Downstream processing

APARNA
JOSHI
What is downstream processing ?
 The various processes used for actual recovery and
purification of biosynthetic products from a
fermentation or any other industrial process together
constitute a downstream processing.
 The various processes used for actual recovery and
purification.
 The purification of biosynthetic products from a
fermentation or any other industrial process together
constitute a downstream processing.
What is downstream processing ?

 Downstream – ‘after the fermentation process’

 Primary ‘unit operations’ of Downstream Processing
Cell recovery/removal
Centrifugation
Dewatering
Ultrafiltration
Precipitation
Spray drying
What is downstream processing ?

 Secondary ‘unit operations’

 Protein purification
 Adsorption chromatography
 Gel permeation chromatography
 Protein processing
 Immobilization
 Beading/Prilling
 Protein packaging
 Sterilization
 Bottling etc.
 Introduction
 Thus downstream processing includes various stages of
processing that occur after the completion of the fermentation
or bioconversion stage, including separation, purification,
and packaging of the product.
Types of products
Introduction
 Downstream processing refers to the recovery and purification of
biosynthetic products, particularly pharmaceuticals, from natural
sources such as animal or plant tissue or fermentation broth,
including the recycling of salvageable components and the proper
treatment and disposal of waste.
 It is an essential step in the manufacture of pharmaceuticals such as
antibiotics, hormones (e.g. insulin and humans growth hormone),
antibodies (e.g. infliximab and abciximab) and vaccines; antibodies
and enzymes used in diagnostics; industrial enzymes; and natural
fragrance and flavor compounds.
Stages in Downstream Processing
Stages in Downstream Processing
Stages in Downstream Processing
Stages in Downstream Processing
 1. Removal of Insolubles
 2. Product Isolation
 3. Product Purification
 4. Product Polishing
 A few product recovery methods may be considered to combine two or
more stages.
 For example, expanded bed adsorption accomplishes removal of insoluble
and product isolation in a single step.
 Affinity chromatography often isolates and purifies in a single step .
Steps involved in downstream processing
Steps involved in downstream processing
 1. Removal of Insolubles
 Capture of the product as a solute in a particulate-free liquid
 Example :-Separation of cells, cell debris or other particulate matter
from fermentation broth containing an
antibiotic.
 Typical Operations
 Filtration
 Centrifugation
 Flocculation
 Floatation
Filtration
 It is a mechanical operation used for the separation of solids from fluids (liquids or
gases) by interposing a medium to fluid flow through which the fluid can pass, but the
solids in the fluid are retained.
 It depends on
 Size and shape of solid particles.
 Solid : liquid ratio
 Scale of operation
 Aseptic condition
 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
Types of Filtration Processes
 Filtration is used at various stages of the downstream processing in the
bioreactor harvest as well as processing of purified products.
 Several filtration processes are used.
 The most common ones are-
 1. Microfiltration - used at the start of the downstream process to
clarify the feed
 2. Ultrafiltration – used between chromatography steps to concentrate
the product and change the buffer conditions
 3. Reverse Osmosis- use of pressure for osmosis
Types of filters
Types of filters
  The basic requirements for filtration equipment are:
Filtr
 mechanical support for the filter medium
ation
 flow accesses to and from the filter medium
 provision for removing excess filter cake.
 In some instances, washing of the filter cake to remove traces
of the solution may be necessary.
 Pressure can be provided on the upstream side of the filter, or
a vacuum can be drawn downstream, or both can be used to
drive the wash fluid through.
Filtration equipments
Filtration-- plate and frame filter press
 In the plate and frame filter press, a cloth or mesh is spread out over
plates which support the cloth along ridges but at the same time
leave a free area, as large as possible, below the cloth for the flow of
the filtrate.
 The plates with their filter cloths may be horizontal, but they are
usually hung vertically with a number of plates operated in parallel
to give sufficient area. that
Filtration-- plate and frame filter press

 In the early stages of the filtration cycle, the pressure drop across
the cloth is small and filtration proceeds at more or less a
constant rate.
 As the cake increases, the process becomes more and more a
constant-pressure one and this is the case throughout most of the
cycle.
 When the available space between successive frames is filled
with cake, the press has to be dismantled and the cake scraped
off and cleaned, after which a further cycle can be initiated.
Filtration-- plate and frame filter press

