IF UNIT-4

PURIFICATION AND CHARACTERIZATION OF PROTEIN:

Protein purification is a series of processes intended to isolate a single type of protein
from a complex mixture.
Protein purification is vital for the characterization of the function, structure and
interactions of the protein of interest. The starting material is usually a biological tissue
or a microbial culture.
Protein purification has an over 200-year history: the first attempts at isolating
substances from plants having similar properties to “egg albumen,” or egg white, were
reported in 1789 by Fourcroy.
Protein characterization involves the use of experimental methods that allow for the
detection and isolation of a protein and its purification, as well as the characterization of
its structure and function.
Protein purification is divided into three stages:
• Preparation of sources
• Primary isolation
• Final purification
Preparation of Sources:
▪ The raw materials from which proteins can be isolated such as microbial culture
or animals or plant sources should be selected.
▪ The amount of protein can be increased by increasing cultivation volume.
▪ By the help of recombinant DNA technology, the amount of protein production can
be increased.
Primary Isolation:
This consists of separation of protein from other cellular components.
For this propose there are different methods:
Filtration:
▪ Different methods can be employed for filtration of extracellular protein.
▪ Ultrafiltration are usually used to filtrate extracellular proteins from cell.
▪ Ultrafiltration is a membrane filtration in which hydrostatic pressure is applied
which causes movement of solution across the semi-permeable membrane.
▪ Water and low molecular weight solute pass while other high molecular weight of
molecules trapped in membrane.
▪ The protein molecules are adsorbed in the membrane surface.
Extraction:
▪ Depending on the source, the protein has to be brought into solution by breaking
the tissue or cells containing it.
▪ There are several methods to achieve this.
▪ Physical method:
 ▪ Homogenizer- used to break down the tissue and release the cellular protein.
▪ Sonication- using high frequency sound waves to breakdown the cells.
▪ Freeze thaw- continuous freezing and thawing break the cells releasing cellular
protein.
▪ Thermolysis: breakdown of cell by the action of heat.
▪ Chemical method:
▪ Detergents- denature the cell membrane.
▪ Chelating agents
▪ Hydroxides and hypo chlorides
▪ Enzymatic method:
▪ Autolysis
▪ Lytic enzyme
After this extraction process soluble protein will be in the solvent, and can be separated
from cell membranes, DNA, etc. by centrifugation.
Precipitation and Solubilisation:
▪ In bulk protein purification, a common first step to isolate proteins is precipitation
with ammonium sulphate (NH4)2SO4.
▪ This is performed by adding increasing amounts of ammonium sulphate and
collecting the different fractions of precipitate protein.
▪ One advantage of this method is that it can be performed inexpensively with very
large volumes.
▪ The precipitated proteins can be separated from the solution mixture by
ultracentrifugation technique.
▪ For solubilization the protein pellet is resuspended in buffer solution.
Final purification:
▪ Chromatography is the usual method for obtaining pure protein.
▪ There are different types of chromatographic methods such as:
Chromatography:
Chromatography is an important biophysical technique that enables the separation,
identification, and purification of the components of a mixture for qualitative and
quantitative analysis.
Chromatography is based on the principle where molecules in mixture applied onto the
surface or into the solid, and fluid stationary phase (stable phase) is separating from each
other while moving with the aid of a mobile phase.
Many different chromatographic methods exist:
Size Exclusion Chromatography:
Protein Purification and Characterization Methods Based on Whole Structure: Size
and Shape.
Chromatography can be used to separate protein in solution or denaturing conditions by
using porous gels. This technique is known as size exclusion chromatography.
Separation occur on the basis of size (MW). Stationary (cross-linked gel particles):
consist of one of two kinds of polymers; the 1st is a carb. polymer (ex. dextran or
agarose); The 2nd is based on polyacrylamide.
Small molecules get trapped in the pores of the stationary phase, while large molecules
flow through the gaps between the beads and have very small retention times. So larger
molecules come out first. In this type of chromatography there isn’t any interaction,
physical or chemical, between the analyte and the stationary phase.

Ion Exchange Chromatography:

