CHAPTER

1
Downstream Process Technology
An Overview

Both downstream processing and analytical separation refer to the isolation and purification of
bioproducts, but they differ in the scale of the operation and the process. Downstream processing
implies the manufacture of a purified product intended for specific use, generally in marketable
quantity. While analytical bioseparation refers to isolation and purification of a bioproduct from a
sample with the sample size as small as single cell for the sole purpose of measuring a component
or components of a mixture. Downstream processing techniques should be selected carefully, as
the cost of process steps generally goes more than 50%, concentration of the product may be very
low compared to impurities present and product may be lost at each unit process. Therefore,
downstream processing unit process is selected carefully involving minimum steps (as possible) to
recover fragile product of interest in purest form with highest yield in a cost effective manner.
Hence, downstream processing plays a major role in the biotechnological process.

1.1 DOWNSTREAM PROCESSING STEPS

Steady as well as unsteady state techniques are the basis for byproduct recovery using downstream
processing steps. Principles of isolation, purification and formulation of the bioproduct depend on
the physicochemical properties of the product, media and ingredients used for the recovery process.
There are six groups of downstream processing operations: removal of insoluble, cell disruption,
extraction, concentration, purification and formulation (Figure 1.1). They are applied in order to
recover bioproducts from its natural state such as component of tissue, cell or fermentation broth
through progressive improvement in purity and concentration taking into consideration of the
yield and the cost.

3
4 DOWNSTREAM PROCESS TECHNOLOGY : A New Horizon in Biotechnology

Figure 1.1 Broad outlines of downstream processing steps in bioproduct recovery.

In the industry, downstream processing starts at media preparation step of upstream

processing because large inputs of impurities reduced at the initial stage itself. We cannot
generalize the unit steps for all the types of the bioproducts and some of these steps are exclusive
for the individual products. Since product of interest has to be isolated, purified and formulated
as per the requirement of the end users, downstream processing is an essential part of the
bioseparation. It is an emerging and challenging area for biotechnologists as the problems involved
in the separation of the biological products are numerous and can be solved using skills of
biologists, chemists and chemical engineers. In olden days the bioproducts like penicillin, ethanol,
 DOWNSTREAM PROCESS TECHNOLOGY: AN OVERVIEW 5

enzymes or organic acids were produced naturally by using living organism such as bacteria,
yeast or moulds by fermentation, and the products of interest were isolated and purified by units
operations which were well described mathematically, and scaled up by pilot plant study. With
the recent advances in biotechnology, genetically modified microorganisms produce products
of interest that are not natural metabolic products and recovery of such products demands precise
techniques of isolation and purification. The selection of specific downstream processing steps
depend basically on the strategies keeping in mind the physicochemical properties of the product,
media and the ingredients of separation, final quality of the product, quantity of the product,
extent of the purity and definitely the cost.

1.1.1 Removal of Insolubles

The first step in the downstream processing is the removal of solids, usually cells, from the liquid
medium. Product of interest may be intracellular or secreted outside, but in either case downstream
processing starts by removal of insolubles as follows:
Filtration
Filtration method separates filamentous fungi such as Aspergillus niger or filamentous bacteria
such as Streptomycetes spp., and often yeast flocks from the fermentation broth. Various methods
used for the removal of insolubles are surface filtration, depth filtration, centrifugal filtration,
cross flow filtration and rotary drum filtration. Rotary drum vacuum filter is most commonly used
in the industry for large-scale separation of filamentous fungi and yeast cells, because of its simple
operation and low cost. Vacuum filter is ideal for the clarification of fermentation broth containing
10–40% solids by volume with particle size of 0.5–10 µm. Plate and frame press filter is ideal
where a relatively dry cake discharge is desirable and seldom used where there are toxic fumes or
biohazards. However, horizontal plate, vertical leaf and candle type filters are ideal for processing
biohazardous feed in small scale. Vertical leaf filters have a relatively high filtration area per volume.
Filtration is the ideal choice when the insolubles are dilute, large and rigid. An advantage of filtration
method is its ability to produce solid cake during separation.
Centrifugation
Centrifugation sometimes could be a better option when filtration is less effective due to the
concentrated broth with lighter solid particles. Eventhough filtration is an efficient method as it
produces cake that are convenient to handle, it is not an effective method to handle most of biological
fluids due to its diverse physicochemical properties. Therefore, the centrifugation is an attractive
alternative to filtration. Three basic types of centrifuges are: tubular bowl centrifuge, disc-bowl
and basket type centrifuge. Tubular bowl is simple and the provision for cooling is very advantageous
in protein work. Disk type is probably the most commonly used type and often can continuously
be used. The stacked conical flask is a disk type centrifuge that allows a larger sedimentation area
to be contained in a relatively compact volume. Basket types are the combination of centrifuge,
commonly used to wash accumulated solids.
Flocculation and floatation
Flocculation is the better choice when the methods such as filtration and centrifugation are inefficient
in separating small bacterial cells from the fermentation broth. Sticking together of cells induced
6 DOWNSTREAM PROCESS TECHNOLOGY : A New Horizon in Biotechnology

