.2… Adsorption/regeneration
The largest throughput per column volume is obtained by using adsorption/regeneration. This
is a multiple-step process. This technique can be used when the desired product A binds better
to the matrix than the contaminant B. In the adsorption step the feed is pumped through the
bed. The fresh beads will mainly adsorb A until the surface is saturated and the small amount
of B adsorbed is flushed out during a washing step. The column is regenerated using an eluent
with different properties that removes the adsorbed component A from the matrix. Then the
cycle starts again. Adsorption fronts are usually sharp and a good separation can be obtained;
during regeneration the diffuse tails make separation more difficult.
5.3… Gel filtration
In gel filtration molecules are separated based upon their difference in size. The gels have a
specific pore size distribution. Small molecules can enter the gel pores but larger molecules
cannot. So smaller molecules are retarded more. Adsorption at the gel surface is minimized:
protein molecules are dissolved in the water in the pores. Gel particles are very open structures
and compressible: this limits the allowable column lengths and velocities.
5.4… Ion exchange
In this sorption process the solid matrix contains charged groups. These can be positive as well
as negative. Positively charged exchangers are quaternary ammonium and negative ones are
sulfonic acid exchangers. Ions with the same charge as the matrix are repelled. The amounts of
free counter ions and charges attached to the matrix are the same. The counter ions can be small
ions but also large molecules like proteins. The counter ions can exchange with other counter
ions in solution.

7. [7]
A positive matrix exchanges negative ions and vice versa. The gels for ion exchange are more
open than for gel filtration: the proteins are not separated by size. Proteins can form a mono
layer around the fibres in the matrix and can occupy up to 10% of the gel volume. The partition
coefficient in ion exchange can be much larger than one. Proteins may have a positive as well
as a negative charge depending on the pH of the solution
5.5… Affinity adsorption/chromatography
Proteins have characteristic shapes, allowing them to fit exactly to counter molecules called
antibodies or ligands. An adsorbent with antibodies on the pore surface can selectively bind
one specific molecule. This is called affinity adsorption. The adsorbent has even larger pores
than ion exchange gels because it has to accommodate the antibody besides the proteins.
Sufficient space has to be available for this antibody to position itself freely with respect to the
protein. So the antibodies are attached to the pore wall by spacers. The gel matrix resembles
that used in gel filtration. The spacer should be chosen with care to allow a minimal interaction
with feed components.
Affinity absorption is an attractive method to isolate a specific component. The column material
is used completely, the product obtained is pure and concentrated and the process needs only a
small amount of eluent. However, it suffers from the drawbacks that for each component a
specific antibody has to be developed, that the adsorbent is expensive and is mostly not very
stable. The spacers can detach leading to a loss of efficiency.
5.6… Electrophoresis
Electrophoresis, the migration of charged molecules under the influence of an electrical field,
is an efficient and inherently mild technique which has found widespread use in both analytical
and small-scale preparative purification of proteins and nucleic acids. There are four basic
techniques in electrophoreosis
 zone electrophoresis
 moving boundary electrophoresis
 isotachophoresis
 isoelectric focusing.
Zone electrophoresis (ZE) resolves the components of a sample on the basis of their relative
electrophoretic mobilities. The mobility is a function of charge and molecular weight for
soluble species and of zeta potential for colloids and particles.
Isoelectric focusing (IEF) separates proteins on the basis of their isoelectric point. A sample is
placed into a support medium, usually a gel, containing a stable pH gradient decreasing from
the cathode to the anode. When an electrical field is applied to the system, each protein migrates
towards the position corresponding to its isoelectric point. When the protein reaches this
position, its net charge falls to zero and its motion stops because the electrical field no longer
exerts a force on it.
Zone electrophoresis is a dynamic separation, as it is based on relative rates of movement,
while IEF is an equilibrium separation which reaches a steady state.

8. [8]
6… Sequencing
To develop a good process, we must get all pieces of equipment to work together. To illustrate
the problem, we consider a process that looks simple in the laboratory, but has to be scaled-up.
The example is the downstream processing of an intracellular enzyme. A large fermentation
capacity is chosen as it clearly shows the problems in the downstream processing. The
fermenter broth consists for the major part of water. Only a small amount of product is present
and a complex mixture of components of secondary metabolites of different sizes and shapes.
For simplicity we consider only the following components:
 Water with salts and other nutrients
 Four different enzymes
 Cells and cell debris.
The four enzymes have different sizes and iso-electrical points. Enzyme 3 is the desired product
and has to be purified. The process starts with cell disruption with a homogenizer because we
are dealing with an intracellular product. A small valve is needed and the costs lie in its energy
consumption for cooling and disruption. The clarification is done by filtration. Because the
debris is so fine it takes a long time to filter. Filtration can be speeded up by adding filter aid.
This consists of large particles that make the filter cake more porous thus increasing the flux.
A thorough washing step is needed to prevent product loss with the filter cake. A rotating drum
vacuum filter can be used with a large filtration area.
Conclusion
All the processes and technologies described above illustrate the important role of downstream
processing and purification processes in the application of biotechnology. It is expected that
this trend will continue, especially with the proliferation of recombinant proteins derived from
the recombinant DNA technology. All of these techniques play a critical role in the downstream
processing and in the effective production of biological products. Most of the early fears related
to the safety aspects of recombinant DNA products have been softened since studies showed
that quantities of DNA (obtained from Chinese hamster ovary cells) at the hundredth of μg
level did not result in the formation of tumors in newborn rats (155).
The science behind the concepts and techniques of bio-separations is exciting: each
fundamental mechanism which is uncovered sets the direction of future development work and
motivates further advances.
On the development side, it is important to recognize that a successful bioprocess leading to a
safe product results from the integration of techniques ingeniously connected with one another.
The fermentation engineer will confer with downstream processing colleagues to design the
fermentation process, since the mode of operation (e.g. fed-batch or continuous) can have
major effects on product stability and response to handling, as well as on the nature of the
impurities which may remain with the product. Looking at a process with an integrated vision
not only minimizes the likelihood that serious mistakes will occur, but it also favors
optimization of each unit operation in the context of the entire process. This plays a significant
role in making a process viable and cost-effective.

9. [9]
REFERENCES
 http://pubs.acs.org/doi/pdf/10.1021/bk-1990-0419.ch001
 http://www.dli.gov.in/rawdataupload/upload/insa/INSA_1/20006177_175.pdf
 www.slideshare.net/chem_engine1/downstream-processing
 en.wikipedia.org/wiki/Downstream_processing
 Basic Biotechnology Third Edition Edited by Colin Ratledge and Bjørn Kristiansen part
1; fundamentals and principles, chapter 9; downstream processing , page 219--250