Protein Purification and Characterization Methods Based on Net Charge
• Ion exchange chromatography separates compounds according to the nature and
degree of their ionic charge.
• The column to be used is selected according to its type and strength of charge.
Anion exchange resins have a positive charge and are used to retain and separate
negatively charged compounds, while cation exchange resins have a negative
charge and are used to separate positively charged molecules.
• Molecules possessing the opposite charge as the resin will bind tightly to the resin,
and molecules having the same charge as the resin will flow through the column
and elute out first.
Affinity chromatography:
Protein Purification and Characterization Methods Based on Bio properties
(Affinity)
• Affinity Chromatography is a separation technique based upon molecular
conformation, which frequently utilizes application specific resins.
• In affinity chromatography, a compound having specific affinity to desired protein
is attached to the resin. For. e.g. Antibody or enzymes against desired protein is
coated on resin.
• The resin is then packed into a column. When mixture of protein is poured, only
those proteins having specific affinity with resin (Ab coated) or enzyme coated
stick on the column.
• All the other protein gets eluted.
• Only the undesired protein gets eluted.
• The protein of interest stuck on the column can be eluted by changing the ionic
strength of the solution so that the desired protein no longer binds to resin and
get eluted.
Immunoaffinity Chromatography:
Immunoaffinity chromatography uses the specific binding of an antibody to the target
protein to selectively purify the protein. The procedure involves immobilizing an
antibody to a column material, which then selectively binds the protein, while everything
else flows through.
Characterization of Protein:
Protein characterization involves the use of experimental methods that allow for the
detection of structure, function and basic properties of protein.
Electrophoresis: (Molecular weight)
• It is the process of separation of charged particles under the influence of electric
field.
• SDS-PAGE is the most widely used method for analysis of protein in the mixture.
• It is useful for monitoring the protein purification.
• SDS-PAGE separates proteins on the basis of molecular weight.
• At first polyacrylamide gel is made and a well is made.
• The protein sample is mixed with beta-mercaptoethanol and sodium dodecyl
sulphate (SDS) and boiled for 5 minutes.
• During boiling, proteins get denatured.
• SDS molecule is highly -vely charged so the protein binds with SDS become -vely
charged.
• When electrophoresis is done, the protein moves towards anode (+ve charge).
• The small size protein migrate faster and large size moves slower forming
different band.
• The band can be visualized by staining with Coomassie brilliant blue (CBB).
2D-PAGE (Isoelectric Point)
• Two-dimensional gel electrophoresis (2DGel) is a successful method used for the
detection and analysis of proteins.
• It has been designed as a combination of the 2DGel, IEF and SDS-PAGE methods,
and is used in the analysis of complex protein mixtures.
• In the first step, protein is separated into its charges with IEF, whereas in the
second step, the protein is separated according to its mass.
Peptide sequencing:
The amino acid composition of the protein was determined by amino acid analyser. The
protein sequence was determined by protein sequencer using Edman degradation or
mass spectrometry.
• Edman degradation, developed by Pehr Edman, is a method of sequencing amino
acids in a peptide.
• In this method, the amino-terminal residue is labeled and cleaved from the peptide
without disrupting the peptide bonds between other amino acid residues.
• The polypeptide is reacted with phenylisothiocyanate under mild alkaline
condition.
• The amino terminal of peptide is converted to phenylthiocarbomyl (PTC).
• Then, under acidic conditions, this derivative of the terminal amino acid is cleaved
as a thiazolinone derivative.
• The thiazolinone amino acid is then selectively extracted into an organic solvent
and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino
acid derivative that can be identified by using chromatography or electrophoresis.
UPSTREAM AND DOWNSTREAM PROCESSING
Basic Steps of Industrial Fermentation
Any industrial fermentation operation can be broken down into three main stages, viz,
upstream processing, the fermentation process and downstream processing.
Upstream processing includes formulation of the fermentation medium, sterilization of
air, fermentation medium and the fermenter, inoculum preparation and inoculation of
