Downstream processing refers to the recovery and purification of products from natural sources like plant/animal tissue or fermentation broth. It involves various unit operations like filtration, centrifugation, chromatography, precipitation etc. to separate and purify the product of interest. Filtration is commonly used to separate biomass from culture filtrate using different types of filters. Cell disruption techniques are employed to break open cells and release intracellular products. Purification methods like liquid-liquid extraction, precipitation, solid-liquid separation, and chromatography are then used to further purify the product.
2. 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.
3. 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.
4. 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.
5. 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
6. 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
7. Filtration is used at various stages of the downstream
processing in the bioreactor harvest as well as
processing of purified products.
Several filtration process are used. 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
8. Cell disruption is an essential part of biotechnology and the
downstream processes related to the manufacturing of
biological products.
The disruption of cells is necessary for the extraction and
retrieval of the desired products, as cell disruption
significantly enhances the recovery of biological products.
10. Bead mill- The main principle requires a
jacketed grinding chamber with a rotating
shaft, running in its center.
Agitators are fitted with the shaft, and
provide kinetic energy to the small beads
that are present in the chamber. That makes
the beads collide with each other causing
disruption
11. Ultrasound- Ultrasonic disruption
is caused by ultrasonic vibrators
that produce a high frequency
sound with a wave density of
about 20 kHz/s.
A transducer then converts the
waves into mechanical
oscillations through a titanium
probe, which is immersed into
the cell suspension. Such a
method is used for both bacterial
and fungal cell disruption.
12. French press and high pressure
homogenizer- In a French press, or
high pressure homogenization, the
cell suspension is drawn through a
valve into a pump cylinder
Then it is forced under pressure of
up to 1500 bar, through a narrow
annular gap and discharge valve,
where the pressure drops to
atmospheric. Cell disruption is
achieved due to the sudden drop in
pressure upon the discharge,
causing the cells to explode.
13. Thermolysis- use of heat to disrupt the cell membrane.
Periplasmic proteins in G(-) bacteria are released when the
cells are heated up to 50ºC.
Cytoplasmic proteins can be released from E.coli within
10min at 90 ºC.
Freezing and thawing of a cell slurry can cause the cells to
burst due to the formation and melting of ice crystals.
14. Decompression- During explosive decompression, the cell
suspension is mixed with pressurized subcritical gas for a
specified time, depending on the cell type.
The gas enters the cell and expends on release, causing the
cell to burst. Gases like carbon dioxide can be used
15. Osmotic shocks- here
cells are first exposed to
either high or low salt
concentration.Then the
conditions are quickly
changed to opposite
conditions which leads to
osmotic pressure and cell
lysis.
16. In addition to physical and mechanical methods, several
chemical methods for cell disruption exist. These methods
rely on utilization of chemical substances or enzymes in
disruption process.
The mechanisms of actions are multiple, but the most widely
used methods act by destroying the cell wall by enzymes,
osmotic pressure, or by interfering or precipitating cell wall
proteins.
17. Detergents-
Detergents that are
used for disrupting
cells are divided
into anionic,
cationic and non-
ionic detergents.
The common thing
for all detergents is
that they directly
damage the cell
wall or membrane,
and this will lead to
release of
intracellular
content.
18. Solvents- Solvents which can be used for cell
lysis include for example some alcohols,
dimethyl sulfoxide, methyl ethyl ketone or
toluene.
These solvents extract cell wall’s lipid
components which leads to release of
intracellular components.
19. Enzymes- Use of
enzymes
depending on the
cell wall
composition
For example,
lysozyme is
commonly used
enzyme to digest
cell wall of gram
positive bacteria.
Lysozyme
hydrolyzes β-1-4-
glucosidic bonds in
the peptidoglycan
20. Liquid -liquid extraction (LLE) is the process
of separation of a liquid mixture of
components where liquid solvents are used
followed by dilution of one or more
components of the initial mixture.
This downstream process is significantly
useful in Bioprocess technology.
This is a unit process which requires the
knowledge of phase behavior and
physicochemical characterization of different
compounds.
21. 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 which is more important
during the extraction or which is the desired
component to be extracted during the
process is transferred to extract.