by inorganic salts, mineral hydrocolloids and inorganic polyelectrolytes is known as flocculation.

By spraying fine gas bubbles through fermentation broth, microbial biomass or long chain fatty
acids get separated from the mixtures by floatation. Flocculation and floatation methods are most
efficient in the recovery of microbial biomass in some single cell protein production system.
Pretreatment of the fermentation broth used to increase the biomass particle size, reduce the
fermentation liquor viscosity, and improve the interaction between the particles, which are essential
for the efficient separation. Besides the flocculating agents, other factors that influence the
flocculation are the physiological state of the cells, ionic environment, temperature and the nature
of the organism. Besides, cationic filter aids also reduce the load of pyrogen, nucleic acid and acid
proteins, which normally foul chromatographic columns. Floatation using fine sprayed bubbles
efficiently separates collector substances such as long chain fatty acids and amines.

1.1.2 Cell Disruption

In bioprocess based industry, diverse range of products are produced using wide varieties of bacterial,
fungal, plant and animal cells, of which some are secreted to the media and many are retained
within the cells. Cell disruption techniques play a major role in the isolation of intracellular product
of interest such as glucose isomerase, phosphatase, b-galactosidase, ethanol dehydrogenase,
NADH/NAD+ and many alkaloids, including the products produced using genetically modified
organisms. Most of the proteins and other products produced and deposited within the cells are
fragile with respect to their biological structure. Isolation of intracellular products requires efficient
methods to prevent the adverse effect of the external environment so that the product recovers in
the purest form with highest yield and activity. Cell disruption method can be used as an important
pretreatment tool in the recovery of such products which is been selected carefully by using the
knowledge of the structure of the cell, physicochemical property of the bioproduct and the basic
principle of the disruption method. Total protein released and the enzyme activity are the criteria
for deciding the suitability of cell disruption method.
Mechanical method
Mechanical methods play a major role in releasing intracellular products by using shear such as
grinding in bead mill, pressure such as homogenizing in homogenizer or pressure release such as
cavitations in ultrasonication. Widely used method of cell disruption is by forcing cell suspension
through a fine nozzle to break the cell by hydrodynamic shear and cavitations. In the industry cell
disruption is carried out by bead mill, high-pressure homogenizer or high-shear mechanical method
carries out disruption. Mechanical method of cell disruption is an important tool to release
intracellular enzymes as enzymes are more complex compared to other products. On the other
hand, enzymes are located in different regions within the cells.
There are many methods of cellular disintegration, for there are many types of cells. Most
cells have particular characteristics which need special attention during disintegration. Animal
tissues vary from the very easily broken erythrocytes to tough collagenous material such as found
in blood vessels and other smooth muscle containing tissue. Plant tissues are generally more difficult
to disrupt than the animal cells because of the cellulosic cell walls. Bacteria vary from fair organism
that can be broken up by digestive enzymes or osmotic shock, to more resistant species with thick
cell wall, which requires vigorous mechanical treatment for disintegration. It is generally not
advisable to use a disruption treatment more vigorous than necessary, as labile enzymes or other
 DOWNSTREAM PROCESS TECHNOLOGY: AN OVERVIEW 7