the medium.
Various steps of Upstream Process:
1. Preparation of Fermentation Medium
2. Sterilization
3. Inoculum preparation
Preparation of Fermentation Medium:
MEDIUM FORMULATION
Medium should offer the necessary amounts of carbon, nitrogen, micronutrients such as
vitamins and trace elements. Variations in concentrations of specific nutrients during
fermentation is important to get desired results.
Fermentation medium consists of macronutrients, micronutrients, trace elements,
dissolved oxygen, vitamins, enzymes, other dissolved gases, and inhibitors. The
components of the medium should accomplish the elemental necessities for metabolite
construction and biomass production with sufficient provision of energy for biosynthesis.
Media formulation: growth medium must have essential nutrients for microbial growth
for successful fermentation process
Two kind media:
Inoculum media: enrich the culture
Production media: contain carbon and nitrogen
Basic components of Medium:
Water
Chief constituent of every fermentation medium is water and it is required in rinsing,
cooling, and heating. It is significant to consider the dissolved salts, contamination,
effluents, and pH, while evaluating the water supply. Water’s mineral contents play an
important function in brewing and are critical in squashing.
Energy Sources
As industrial microbes are chemoorganotrophs, so their source of energy is carbon
source in form of lipids, proteins, and carbohydrates. While, in some cases, methanol or
hydrocarbons may be used by some microorganisms as a source of energy or carbon.
Carbon Source
Carbon is considered as a main product of a fermentation process. If 60-70% of
production cost is raw materials during single-cell protein or ethanol production.
Examples of Carbon Sources
I. Carbohydrates
Starch obtained from cereals, potatoes, and maize, is easily available as a source of
carbohydrates and are extensively used in fermentation of alcohol. Grains (maize etc) are
used in the form of powder and also as a source of carbohydrates. Cheapest source of
carbohydrates is molasses, and used in organic acid, amino acid, single-cell protein, and
alcohol fermentations.
II. Fats and Oils
Oils were firstly used as antifoaming agents in antibiotic processes. Oils provide
maximum energy per weight than sugars. Oils possess anti-foaming qualities but are used
as additives. These may also be used for their high content of the fatty acids.
Nitrogen Sources
Industrially used microorganisms have ability to use organic as well as inorganic means
of nitrogen. Inorganic source of nitrogen is supplied as ammonium salts, ammonia gas
and nitrates. Inorganic substrates which can be used as a source of nitrogen includes urea,
ammonium salts, and ammonia. Ammonia is used to control pH during fermentation
process.
Minerals
Essential minerals which are used in all media formulation include potassium, sulphur,
chlorine, phosphorous, magnesium, and calcium. We require a minute amounts other
mineral such as cobalt, manganese, zinc, iron, copper and molybdenum and they exist as
impurities. The specific concentration of these all elements depends on the micro-
organism.
Growth Factors
Few of the microbes cannot produce complete complement of components of the cell and
consequently requisite some of the preformed components known as growth factors.
Growth factors includes amino acids, vitamins, sterols and fatty acids. Some natural
sources such as nitrogen and carbon are used in growth medium formulations having
required growth factors.
Buffers
pH has great influence on microbial growth. pH of the growth media can be maintained
by addition of buffers that would resist pH changes. Many microorganisms have optimum
pH range 7.0. Some of the examples of buffers that are commonly used include; ammonia
sodium hydroxide and calcium carbonate.
ADDITION OF METABOLIC REGULATORS
Precursors
Many precursor molecules are used as metabolic regulators that improves product yield.
Some of the chemicals are used as precursors and induced to cultivation processes that
incorporate into the product formed by fermentation process.
Inhibitors
A specific product or metabolic intermediate is formed by the addition of specific
inhibitors to the fermentation. Earliest substrate includes glycerol, which is produced due
to the microorganisms. Glycerol production is possible after modification of ethanol by
removing acetaldehyde. Acetaldehyde is formed as sodium bisulfite is added to broth. As
acetaldehyde is replaced by dihydroacetone phosphate as it is no longer available for the
reoxidation of NADH2, which is produced in glycolysis process. Product of this reaction
(Glycerol-3-phosphate) is teansformed into glycerol.