 The plate and frame filter press is cheap but it is difficult to mechanize
to any great extent.
 Filtration can be done under pressure or vacuum.
 The advantage of vacuum filtration is that the pressure drop can be
maintained whilst the cake is still under atmospheric pressure and so
can be removed easily.
 The disadvantages are the greater costs of maintaining a given pressure
drop by applying a vacuum and the limitation on the vacuum to about
80 kPa maximum
plate and frame filter press
rotary filters
In rotary filters, the flow passes through a rotating cylindrical cloth
from which the filter cake can be continuously scraped.
 Either pressure or vacuum can provide the driving force, but a
particularly useful form is the rotary vacuum filter.
Centrifugal Filters

 Centrifugal force is used to provide the driving force in some

filters.
 These machines are really centrifuges fitted with a perforated
bowl that may also have filter cloth on it.
 Liquid is fed into the interior of the bowl and under the
centrifugal forces, it passes out through the filter material.
Cross flow filters

 In this method, the feed flow is driven within the filter in

downward spiralling direction.
 When flow is parallel to the filter surface, it is being
pressurized against the filter surface to obtain the filtrate.
Cross flow filters
Centrifugation
 Centrifugation is used to separate particles of 100 – 0.1 micrometer
from liquid by gravitational forces.
 It depends on particles size , density difference between the cells
and the broth and broth viscosity.
 Use of the centrifugal force for the separation of mixtures
 More-dense components migrate away from the axis of the
centrifuge
 Less-dense components migrate towards the axis.
 Types of centrifuges used are Tubular bowl centrifuge,
multichamber centrifuge , disc bowl centrifuge etc.
Centrifugation
 Centrifugal force is used to provide the driving force in some filters.
 These machines are really centrifuges fitted with a perforated bowl that
may also have filter cloth on it.
 Liquid is fed into the interior of
the bowl and under the
centrifugal forces, it passes
out through the filter material.
 Centrifuge equipment
 1. Tubular centrifuge
 The bowl is tall and has a narrow diameter
 Such centrifuge, known as super-centrifuges,
develop a force about 13000 times the force
of gravity.
 Some narrow, centrifuges. Having a diameter
of 75 mm and very high speeds or so rev/min,
are known as ultracentrifuges
 These centrifuges are often used to
separate liquid-liquid emulsions
 Centrifuge equipment
 2. Disk bowl centrifuge
 The feed enters the actual compartment at the bottom and travels upward
through vertically spaced feed holes, filling the spaces between the disks .
 The holes divide the vertical assembly into an inner section, where mostly
light liquid is present, and an outer section, where mainly heavy liquid is
present.
 The heavy liquid flows beneath the
underside of a disk to the
periphery of the bowl .
Centrifuge equipment

 The light liquid flows over the upper side of the disks and toward
the inner outlet
 Any small amount of heavy solids is thrown outer wall
 Periodic cleaning is required to remove solids deposited
 Disk bowl centrifuges are used in starch-gluten separation,
concentration of rubber latex, and cream separation
Centrifuge equipment
 3.Decanter centrifuge
 Only sedimentation centrifuge designed to handle more solid
concentration in feed suspension.
 Also obtains good degree of clarification of liquid conc.
 Itconsists of a horizontal, cylindrical bowl rotating at a high speed
within a helical extraction screw.
Sedimentation
 Sedimentation
 It is applicable only for large particles greater than 100 micro
meter flocs.
 It is a slow process and takes ~3 hours.
 It is used in processes like activated sludge effluent
treatment.
 It’s a free-settling process that depends only on gravity.
 Particle settling is a high particle density
suspension(hindered settling).
Flocculation
 Process where a solute comes out of solution in the form of
flocs or flakes.
 Particlesfiner than 0.1 µm in water remain continuously in
motion due to electrostatic charge which causes them to repel
each other
 Once their electrostatic charge is neutralized (use of coagulant)
the finer particles start to collide and combine together .
 These larger and heavier particles are called flocs.
■ 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.
■ Sometimes flocculating agents are also used to achieve
appropriate flocculation.
■ Some flocculating agents are – inorganic salts,
 organic polyelectrolyte,
 mineral hydrocolloid.