22.
23. The extraction is carried out in two ways of
mixing; countercurrent 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.
24.
25. 1. FERMENTAION AND ALGAE BROTH
2. REMOVAL OF HIGH ORGANIC WASTES FROM
WASTEWATER
3. REMOVAL OF CARBOXYLIC ACID
4. PROTEIN SEPERATION AND PURIFICATION
5. ESSENTIAL OIL EXTRACTION
6. FOOD INDUSTRY APPLICATION
26. PRECIPITATION
■ 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.
■ It is the most commonly used technique in
industry for the concentration of
macromolecules.
■ It can also be employed for the removal of
certain unwanted by-products.
■ Neutral salts, organic salts, alteration in
temperature and pH are used in precipitation.
27. ■ 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
Protein specific precipitation – e.g.- affinity precipitation
- ligand precipitation
28. 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
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.
29. METHODS OF PRECIPITATION
■ Salting out
■ Isoelectric precipitation
■ Precipitation with miscible solvents
■ Non-ionic hydrophilic polymers
Polymers such as dextrans and
polyethylene glycol
■ Flocculation by pyroelectrolytes
Alginate, carboxymethylcellulose, tannic acid
polyacrylic acid and phosphatases are used
■ Polyvalent metallic ions
Ca2+, Mg2+, Mn2+ are used
■ Increase in temperature
■ Change in pH
30. SOLID-LIQUID SEPARATION
■ the separation of cells from the culture broth,
removal of cell debris, collection of protein
precipitate, etc.
■ The term harvesting of microbial cells are used
for the separation of cells from the culture
medium.
■ Several methods used for solid-liquid
separation are –
flotation
flocculation
filtration
centrifugation
31. FLOTATION
■ When gas is introduced into the liquid broth, it
forms bubbles.
■ The cells and other solid particles get absorbed
on gas bubbles.
■ These bubbles rise to the foam layer which can
be collected and removed.
■ Certain substances called as collector
substances are used to facilitate stable foam
formation.
■ Collector substances used are like – long chain
fatty acids
- amines
32. 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.
■ Sometimes flocculating agents are also used to achieve appropriate
flocculation.
■ Some flocculating agents are – inorganic salts,
organic polyelectrolyte,
mineral hydrocolloid.
33. FILTRATION
■ Filtration is the most commonly used technique for separating the
biomass and culture filtrate.
■ The mixture goes through a filter which retains the particles according to
size while allows the passage of fluid through the filter.
■ The efficiency of filtration depends on many factors – size of the
organism,
viscosity of the
medium,
temperature.
■ Several filters are in use like – depth filter
absolute filter
rotary drum vacuum filter
membrane filter
35. CENTRIFUGATION
■ Separation by means of the accelerated gravitational
force achieved by a rapid rotation.
■ Relies on the density difference between the particles
and the surrounding medium.
■ Most effective when the particles to be separated are
large, the liquid viscosity is low and the density
difference between particles and fluid is great.
■ Batch centrifuge is common in the labs but the low
processing capacity limits its use in large scale.
■ Continuous centrifuges are common in large-scale
processing in which the deposited solids are removed
continuously or intermittently.
36. TUBULAR BOWL CENTRIFUGE
■ High speed
■ Length diameter ratio 4.8
■ 15000r.p.m.
■ Used widely in emulsion
■ Used in solid with small amount
■ Can be run in both batch or continuous mode
37. DISC CENTRIFUGE
■ Contain conical sheets of metal (discs) which
are stacked with clearances.
■ Disc size – 0.3mm
■ The discs rotate with the bowl to split the liquid
into thin layers.
■ The slurry is fed through a central tube.
■ The clarified fluid moves upward while the
solids settle at the lower surface.
38. MULTICHAMBER CENTRIFUGE
■ Modification of tubular bowl centrifuge.
■ Consist of several chambers in such a way that
feed flows in a zigzag fashion.
■ Particle size – 0.1 to 200 micrometre diameter.
■ Variation in centrifugal force in different
chambers.
■ Force is higher in the periphery chambers.
■ Smallest particle settle down in the outermost
chamber.