products may be inactivated once liberated into solution. On occasions when an enzyme is present
in an organelle, the method may still be suitable in that they also disrupt the organelles. On the
other hand, it may be desired to isolate the organelles, for the preparation of mitochondria from
tissues such as skeletal or cardiac muscle using this procedure. However, this is applicable on a
small scale and more suited for metabolic studies on the organelles than the enzyme purification.
Yield of purified organelles may be very low, and it is often better procedure to do complete tissue
disruption and then approach the problems of isolating the proteins required from the complex
mixture of proteins in the extract. Hence, mitochondrial and chloroplast enzymes are often be
prepared from a complete tissue homogenate rather than from the isolated organelles.
Bead mill disruption: The vertical or horizontal bead mills are commonly used disruption
methods using grinding beads such as glass, titanium carbide, zirconium oxide or zirconium silicate.
Horizontal bead mills are efficient for disrupting cells of smaller size using high load of up to 80%
beads at lower speeds and lower energy output in bead mills. Since rate and degree of cell disruption
depend on the nature of the microorganisms, product location within the cell, the type of bead mill,
bead size, resident time and temperature, the careful selection of the method of cell disruption
results in highest yield. Mechanical method may be harsh to some enzymes. Vertical bead mills
have the advantage of higher loading of beads of up to 80% of smaller size, uniform distribution of
beads for good grinding at lower speed and energy output. Bead mills are efficient for disrupting
bacterial cells, yeast, algae and filamentous fungi.
High-pressure homogenizer: High-pressure homogenizer is ideal to disrupt all types of cells
such as bacterial, animal or plant cells including spores. In this process, cell-suspension is pressurized
through the adjustable discharge valve with restricted orifice using a high-pressure positive
displacement pump. Cell disruption occurs due to various stresses developed in the fluid. Scale up
of the homogenizer is relatively simple as only a bigger plunged pump and discharge valve are
required, provided all other variables are maintained constant.
High-shear mechanical methods: High-shear mechanical methods for cell disruption can be
categorized into three main types: rotor-stator disruptors, valve-type processors, and fixed-geometry
processors. These fluid processing systems are also used extensively for homogenization and
disaggregation of wide range of materials. These processors work by placing the bulk aqueous
media under shear forces that literally pull the cells apart. These systems are especially useful for
larger scale laboratory experiments of over 20 ml, and offer the option for large-scale production.
Rotor-stator processors are the most commonly used to disrupt tissues.
Non-mechanical method
Non-mechanical method of cell disruption includes those methods other than the use of mechanical
means. Of these osmotic shock, detergent solubilization and lipid dissolution methods play major
role in cell disruption.
Detergent solubilization: Anionic detergents such as sodium dodecyl sulphate (SDS), sodium
sulphate and sodium taurocholate, cationic detergents such as cetyl trimethyl ammonium bromide
(CTAB) and non-ionic detergents such as Triton X-100 used by adding concentrated solutions of
detergents to about half the solutions volume of cells to disrupt the cell wall. In the case of
mammalian cells, non-ionic detergents such as saponin or steroid b-hydroxy-sterols that are capable
8 DOWNSTREAM PROCESS TECHNOLOGY : A New Horizon in Biotechnology

of complexing membrane cholesterol, have been used to release intracellular proteins by