 Floatation
■ When gas is introduced into the liquid broth, it
forms bubbles.
■ The cells and other solid particles get absorbed by
gas bubbles.
■ These bubbles rise to the foam layer which can be
collected and removed.
■ Certain substances called collector substances are
used to facilitate stable foam formation.
■ Collector substances used are like – long chain
 fatty acids
- amines
2. Product Isolation
 Product Isolation
 Removal of those components whose properties vary markedly
from that of the desired product.
 Water is the chief impurity
 a) Isolation steps are designed to remove it ( i.e. dialysis)
 b) Reducing the volume
 c) Concentrating the product.
 d) Liquid –liquid extraction, adsorption, ultrafiltration, and
precipitation are some of the unit operations involved.
 2. Product Isolation-Liquid -Liquid extraction

 Liquid -Liquid extraction

 It is a separation process that takes the advantage of the relative
solubility of solute in immiscible solvents.
 Solute is dissolved more readily and becomes more concentrated
in the solvent in which it has a higher solubility.
 A partial separation occurs when a number of solutes have
different relative solubility in the two solvents used.
 Solvent should be non toxic, selective, inexpensive and
immiscible with broth and should have a high distribution
coefficient for the product.
2. Product Isolation-Liquid -Liquid extraction

 Liquid -liquid extraction (LLE) is the process of separating a liquid

mixture of components where liquid solvents are used followed by
diluting one or more components of the initial mixture.
 This downstream process is significantly useful in Bioprocess
technology.
  This unit process requires knowledge of phase behaviour and
physicochemical characterization of different compounds. Liquid-
liquid
2. Product Isolation-Liquid -Liquid extraction

 In liquid-liquid extraction, components in the fed material,

consisting of liquid phases are separated when third liquid also
known as solvent is added to the process.
 By adding this new component which is insoluble in the feed,
a new phase is formed.
 The component that is more important during the extraction or
which is the desired component to be extracted during the
process is transferred to extract.
2. Product Isolation-Liquid -Liquid extraction

 The extraction is carried out in two ways of mixing; counter

current and co-current mixing.
 The co-current flow is limited to one stage per extraction,
whereas, counter current is controlled as multi stages per unit.
  Depending on the density of the solvent to the carrier liquid
the counter-current extraction can be carried out on two ways
  If the solvent is less dense than carrier liquid, solvent is fed
from the bottom.
  The reverse phenomenon happens if the solvent is denser
than the carrier liquid.
Applications of Liquid Liquid Extraction

 1. Fermentation And Algae Broth

 2. Removal Of High Organic Wastes From Wastewater
 3. Removal Of Carboxylic Acid
 4. Protein Separation And Purification
 5. Essential Oil Extraction
 6. Food Industry Application
2. Product Isolation- Adsorption

 Adsorption
 It is a surface phenomenon
 It is the binding of molecules to the surface and different from
absorption.
 The binding to the surface is weak and reversible.
 Compounds containing chromogenic group are usually
strongly adsorbed on activated carbon.
 Common adsorbent used are activated carbon , silica gel ,
alumina because they present enormous surface areas per unit
weight.
 2. Product Isolation- Adsorption
 Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or
dissolved solid to a surface.
 This process creates a film of the adsorbate on the surface of the adsorbent.
 This process differs from absorption, in which a fluid (the absorbate) is
dissolved by or permeates a liquid or solid (the absorbent), respectively.
 Adsorption is a surface phenomenon, while absorption involves the whole
volume of the material.
 The term sorption encompasses both processes, while desorption is the reverse
of it.
 The separation is based on the interaction of the adsorbate with the adsorbent.
 The adsorbent is the surface and adsorbate is the molecules of interest which are
getting adsorbed on the adsorbent.
 2. Product Isolation - Ultrafiltration

 Ultrafiltration
 It is basically a pressure-driven separation process.
 The operating pressure is usually between 0.1 and 1 MPa.
 Ultrafiltration is governed by a screening principle and dependent on particle size.
 Ultrafiltration 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.
 2. Product Isolation - Ultrafiltration
 Ultrafiltration (UF) is a pressure-driven barrier to suspended solids, bacteria, viruses,
endotoxins and other pathogens to produce water with very high purity and low silt
density.
 Ultrafiltration (UF) is a variety of membrane filtration in which hydrostatic pressure
forces a liquid against a semi permeable membrane.
 The variety of membrane filtration in which forces like pressure or concentration
gradients lead to a separation through a semipermeable membrane.
 Suspended solids and solutes of high molecular weight are retained in them called
retentate, while water and low molecular weight solutes pass through the membrane in
the permeate (filtrate).
 This separation process is used in industry and research for purifying and concentrating
macromolecular (103 - 106 Da) solutions, especially protein solutions.
2. Product Isolation - Precipitation