Inducers
Inducible enzymes are of interest in industry. Inducers are substrates which include
pectin for pectinases, maltose for pullulanase, and starch or dextrin for amylases.

Sterilization:
For successful fermentation, it is absolutely essential to ensure:
• Sterility of the media containing the nutrients.
• Sterility of incoming and outgoing air.
• Sterility of the bioreactor.
Prevention of contamination during fermentation.
A bioreactor can be sterilized by destroying the organisms by heat/chemicals/radiation
or sometimes by physical procedures such as filtration.
Sterilization of Culture Media:
The constituents of culture media, water and containers contribute to the contamination
by vegetative cells and spores. The media must be free from contamination before use in
fermentation. Sterilization of the media is most commonly achieved by applying heat and
to a lesser extent by other means (physical methods, chemical treatment, and radiation).
Heat sterilization:
Heat is the most widely used sterilization technique. The quality and quantity of
contamination (i.e., the type and load of microorganisms), composition of the media and
its pH and size of the suspended particles are the important factors that influence the
success of heat sterilization.
Physical methods:
The physical methods such as filtration, centrifugation, and adsorption (to ion-
exchangers or activated carbon) are in use. Among these, filtration is most widely used.
Certain constituents (vitamins, blood components, antibiotics) of culture media are heat
labile and therefore, are destroyed by heat sterilization. Such components of the medium
are completely dissolved (absolutely essential or else they will be removed along with
microorganisms) and then subjected to filter sterilization.
Batch sterilization:
The culture media are subjected to sterilization at 121°C in batch volumes, in the
bioreactor. Batch sterilization can be done by injecting the steam into the medium (direct
method) or injecting the steam into interior coils (indirect method). For the direct batch
sterilization, the steam should be pure, and free from all chemical additives (that usually
come from steam manufacturing process).
There are two disadvantages of batch sterilization:
1. Damage to culture media:
Alteration in nutrients, change in pH and discolouration of the culture media are common.
2. High energy consumption:
It takes a few hours (2-4 hrs.) for the entire contents of the bioreactor to attain the
requisite temperature (i.e. 120°C). Another 20-60 minutes for the actual process of
sterilization, followed by cooling for 1-2 hours. All this process involves wastage of
energy, and therefore batch sterilization is quite costly.
Continuous sterilization:
Continuous sterilization is carried out at 140°C for a very short period of time ranging
from 30 to 120 seconds. (This is in contrast to the batch fermentation done at 121°C for
20-60 minutes). This is based on the principle that the time required for killing
microorganisms is much shorter at higher temperature. Continuous sterilization is
carried out by directly injecting the steam or by means of heat exchangers.
Air sterilization:
In general, the industrial fermentations are carried out under vigorous and continuous
aeration. For an effective fermentation, the air should be completely sterile, and free from
all microorganisms and suspended particles. There is a wide variation in the quantity of
suspended particles and microbes in the atmospheric outdoor air.
Among the microorganisms present in the air, the fungal spores (50%) and Gram-
negative bacteria (40%) dominate. Air or other gases can be sterilized by filtration, heat,
UV radiation and gas scrubbing. Among these, heat and filtration are most commonly
used.
(a) Air sterilization by heat:
In the early years, air was passed over electrically heated elements and sterilized. But this
is quite expense, hence not in use these days.
(b) Air sterilization by filtration:
Filtration of air is the most commonly used sterilization in fermentation industries.
Membrane cartridge filters:
These are removable pleated membrane filters made up of cellulose ester, nylon or
polysulfone. Membrane cartridge filters are smaller in size, simpler for operation and
replacement