permiabilizing the plasma membrane alone without effecting the organelle membrane.
Osmotic shock: Cell disruption by osmotic shock is simpler than the detergent solubilization,
just by dumping a given volume of cells into pure water often about twice the volume of cells.
Susceptibility of cells to get disrupted depends strongly on their type. Red blood cells are easily
disrupted by osmotic shock, however, animal cells require mechanical mincing or homogenization.
Plant cells are much more difficult to disrupt, for their cell walls often contain strong woody
material that is relatively impermeable to osmotic flow.
Osmotic shock effect is often minimal on bacterial cells. However, the method is useful
particularly if the desirable products like enzymes are located in the periplasmic region. This
method is used for releasing hydrolytic enzymes and membrane bound proteins from number of
gram-negative bacteria especially Escherichia coli and Salmonella typhimurium.
Alkali treatment: Even though this is a very harsh method, it is a cheap and effective method.
Alkali treatment is ideal for isolating alkaline phosphatase because it is inactivated at pH 11–12.
Alkaline treatment is efficient for isolating pyrogen-free therapeutic enzymes such as L-asparaginase.
Organic solvents: Organic solvents such as toluene especially at 1–3% permeabilize cell wall.
This method is useful in retaining the content of cells for sequential release of desired products,
and these permeabilized cells can be used as porous bags of catalysts. Toluene permeabilization of
Agrobacterium radiobacter cell wall to isolate commercial enzymes like hydantoinase and
N-carbamylamideohydrolase commonly used for the enzymatic conversion of hyadantoin and
substituted hyadantoin to optically pure D-phenyl glycine and D-p-hydroxyphenyl glycine required
for the manufacture of semi-synthetic penicillin. Toluene permeabilization of yeast cells releases
histydyl-t-RNA synthase and enolase. Ethyl acetate releases the periplasmic invertase and
a-glucosidase by cell wall permeabilization. Dimethyl sulphoxide (DMSO) are used to release
intracellular circulatory drug, ajmalicine from Catharanthus roseus plant cell wall by
permeabilization. A selective liberation of enzymes from periplasmic space is possible by treatment
with water miscible solvents such as methanol, ethanol, isopropanol or tert-butanol, but requires
fire-proof equipments and special precautions.
Enzyme digestion: Even after being an expensive product, enzymes can be used selectively and
effectively to recover commercially important enzymes such as glucose isomerase from
Streptomyces spp. By enzymatic digestion coupled with EDTA, it is possible to produce porous
Escherichia coli spheroplasts. Other bacteriolytic enzymes such as glycosidase, acetyl
muramyl-L-alanineamidase and endopeptidase also break bacterial cell wall. Mixture of different
enzymes such as glucanase, protease, mannanase and chitinase in combination breaks tough yeast
cell walls. Cellulase and pectinase effectively disrupt plant cells. Controlled and sequential
degradation of cell walls by combinations of enzymes selectively releases enzymes without
contamination and denaturation. Cell disruption using enzyme mixture effectively releases products
such as recombinant proteins, pigments, specialized lipids, ethanol, etc. The yeast cell wall
degradation enzyme from the fungus Rhizoctonia spp. liberates cell bound invertase from
Saccharomyces cerevisiae. The advantages of using enzymes besides their selectivity during product
release are their ability to work at mild conditions at very low concentration. Lysozyme is a
commonly used enzyme to disrupt cell wall in large scale. A combination of enzymatic or chemical
 DOWNSTREAM PROCESS TECHNOLOGY: AN OVERVIEW 9

lyses with mechanical disruption increases the efficiencies of the respective methods with the
option of energy, time and money savings.

1.1.3 Isolation
Extraction
Compound or group of compounds extracted from a mixture or from cells into a solvent phase.
This usually achieves isolation as well as purification of the bioproducts. In the case of antibiotic
recovery, extraction is used as an early step especially for the recovery of lipophilic compounds.
Presently extraction is a major method of product isolation, many of which have been antibiotics,
either by changing solvent or changing solute via ion pairs or pH. These extractions take a clarified
beer or cell fraction containing perhaps milligrams per litre and produce an extract whose
concentration may be several kg by weight, where applicable extractions can be a cornerstone of
biological separations.
Liquid–liquid extraction: In the biotechnology industry, this method achieves both concentration
and purification in large scale. Liquid–liquid extraction method extracts the product by multistage
parallel-flow extraction or by counter current extraction to extract protein that is differentially
soluble in two phases. Usually, successively smaller volumes of the solvent by repeated extractions
of a given sample is carried out, and along with this back extraction also tends to increase the
selectivity of extraction. Even though counter current extraction is most complex, it is most effective.
Liquid–liquid extraction plays a major role in the isolation of proteins from crude homogenate and
it is a very important tool to separate proteins from highly viscous solution with heterogeneous
distribution of particle sizes. It has the advantages of handling mixtures in high capacity that is
easy to scale-up. Low molecular weight products can be extracted using organic solvents by physical,
dissociative or reactive extraction. Extractive methods are effectively extract non-ionizing
compounds. Solvent that gives maximal difference of K (extraction constant) value for different
mixture in the crude sample separates non-ionizing compounds. Dissociative extraction extracts
penicillin and some other antibiotics by exploiting dissociative constants of ionizable components
to overcome the adverse ratio of partition coefficients. Reactive extraction method extracts compound
by adding aliphatic amines or a phosphorus compounds to the organic solvents that forms selective
salvation bonds or stoichiometric complexes that are also soluble in aqueous phase.
Supercritical fluid extraction: This method is gaining importance particularly for extracting
highly labile byproducts such as food aroma components and flavours. It is an important tool as it
is controllable, economical, safe, and recoverable with highest yield, residueless, non-toxic, efficient,
less energy demanding and effective. This method handles varieties of samples such as solids,
semisolids and liquids of different chemical nature. Commonly used for the extraction of bitter
flavours from hops, caffeine from coffee beans, flavour from fruits, oleoresins from spices,
b-carotene and oils from seeds, monoglycerides from vegetable oils and common organic chemicals
such as alcohols, ketones, carboxylic acids and esters from aqueous media.
Aqueous two-phase extraction: Two-phase extraction method is a common method for the
recovery of fumerase and penicillin acylase. Aqueous two-phase extraction in four-stage process
can achieve about 10-fold concentration and about 70% purity of formate dehydrogenase. This
10 DOWNSTREAM PROCESS TECHNOLOGY : A New Horizon in Biotechnology