 It is the most commonly used technique in industry for the concentration of

macromolecules such as proteins and polysaccharides .
 It can also be employed for the removal of certain unwanted by products 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.
 Precipitation of protein is widely used in downstream processing in order to
concentrate proteins and purify them from various contaminants.
2. Product Isolation - Precipitation

 Neutral salts, organic salts, alteration in temperature and pH are used in

precipitation.
 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.
 Protein precipitation can be – non-specific protein precipitation - protein specific
precipitation Protein specific precipitation – e.g.- affinity precipitation - ligand
precipitation
2. Product Isolation - Precipitation

 METHODS OF PRECIPITATION
 Salting out
 Isoelectric precipitation
 Precipitation with miscible solvents
 Non-ionic hydrophilic polymers Polymers such as dextrans and polyethylene
glycol
 Flocculation by pyro electrolytes Alginate, carboxy methyl cellulose, tannic
acid polyacrylic acid and phosphatases are used
 Polyvalent metallic ions Ca2+, Mg2+, Mn2+ are used
 Increase in temperature
 Change in pH
2. Product Isolation - Precipitation

 Precipitation of protein is widely used in downstream

processing in order to concentrate proteins and purify
them from various contaminants.
 Protein precipitation can be –
 non-specific protein precipitation –
 protein specific precipitation–
 e.g.- affinity precipitation - ligand precipitation
2. Product Isolation - Precipitation
 AFFINITY PRECIPITATION
 In affinity precipitation, the protein is free in solution,
rather than bound to an insoluble support.
 Ligand binding gives rise to the precipitation of the protein
from the solution, which is then followed by
centrifugation.
 The pellet contains the protein of interest and the ligand,
whereas the other components of the mixture remain in the
supernatant, allowing easy separation.
3. Product Purification

 This can be achieved by

Gel filtration chromatography
Ion exchange chromatography
Affinity chromatography
Hydrophobic interactions.
3. Product Purification
3. Product Purification
 CHROMATOGRAPHY
 ‘Chromatography’ is an analytical technique commonly used for separating a mixture
of chemical substances into its individual components, so that the individual
components can be thoroughly analyzed.
 The mixture is dissolved in a fluid called the mobile phase, which carries it through a
structure holding another material called the stationary phase.
 Chromatography is used in downstream processing to effectively purify the
biological products (proteins, pharmaceuticals, diagnostic compounds and research
materials)
 There are many types of chromatography e.g., liquid chromatography, gas
chromatography, ion-exchange chromatography, affinity chromatography, but all of
these employ the same basic principles.
 3. Product Purification

 PRINCIPLE
 Chromatography is based on the principle of separation of compounds into different
bands (color graphs) and then identification of those bands.
 The preferential separation is done due to differential affinities of compounds towards
stationary and mobile phase.
 After separation of the compounds, they are identified by suitable detection methods.
 The differences in affinities arise due to relative adsorption or partition coefficient in
between components towards both the phases.
 Gel-filtration chromatography
 This is also referred to as size-exclusion chromatography. In this technique, the
separation of molecules is based on the size, shape and molecular weight.
 The sponge-like gel beads with pores serve as molecular sieves for separation of
smaller and bigger molecules.
 A solution mixture containing molecules of different sizes (e.g. different proteins)
is applied to the column and eluted.
 The smaller molecules enter the gel beads through their pores and get trapped.
 On the other hand, the larger molecules cannot pass through the pores and therefore
come out first with the mobile liquid .
 At the industrial scale, gel-filtration is particularly useful to remove salts and low
molecular weight compounds from high molecular weight products.
Gel-filtration chromatography
 Ion-exchange chromatography
 It involves the separation of molecules based on their surface charges.
 Ion-exchangers are of two types (cation- exchangers which have negatively charged groups
like carboxymethyl and sulfonate, and anion- exchangers with positively charged groups
like diethylaminoethyl (DEAE).
 The most commonly used cation-exchangers are Dowex HCR and Amberlite IR, the anion-
exchangers are Dowex SAR and Amberlite IRA.
 In ion-exchange chromatography, the pH of the medium is very crucial, since the net charge
varies with pH.
 In other words, the pH determines the effective charge on both the target molecule and the
ion-exchanger.
 The ionic bound molecules can be eluted from the matrix by changing the pH of the eluant
or by increasing the concentration of salt solution.
 Ion-exchange chromatography is useful for the purification of antibiotics, besides the
purification of proteins.
 Affinity chromatography
 This is an elegant method for the purification of proteins from a complex mixture.
 Affinity chromatography is based on an interaction of a protein with an immobilized
ligand.
 The ligand can be a specific antibody, substrate, substrate analogue or an inhibitor.
 The immobilized ligand on a solid matrix can be effectively used to fish out
complementary structures.
 The protein bound to the ligand can be eluted by reducing their interaction.
 This can be achieved by changing the pH of the buffer, altering the ionic strength or by
using another free ligand molecule.
 The fresh ligand used has to be removed in the subsequent steps
4. Product Polishing
• Final processing steps which end with
packaging of the product in a form that is
stable, easily transportable, and
convenient.