Inoculum preparation
It is essential that the culture used to inoculate a fermentation satisfies the following
criteria:
1. It must be in a healthy, active state thus minimizing the length of the lag phase in the
subsequent fermentation.
2. It must be available in sufficiently large volumes to provide an inoculum of optimum
size.
3. It must be in a suitable morphological form.
4. It must be free of contamination.
5. It must retain its product-forming capabilities.

Inoculum build up is the preparation of the seed culture in amounts sufficient to be used
in the large Fermenter vessel.
This involves growing the microorganisms obtained from the pure stock culture in
several consecutive Fermenter. This process cuts down the time required for the growth
of microorganisms in the Fermenter, thereby increasing the rate of productivity.
Then the seed culture obtained through this process is used to inoculate the fermentation
medium.
Downstream Process:
Downstream process can be defined as the various stages of processing that occur after
the completion of the fermentation or bioconversion stage, including separation,
purification, and packaging of the product.
Stages in Downstream Processing
▪ Removal of Insoluble or Cell separation or solid liquid separation
▪ Product Isolation
▪ Product Purification
▪ Product formulation
# REMOVAL OF MICROBIAL CELLS FROM BIOREACTORS
The first step in product recovery is the separation of whole cells (cell biomass) and other
insoluble ingredients from the culture broth.
These include flotation, flocculation, filtration and centrifugation.
Flotation:
When a gas is introduced into the liquid broth, it forms bubbles. The cells and other solid
particles get adsorbed on gas bubbles. These bubbles rise to the foam layer which can be
collected and removed.
Flocculation:
In flocculation, the cells (or cell debris) form 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.
Filtration:
A mechanical operation used for the separation of solids from fluids (liquids or gases) by
interposing a medium to porous membrane through which the fluid can pass, but the
solids in the fluid are retained.
Centrifugation:
The technique of centrifugation is based on the principle of density differences between
the particles to be separated and the medium. Thus, centrifugation is mostly used for
separating solid particles from liquid phase (fluid/particle separation). Unlike the
centrifugation that is conveniently carried out in the laboratory scale, there are certain
limitations for large scale industrial centrifugation.
# PRODUCT ISOLATION:
As already stated, there are several biotechnological products (vitamins, enzymes) which
are located within the cells. Such compounds have to be first released (maximally and in
an active form) for their further processing and final isolation. The microorganisms or
other cells can be disintegrated or disrupted by physical, chemical or enzymatic methods.
Cell Disruption:
Physical methods of cell disruption:
The microorganisms or cells can be disrupted by certain physical methods to release the
intracellular products.
Ultra sonication:
Ultrasonic disintegration is widely employed in the laboratory. However, due to high cost,
it is not suitable for large-scale use in industries.
Osmotic shock:
This method involves the suspension of cells (free from growth medium) in 20% buffered
sucrose. The cells are then transferred to water at about 4°C. Osmotic shock is used for
the release of hydrolytic enzymes and binding proteins from Gram-negative bacteria.
Heat shock (thermolysis):
Breakage of cells by subjecting them to heat is relatively easy and cheap. But this
technique can be used only for a very few heat-stable intracellular products.
High pressure homogenization:
This technique involves forcing of cell suspension at high pressure through a very narrow
orifice to come out to atmospheric pressure. This sudden release of high pressure creates
a liquid shear that can break the cells.
Chemical methods of cell disruption:
Treatment with alkalies, organic solvents and detergents can lyse the cells to release the
contents.
Alkalies:
Alkali treatment has been used for the extraction of some bacterial proteins. However,
the alkali stability of the desired product is very crucial for the success of this method e.g.,
recombinant growth hormone can be efficiently released from E. coli by treatment with
sodium hydroxide at pH 11.
Organic solvents:
Several water miscible organic solvents can be used to disrupt the cells e.g., methanol,
ethanol, isopropanol, butanol. These compounds are inflammable; hence require
specialised equipment for fire safety. The organic solvent toluene is frequently used. It is
believed that toluene dissolves membrane phospholipids and creates membrane pores
for release of intracellular contents.
Detergents:
Detergents that are ionic in nature, cationic-cetyl trimethyl ammonium bromide or
anionic-sodium lauryl sulfate can denature membrane proteins and lyse the cells. Non-
ionic detergents (although less reactive than ionic ones) are also used to some extent e.g.,
Triton X-100 or Tween. The problem with the use of detergents is that they affect
purification steps, particularly the salt precipitation. This limitation can be overcome by
using ultrafiltration or ion-exchange chromatography for purification.
Enzymatic methods of cell disruption:
Cell disruption by enzymatic methods has certain advantages i.e., lysis of cells occurs
under mild conditions in a selective manner. This is quite advantageous for product
recovery. Lysozyme is the most frequently used enzyme and is commercially available
(produced from hen egg white). It hydrolyses β-1, 4-glycosidic bonds of the mucopeptide
in bacterial cell walls. The Gram- positive bacteria (with high content of cell wall
mucopeptides) are more susceptible for the action of lysozyme.
# CONCENTRATION:
The filtrate that is free from suspended particles (cells, cell debris etc.) usually contains
80-98% of water. The desired product is a very minor constituent. The water has to be
removed to achieve the product concentration. The commonly used techniques for
concentrating biological products are evaporation, liquid-liquid extraction, membrane
filtration, precipitation and adsorption.
Liquid -Liquid extraction
The concentration of biological products can be achieved by transferring the desired
product (solute) from one liquid phase to another liquid phase, a phenomenon referred
to as liquid-liquid extraction. Besides concentration, this technique is also useful for
partial purification of a product.
Evaporation:
Water in the broth filtrate can be removed by a simple evaporation process. The
evaporators, in general, have a heating device for supply of steam, and unit for the
separation of concentrated product and vapour, a condenser for condensing vapour,
accessories and control equipment. The capacity of the equipment is variable that may
range from small laboratory scale to industrial scale.
Adsorption:
The biological products of fermentation can be concentrated by using solid adsorbent
particles. In the early days, activated charcoal was used as the adsorbent material. In
recent years, cellulose-based adsorbents are employed for protein concentration.
The binding to the surface is weak and reversible.
Membrane Filtration:
Membrane filtration has become a common separation technique in industrial
biotechnology. It can be conveniently used for the separation of biomolecules and
particles, and for the concentration of fluids. The membrane filtration technique basically
involves the use of a semipermeable membrane that selectively retains the
particles/molecules that are bigger than the pore size while the smaller molecules pass
through the membrane pores.
Ultrafiltration
UF is governed by a screening principle and dependent on particle size. UF membranes
have a pore size between 1 nm and 100 nm (10 and 2000 Å), thus allowing retention of
compounds with a molecular weight of 300 to 500 000 Dalton.
Typically, the process is suitable for retaining biomolecules, bacteria, viruses, polymers,
colloidal particles and sugar molecules.
Precipitation
Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides. Further, precipitation technique
can also be employed for the removal of certain unwanted byproducts e.g. nucleic acids,
pigments.
Formation of a solid in a solution during a chemical reaction. Solid formed is called the
precipitate and the liquid remaining above the solid is called the supernate.
Salts such as ammonium & sodium sulphate are used for proteins to precipitate. Organic
solvents methanol used to precipitate dextrans. Chilled ethanol and acetone used for
protein precipitation. Non ionic polymer such as polyethylene glycol used in
precipitation.
# Product Purification:
The biological products of fermentation (proteins, pharmaceuticals, diagnostic
compounds and research materials) are very effectively purified by chromatography. It
is basically an analytical technique dealing with the separation of closely related
compounds from a mixture.
Chromato-graphy usually consists of a stationary phase and mobile phase. The stationary
phase is the porous solid matrix packed in a column (equilibrated with a suitable solvent)
on to which the mixture of compounds to be separated is loaded. The compounds are
eluted by a mobile phase.
Size Exclusion Chromatography:
Product Purification Methods Based on Whole Structure: Size and Shape.