method is also used for the recovery and purification of alkaline protease from whole broths of
Bacillus licheniformis, recovery of bulk intracellular proteins from wastes, separation of yeast
from brewing operations, and isolation of b-galactosidase or lactase from homogenized yeast.
Amino acids such as lysine, glutamic acid, and phenylalanine, in pure solution or in fermentation
broth, extracted with the aqueous two-phase system consisting of polyethylene glycol and salts,
giving a very sharp separation. Type and the amount of salts used, pH and components of the broth
influence the partition. Use of affinity partitioning based on molecular recognition of the desired
proteins or enzyme by a ligand covalently bound to one of the phase forming polymers significantly
enhance the selectivity and yield of aqueous two-phase polymer extractions. This is a very important
method as the interfacial tension between two phases is significantly lower than in water-organic
solvent systems and presently suitable systems for proteins. Enzyme separated from cells or cell
debris in an aqueous polyethylene glycol-dextrane mixture, which forms separate phase, is rather
easy and free from some of the difficulties encountered in centrifugation.
Reverse micelles: This extraction system is an important tool in the downstream processing of
proteins using thermodynamically stable aggregates of surfactant molecules and water in organic
solvents. These systems play a major role in biotechnological process as they can be used to
solubilize enzymes without the loss of activity, and can also be used for the separation and recovery
of proteins by extraction from aqueous feed.
Concentration
Even though concentration occurs to some extent by extraction, other methods such as evaporation,
membrane filtration, ion exchange methods and adsorption method achieve further concentration.
Evaporation: Continuous flow evaporators, falling film evaporators, thin film evaporators,
centrifugal thin film evaporators and spray dryers are efficient and cost effective to evaporate the
solvents from the product extracted by the extraction method. Evaporation of the whole broth
using spray dryers for low-grade products is common. Even though it is energy consuming process,
this method is common because of its simplicity and reliability. In biotechnological process, the
evaporation step should be able to work as multipurpose process and the equipment should be able
to handle broad range of product viscosity, and heat sensitive products with minimal scale formation.
Falling film evaporators, suited for concentrating viscous product, is common in fermentation
industry. Plate evaporators with large heating surface per unit volume concentrate less viscous and
dilute mixtures. Forced film evaporators produce very dry products with minimum resident time
of few seconds for heat resistant products. Centrifugal forced-film evaporators allow us to reduce
the residence time so that even the heat labile substances can be concentrated under gentle conditions.
Membrane filtration: Membrane filtration achieves isolation, concentration and purification
of the product based on the size of molecule by microfiltration, ultrafiltration, reverse osmosis and
electrodialysis. Microfiltration and ultrafiltration work as sieves and separates molecules of different
sizes, but reverse osmosis can separate molecules of similar size. Microfiltration is common in the
concentration of bacteria and viruses, harvesting of the cells, and characterization of the fermentation
broth. Ultrafiltration is widely used for the fractionation of biomolecules, desalting, production of
enzymes and processing of whey. Hyperfiltration is increasingly been used for the concentration
of pharmaceuticals, production of lactose and part desalination of solutions. Electrodialysis purifies
 DOWNSTREAM PROCESS TECHNOLOGY: AN OVERVIEW 11