• Crystallization, desiccation, lyophilization,

and spray drying are typical unit
operations
 Lyophilization
Crystallization
• freezing the material
• process of formation of solid
crystals precipitating from a
solution, melt or more rarely
• reducing the surrounding
deposited directly from a gas. pressure and adding enough
heat to allow the frozen water
• chemical solid-liquid in the material to sublime
separation technique, in directly from the solid phase
which mass transfer of a
solute from the liquid
to gas.
solution to a pure solid
crystalline phase occurs.
Lyophilization
A stabilizing process in which a substance is first
frozen and then the quantity of the solvent is reduced,
first by sublimation (primary drying stage) and then
desorption (secondary drying stage) to values that will
no longer support biological activity or chemical
reactions.
 It is a drying process applicable to the
manufacture of certain pharmaceuticals and
biologicals that are thermolabile or otherwise
unstable in aqueous solutions for prolonged storage
periods, but that are stable in the dry state
Lyophilization- Principle
 Lyophilization is based on a simple principle of physics
called “SUBLIMATION”. Sublimation is the process of the
transition of a substance from a solid to a vapor state
without passing through an intermediate liquid phase.
 Lyophilization is performed at temperature and pressure
conditions below the triple point, to enable sublimation of
ice.
 The material to be dried is first frozen and then subjected
under a high vacuum to heat (by conduction or radiation or
by both) so that frozen liquid sublimes leaving only solid,
dried components of the original liquid.
 A
Fundamental process steps are:
1.Freezing: the product is frozen. This provides a necessary
condition for low-temperature
2.Vacuum: after freezing, the product is placed under vacuum. This
enables the frozen solvent in the product to vaporize without passing
through the liquid phase, a process known as SUBLIMATION.
3.Heat: Heat is applied to the frozen product to accelerate
sublimation.
4.Condensation: Low-temperature condenser plates remove the
vaporized solvent from the vacuum chamber by converting it back to a
solid. This completes the separation process.
5.Resulting product has a very large surface area thus promoting
rapid dissolution of dried product.
 Applications
⮚ PHARMACEUTICAL AND BIOTECHNOLOGY
1.Pharmaceutical companies often use freeze-drying to increase the shelf life of
products,
such as vaccines and other injectables.
2. By removing the water from the material and sealing the material in a vial, the material
can be easily stored, shipped, and later reconstituted to its original form for injection.
⮚ FOOD INDUSTRY
1.Freeze-drying is used to preserve food and make it very lightweight.
2. The process has been popularized in the form of freeze-dried ice cream, an example of
astronaut food.
⮚ TECHNOLOGICAL INDUSTRY
1.In chemical synthesis, products are often freeze-dried to make them more stable, or
easier to dissolve in water for subsequent use.
2. In bio-separations, freeze-drying can be used also as a late-stage purification procedure,
because it can effectively remove solvents.
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.
 It is
a kind of continuous atmospheric dryer which can
be used to dry materials such as fuel, intermediates,
soap powder, or inorganic salts, etc.
 Applications
 Spray dryers are used for the drying of liquid materials like emulsion,
suspension,
 solution, slurries, thin pastes, etc.
  Spray drying can be used to dry materials that are sensitive to
heat or oxidation without degrading them, even when high temperature
air is employed.
  The liquid feed is dispersed into droplets, which are dried in
seconds because of their high surface area and intimate contact with
the drying gas.
  The product is kept cool by the vaporization of the enveloping
liquid, and the dried product is kept from overheating by rapid removal
from the drying zone.
  The improvement in flow and reduction of air entrapment makes
the spray-dried material suitable for use in the manufacturing of tablets
and capsules. high-temperature