Chromatography can be used to separate protein in solution or denaturing conditions by

using porous gels. This technique is known as size exclusion chromatography.
Separation occur on the basis of size (MW). Stationary (cross-linked gel particles):
consist of one of two kinds of polymers; the 1st is a carb. polymer (ex. dextran or
agarose); The 2nd is based on polyacrylamide.
Small molecules get trapped in the pores of the stationary phase, while large molecules
flow through the gaps between the beads and have very small retention times. So larger
molecules come out first. In this type of chromatography there isn’t any interaction,
physical or chemical, between the analyte and the stationary phase.

Ion Exchange Chromatography:

Product Purification Methods Based on Net Charge

• Ion exchange chromatography separates compounds according to the nature and

degree of their ionic charge.
• The column to be used is selected according to its type and strength of charge.
Anion exchange resins have a positive charge and are used to retain and separate
negatively charged compounds, while cation exchange resins have a negative
charge and are used to separate positively charged molecules.
• Molecules possessing the opposite charge as the resin will bind tightly to the resin,
and molecules having the same charge as the resin will flow through the column
and elute out first.
Affinity chromatography:
Product Purification Methods Based on Bio properties (Affinity)

• Affinity Chromatography is a separation technique based upon molecular

conformation, which frequently utilizes application specific resins.
• In affinity chromatography, a compound having specific affinity to desired protein
is attached to the resin. For. e.g. Antibody or enzymes against desired protein is
coated on resin.
• The resin is then packed into a column. When mixture of protein is poured, only
those proteins having specific affinity with resin (Ab coated) or enzyme coated
stick on the column.
• All the other protein gets eluted.
• Only the undesired protein gets eluted.
• The protein of interest stuck on the column can be eluted by changing the ionic
strength of the solution so that the desired protein no longer binds to resin and
get eluted.

Immunoaffinity Chromatography:
Immunoaffinity chromatography uses the specific binding of an antibody to the target
protein to selectively purify the protein. The procedure involves immobilizing an
antibody to a column material, which then selectively binds the protein, while everything
else flows through.
Hydrophobic interaction chromatography (HIC):
This is based on the principle of weak hydrophobic interactions between the hydrophobic
ligands (alkyl, aryl side chains on matrix) and hydrophobic amino acids of proteins. The
differences in the composition of hydrophobic amino acids in proteins can be used for
their separation. The elution of proteins can be done by lowering the salt concentration,
decreasing the polarity of the medium or reducing the temperature.

Product Formulation or Product Polishing:

Formulation broadly refers to the maintenance of activity and stability of a
biotechnological products during storage and distribution. The formulation of low
molecular weight products (solvents, organic acids) can be achieved by concentrating
them with removal of most of the water. For certain small molecules, (antibiotics, citric
acid), formulation can be done by crystallization by adding salts. Proteins may be
formulated in the form of solutions, suspensions or dry powders.
Drying:
Drying is an essential component of product formulation. It basically involves the transfer
of heat to a wet product for removal of moisture. Most of the biological products of
fermentation are sensitive to heat, and therefore require gentle drying methods.
Spray drying:
Spray drying is used for drying large volumes of liquids. In spray drying, small droplets
of liquid containing the product are passed through a nozzle directing it over a stream of
hot gas. The water evaporates and the solid particles are left behind.
Freeze-drying:
Freeze-drying or lyophilization is the most preferred method for drying and formulation
of a wide-range of products—pharmaceuticals, foodstuffs, diagnostics, bacteria, viruses.
This is mainly because freeze-drying usually does not cause loss of biological activity of
the desired product.
Lyophilization is based on the principle of sublimation of a liquid from a frozen state. In
the actual technique, the liquid containing the product is frozen and then dried in a freeze-
dryer under vacuum. The vacuum can now be released and the product containing vials
can be sealed e.g., penicillin can be freeze dried directly in ampules.