charged small molecules such as organic acids. Pervaporation plays a major role in the selective
removal of solvents such as ethanol, acetone-butanol during fermentation and purification of solvents
from azeotropic mixtures. Perstraction is very important tool for the extraction of small molecules
from aqueous or organic solutions. Membrane absorbers efficiently adsorb proteins with high
resolution from the clarified feed.
Ion exchange resins: Solid or liquid polymers with attached ionizable groups either packed in
columns or added to extract to recover product of interest. Ion exchange resins can recover some
of the antibiotics directly from the whole broth.
Adsorption columns: Porous polar and semipolar polymers without ionization groups recover
many compounds by adsorbing it to the resins in non-ionizable state. Solvent extraction method or
elution with changed pH recovers products adsorbed on the resins. High-capacity solid adsorbent
particles are effective in concentrating particular molecules from crude extract. A wide range of
adsorbents matrices such as polystyrene, methacrylate and acrylates-based matrices are important
for industrial process of concentration of low molecular weight compounds such as antibiotics,
vitamins and peptides. Cellulose-based adsorbents are helpful for protein concentration.

1.1.4 Product Purification

Product purification is the key to any successful bioseparation. Major purification steps are
precipitation, crystallization and chromatography.
Precipitation
Precipitation is an easy straightforward method of purifying biological solutes in a non-crystalline
state. Since the precipitated product is impure, this method is more appropriate as a concentration
step than the purification, and this method is important in the preliminary stages of downstream
processing of biological products, and well established for the recovery of bulk proteins. It has the
inherent advantages of being a simple method, requiring very simple equipments with the option
of wide varieties of precipitating agents. This method is an important purification step in fractionation
of blood plasma proteins. Adding salts precipitate most enzymes and organic solvents
advantageously fractions most of the enzymes. Non-ionic polymers precipitate proteins such as
plasma proteins, fibrinogen and g-globulins without denaturation and can be used at room
temperature. Ionic polyelectrolytes are gaining importance in industry for the recovery of enzymes
and food proteins. Polyacrylic acids as calcium salts can precipitate basic proteins such as lysozymes,
cytochrome C, prolamines and trypsin with 90% yield. It is a well-established large scale industrial
separation process used efficiently to recover proteins, polysaccharides, and undesirable components
such as nucleic acids, pigments and other residual components form a crude extract. Affinity
precipitation using homobifunctional ligands precipitates multimetric proteins as large complexes,
and even heterobifunctional ligands are helpful in a more general mode.
Crystallization
Generally during penicillin G recovery process, the broth is filtered and washed, and extracted to
butyl acetate or methyl isobutyl ketone after acidification of the filtrate. The solvent is then
decolourized with the help of active carbon and the penicillin G acid is back-extracted to water
12 DOWNSTREAM PROCESS TECHNOLOGY : A New Horizon in Biotechnology

upon neutralization with an aqueous potassium salt solution such as potassium acetate. The resulting
mixture is mixed with sufficient quantity of butanol, and the water content is reduced using
evaporation of the butanol-water azeotrope. The penicillin G crystals formed are recovered by
filtration and subsequent washing and drying. In another process, the crystalline material is directly
obtained from the organic solution by adding potassium acetate or other potassium salts and
evaporation of the azeotrope, whereupon the crystals are filtered, washed with butanol, filtered
and dried. Crystallization is the final stage in purification of products like citric acid and sodium
glutamate. Well formed crystals are expected to be pure because each molecule or ion must fit
perfectly into the lattice as it leaves the solution. Impurities would normally not fit well in the
lattice, and thus remain in solution preferentially. Hence, molecular recognition is the principle of
purification in crystallization.
Chromatographic methods
Group of high resolution closely related separation methods based on the distribution between
mobile and stationary phase find use in separation, purification and identification of compounds.
Chromatographic methods purify low molecular weight compound from the mixture of similar
molecules such as homologous antibiotics and of macromolecules, especially enzymes that are
similar in properties. Different chromatographic procedures such as adsorption, ion exchange, gel
filtration, hydrophobic, affinity, covalent and partition chromatography are used widely for isolation,
purification and characterization of molecules.
Size exclusion chromatography: Size exclusion chromatography is efficient for the partitioning
of proteins from aggregates or degradation products between stationary liquid held by pores of the
gel particles and the mobile liquid in the void volume between the particles. It is more efficient in
small scale and hence used in the final protein-polishing step in purification protocol.
Adsorption chromatography: In adsorption chromatography, resolution of the macromolecules
is a surface mediated process. In adsorption chromatography, principal adsorbents for proteins are
ion exchangers, hydrophobic materials, inorganic such as calcium sulphate gels, chemically
synthesized ligand adsorbents. Chemically synthesized adsorbents are ligand of mixed functional
characteristics attached to a matrix or biological compounds such as substrates, enzyme inhibitors,
or antibodies, which constitute affinity adsorbents. Adsorptive chromatographic methods are efficient
in purifying wide varieties of products.
Ion exchange chromatography: Ion exchange chromatography is most widely used method
because of its general applicability, good resolution and high capacity. Ion exchange chromatography
is effective in the initial stages of downstream processing as it is insensitive to the sample volume.
Ion exchangers are most widely used may be either as an anionic exchangers like
dodecylethylaminoethyl (DEAE) and quaternary amino (Q) or cationic exchangers like
carboxymethyl (CM) and sulphonate (S).
Hydrophobic interaction chromatography: Hydrophobic adsorbents as in the case of
hydrophobic interaction chromatography exploit the variability of external hydrophobic amino
acid residues on different proteins, and interact with the proteins by virtue of the fact that in
aqueous solvents hydrophobic patches on proteins seek out other hydrophobic surface preferentially.
It is a robust and high capacity method for both concentration and purification of proteins.
 DOWNSTREAM PROCESS TECHNOLOGY: AN OVERVIEW 13

Hydrophobic interaction chromatography is widely used for the purification of commercially

important products such as protein and plasmids. The application of hydrophobic interaction
chromatography used for the purification of the enzymes of secondary compound biosynthesis,
which is difficult to obtain in pure form, by size-exclusion and ion exchange techniques alone as in
the case of tryptophan decarboxylase, strictosidine synthase and geraniol-10-hydroxylase.
Reverse phase chromatography: Reverse phase chromatography for proteins has been highly
successful when dealing with small, structurally sturdy proteins, up to about 30,000 Da in size. It
has its greatest application in the separation of peptides, such as those generated by enzymatic
digestion of proteins and for the purification of acid phosphatase from bovine cortical bones.
Affinity adsorbents are highly specific for proteins. Antibody adsorbents are used to separate protein
using specific antibodies or antibodies using specific ligands.
Affinity chromatography: Affinity chromatography is efficient for the recovery of human
leucocytes in high yield and in high purity using monoclonal antibody immobilized in a sepharose
column. Human very low-density lipoproteins (VLDL) purifies very efficiently and relatively quickly
by chromatofocussing. It has also been used for the preparative separation of molecular and
heterogeneous forms of ferredoxin-NADP oxidoreductase from nitrogen fixing Cyanobacterium,
and purifying various steroid receptor protein isoforms for the female sex harmone estradiol. Affinity
chromatography is efficient for the purification of harmone, antigens, antibodies, enzymes,
glycoprotein, lectins, immunoglobulin, coagulation factors, protein kinase, dehydrogenase, etc.

1.1.5 Product Formulation

Maintenance of the product activity and stability of the product during distribution and storage are
crucial factors for the commercial viability of any of the biotechnological products. After the
removal of water most of the low molecular weight products such as bulk solvents and bulk organic
acids are formulated as a concentrated solutions. Small molecules such as antibiotics, citric acid,
and sodium glutamate are crystallized from solution by the addition of salts once they reach the
required level of purity, as they are required in pure form. Protein products formulated as solutions,
suspensions or dry powders are less prone to oxidation, temperature and presence of proteases.
Along with this variety of stabilizing additives such as sugars, salts, polyhydric alcohols or polymers
are included in the formulations in order to prolong the product shelf life. Presently bulk enzymes
are available in liquid form, but preferred in dry powdered form as enzymes are prone to denaturation
in aqueous solution, and the volume is less in dry form. Most of the byproducts are commonly
formulated using contact dryer, convection dryer or radiation dryer as it exerts uniform thermal
stress on the products, high throughput, short drying period and possibility for development of
continuous process. Spray drying of the product through a nozzle as an aerosol into a stream of hot
gas dries large quantity of liquids, and is used for formulating enzymes, antibiotics, and food
products. One of the least harsh methods of product drying is freeze-drying or lyophilization of
products such as enzymes, pharmaceutical products, diagnostics, vaccines, plasma fractions,
foodstuffs, viruses, and bacteria. Various lyoprotectants are included during lyophilization to protect
protein from product blow and to enhance product solubility.