CENTRIFUGATION:
A centrifuge is a device for separating particles of 100-0.1 micrometer from liquid by
gravitation forces. It depends according to particle size, shape and density, viscosity of
the medium and rotor speed. More dense components migrate away from the axis of
centrifuge and less dense components migrate towards the axis. It is used to separate
proteins, bacteria etc. from broth preferably when filtration does not give desired results.
Most common types of centrifuges used are
1. Basket centrifuge
2. Tubular bowl centrifuge
3. Solid bowl Scroll centrifuge
4. Multichamber centrifuge
5. Disc-bowl centrifuge
Basket centrifuges In basket centrifuges, the solids & liquids are separated by
centrifugal force using a filter media (usually a cloth) mounted over supporting mesh,
which are together supported inside the rotating basket.
The slurry to be filtered is fed through the feed nozzles to the basket and due to
centrifugal force the liquid is forced out through the filter media while solids are retained
within the filter media inside the basket. These solids then separated or discharged by
various discharging methods namely – manually, bag lifting basket, through scrapper,
operated manually pneumatically / hydraulically, for which different models are
available.
Tubular bowl centrifuge It is used for liquid/liquid and solid/liquid separation.
However, the advantage of conical plate centrifuge over tubular bowl centrifuge is that
solid discharge is possible in conical plate but recovery of solids in tubular bowl is difficult
and there is limited solid capacity.
Solid bowl centrifuge It can be used for liquid/liquid and solid/liquid separation. The
chamber bowl has a high capacity for solids but there is no solid discharge. Bowl cooling
is possible for both conical plate centrifuge and chamber bowl centrifuge. However,
cleaning is easier as well as better sludge dewatering in chamber bowl centrifuge as
compared to conical plate centrifuge.
Disc bowl centrifuge The interior of the bowl consists of a stack of coaxial conical discs
(approximately 100) that are separated by a distance between 0.3 to 0.4 mm. This divides
the bowl into a series of fine layers between which the suspension circulates. Holes
perforate the discs in the stack, thus forming a series of vertical channels at the level of
the liquid interphase. The light liquid phase circulates centripetially and travels along the
upper disc faces until it reaches its evacuation point at the center. The heavy liquid
phase‟s centrifugal circulation causes it to travel along the lower face of the disc until it
surpasses the maximum level of fluid at the top, after which it is evacuated at the center
of the bowl.
Multi-chamber separator It is made up of a number of separating chambers with
annular clearances composed of many concentric circular cylinders. The radius of every
concentric circular cylinder is determined based on the equal sectional area of ring
clearance. The suspension enters the rotating drum through the central feed pipe and
further flows through every separating chamber from the center to the outward chamber.
Under the action of gradually increasing centrifugal force field, the fine particles settle on
the cylinder wall of the outward separating chamber.

ULTRACENTRIFUGATION:
It is an important tool in biochemical research. Which through rapid spinning imposes
high centrifugal forces on suspended particles, or even molecules in solution, and causes
separations of such matter on the basis of differences in weight.
• Its rotational speed up to 150,000 rpm.
• It is creating a centrifugal force up to 900,000 x g.
Ultracentrifugation is basically carried out in two ways: ‘Preparative’ centrifugation aims
to isolate and purify specific particles such as subcellular organelles, while the object of
‘analytical’ centrifugation is to study molecular interactions between macromolecules or
to analyse the properties of sedimenting particles such as their apparent molecular
weights.
FILTRATION OF FERMENTIVE BROTH:
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 the organism,
presence of other organisms, viscosity of the medium, and temperature. Several filters
such as depth filters, absolute filters, rotary drum vacuum filters and membrane filters
are in use.
Depth Filters:
They are composed of a filamentous matrix such as glass wool, asbestos or filter paper.
The particles are trapped within the matrix and the fluid passes out. Filamentous fungi
can be removed by using depth filters.
Absolute Filters:
These filters are with specific pore sizes that are smaller than the particles to be removed.
Bacteria from culture medium can be removed by absolute filters.
Rotary Drum Vacuum Filters:
These filters are frequently used for separation of broth containing 10-40% solids (by
volume) and particles in the size of 0.5-10µm. Rotary drum vacuum filters have been
successfully used for filtration of yeast cells and filamentous fungi. The equipment is
simple with low power consumption and is easy to operate. The filtration unit consists of
a rotating drum partially immersed in a tank of broth . As the drum rotates, it picks up
the biomass which gets deposited as a cake on the drum surface. This filter cake can be
easily removed.

Membrane Filters:
In this type of filtration, membranes with specific pore sizes can be used. However,
clogging of filters is a major limitation. There are two types of membrane filtrations—
static filtration and cross-flow filtration. In cross-flow filtration, the culture broth is
pumped in a crosswise fashion across the membrane. This reduces the clogging process
and hence better than the static filtration.
Liquid-Liquid Extraction:
Liquid–liquid extraction, sometimes also called solvent extraction, is an important
separation technology for a variety of applications in the chemical process industry,
including the petrochemical, food, pharmaceutical, and metal industries.
The concentration of biological products can be achieved by transferring the desired
product (solute) from one liquid phase to another liquid phase, a phenomenon referred
to as liquid-liquid extraction. Besides concentration, this technique is also useful for
partial purification of a product.
GENERAL DESCRIPTION OF THE PROCESS
Whereas distillation takes advantage of different volatilities means different
distributions of a product in the liquid and the gas phase the liquid/liquid extraction is
based on different solubilities means different distributions of a product in 2 co-existing
liquid phases.
For the extraction of a product out of the so called feed a suitable solvent has therefore to
be found. The first step of an extraction process is mixing for an intensive contact of both
liquid phases to enable the mass transfer of the product from the feed liquor into the
solvent. The second step is the phase separation or settling of the 2 liquid phases. After
the extraction of the product the feed liquor is called raffinate whereas the solvent
containing the product is called extract. For the recovery of the product the solvent has
to be separated in a subsequent third step from the product which is mostly done by
distillation.
Purpose of Extraction
• To separate closed-boiling point mixture
• Mixture that cannot withstand high temperature of distillation
Example:
• recovery of penicillin from fermentation broth solvent: butyl acetate
• recovery of acetic acid from dilute aqueous solutions solvent: ethyl-acetate
Basic Design Of a Fermentation system:
Fermentation can be defined as a metabolic process in which cheap raw materials such
as sugar or carbohydrates are converted into economically important products like acids,
gases and alcohols by micro-organism. This process is carried out in a equipment called
as fermenter.
What is a Fermenter?
A Fermenter can be defined as a vessel in which sterile nutrient media and pure culture
of micro-organism are mixed and fermentation process is carried out under aseptic and
optimum condition.
Fermenter provides a sterile environment and optimum condition that are important for
growth of micro-organisms and synthesis of desired product.
A fermenter should be constructed in such a way that it can make provisions for
the below activities:
• Sterilization
• Temperature control
• pH control
• Foam control
• Aeration and agitation
• Sampling point
• Inoculation points for micro-organisms, media and supplements
• Drainage point for drainage of fermented media
• Harvesting of product
• Cleaning
• Facility of providing hot, cold water and sterile compressed air.

Major parts of fermenter and their function.

1. Material used for fermentor

2. Impellers
3. Baffles
4. Inoculation port
5. Sparger
6. Sampling point
7. pH control device
8. Temperature control system
9. Foam control device
10. Bottom drainage system

Material used for fermenter

The material used for designing of a fermentor should have some important functions.
• It should not be corrosive
• It should not add any toxic substances to the fermentation media.
• It should tolerate steam sterilization process.
• It should be able to tolerate high pressure and resist pH changes.
A fermentor should provide the facility to control and monitor various parameters for a
successful fermentation process.
Impellers
• Impellers are an agitation device. They are mounted on the shaft and introduced
in the fermentor through its lid.
• They are made up of impeller blades and the position may vary according to its
need.
• These impellers or blades are attach to a motor on lid.
• The important function of an impeller is to mix micro-organisms, media and
oxygen uniformly.
• Impeller blades reduce the size of air bubbles and distributes these air bubbles
uniformly into the fermentation media.
• Impellers also helps in breaking foam bubbles in the head space of fermentor.
This foam formed during fermentation process can cause contamination problem
and this problem is avoided by the use of impellers.
Baffles
• Baffles are mounted on the walls of a fermentor.
• The important function of baffles is to break the vortex formed during agitation
process by the impellers.
• If this vortex is not broken, the fermentation media may spill out of fermentor
and this may result in contamination as well as can lead to different problems. So
it is important to break the vortex formed by using a barrier.
• Baffles acts as a barrier which break the vortex.
Inoculation Port
• Inoculation port is a device from which fermentation media, inoculum and
substrate are added in the fermentation tank.
• Care should be taken that the port provides aseptic transfer.
• The inoculation port should be easy to sterilize.
Spargers
• A Sparger is an aeration system through which sterile air is introduced in the
fermentation tank.
• Spargers are located at the bottom of the fermentation tank.
• Glass wool filters are used in a sparger for sterilization of air and other gases.
• The sparger pipes contain small holes of about 5-10 mm. Through these small
holes pressurized air is released in the aqueous fermentation media.
• The air released is in the form of tiny air bubbles. These air bubbles helps in
mixing of media.
Sampling point
• Sampling point is used for time to time withdrawal of samples to monitor
fermentation process and quality control.
• This sampling point should provide aseptic withdrawal of sample.
pH Control device
• The pH controlling device checks the pH of media at specific intervals of time and
adjusts the pH to its optimum level by addition of acids or alkalis.
• Maintaining pH to its optimum level is very important for growth of micro-
organism to obtain a desired product.
Temperature control
• Temperature control device generally contains a thermometer and cooling coils
or jackets around fermentor.
• During the fermentation process, various reactions take place in the fermentor.
Heat is generated and released in the fermentation media. This increase in
temperature is detrimental to the growth of micro-organisms, which may slow
down the fermentation process.
• So, it is necessary to control this rise in temperature. This is done by passing cool
water through the coils or jackets present around fermentor.
Foam controlling device
• A Foam controlling device is placed on the top of fermentor with a inlet into
fermentor. This device contains a small tank containing anti-foaming agent.
• Foam is generated during fermentation. It is necessary to remove or neutralize
this foam with the help of anti-foaming agents, lest the media may spill out of
fermentor and lead into contamination and a mess.
Bottom drainage system
• It is an aseptic outlet present at the bottom of fermentor for removal of
fermented media and products formed.