39. SCROLL CENTRIFUGE OR
DECANTER
■ Composed of a rotating horizontal bowl tapered at one end.
■ Used to concentrate fluid with high solid concentration.
■ Solids are deposited on the wall of the bowl.
40. 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.
41. 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.
42. CHROMATOGRAPHIC TECHNIQUES
CHROMATOGRAPHY
GEL – FILTRATION ( size exclusion)
ION EXCHANGE
CHROMATOFOCUSSING
AFFINITY
HYDROPHOBIC INTERACTION
IMMOBILIZED METAL ION-AFFINITY
PRINCIPLE
SIZE AND SHAPE
NET CHARGE
NET CHARGE
BIOLOGICAL AFFINITY AND MOLECULAR
RECOGNITION
POLARITY
METAL ION BINDING
43. APPLICATIONS OF CHROMATOGRAPHY
The chromatographic technique is used for the separation of amino acids,
proteins & carbohydrates.
It is also used for the analysis of drugs,hormones,vitamins .
Helpful for the qualitative & quantitative analysis of complex mixtures.
The technique is also useful for the determination of molecular weight of
proteins
44. DRYING DEVICES
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
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.
45. PRINCIPLE:-
Lyophilization is based on a simple principle of physics called “SUBLIMATION”.
Sublimation is the process of transition of a substance from solid to the 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.
46. Equipment used for Lyophilization – LYOPHILIZER
A lyophilizer consists of a vacuum chamber containing product shelves which are
capable of cooling and heating containers and their contents.
A vacuum pump, a refrigeration unit, which is associated controls are
connected to the vacuum chamber.
47. How does it work
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 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. Resulting product has a
very large surface area thus promoting rapid dissolution of dried product.
49. FREEZING PRIMARY DRYING
STAGE
SECONDARY
DRYING STAGE
PACKING
• The product
must be frozen
to a low enough
temperature to
be completely
solidify and be
adequately pre-
frozen.
• Decrease the
shelf
temperature to
-50⁰c.
• Low temperature
and low
atmospheric
pressure are
maintained
• Formation of ice
crystals occurs.
• Heat is
introduced from
shelf to the
product under
graded control
by electrical
resistance coils
or circulating
silicone.
• The temperature
and pressure
should be below
the triple point
of water i.e.,
0.0098°C and
4.58mmHg.
• Easily removes
moisture up to
98% to 99%.
• The temperature
is raised to 50°C
– 60°C and
vacuum is
lowered about
50mmHg.
• Bound water is
removed.
• Rate of drying is
low.
• It takes about
10-20 hrs
• This process is
called
‘Isothermal
Desorption’ as
the bound water
is desorbed from
the product.
• After drying the
vacuum is
replaced by
filtered dry air
or nitrogen to
establish
atmospheric
pressure
• Vials and bottles
are sealed with
rubber closures
and aluminum
caps
• e.g., penicillin
can be freeze
dried directly in
ampules.
50. 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 forms 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.
51. SPRAY DRY TECHNOLOGY
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.
52. HOW DOES IT WORK?
A spray dryer uses the spray method to transform the material into fog droplets
in order to be dispersed into the hot gas stream.
The material connects with the hot air in a co-current, countercurrent, or
mixed flow manner so that the water can evaporate quickly to achieve the
drying effect.
The spray dryer provides a large surface area for heat and mass transfer by
atomizing the liquid to small droplets. These are sprayed into a stream of hot
air, so that each droplet dries to a solid particle. The drying chamber
resembles the cyclone ensuring good circulation of air, to facilitate heat and
mass transfer, and that dried particles are separated by the centrifugal
53.
54. 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 make the spray-dried
material suitable for use in the manufacturing of tablets and capsules.
56. DEFINITION
▪ Self-contained integrated device that is capable of providing
specific qualitative or semi-quantitative analytical information
using a biological recognition element which is in direct-spatial
contact with a transduction element. (IUPAC,1998)
▪ In simple words, Biosensors detect analytes of interest by
combining a biological component with a physiochemical detector
via electronic signals.
▪ They can be Nano Biosensors, Amperometric Biosensors, Blood
Glucose Biosensors, Quantum Mechanical- Based Biosensors etc.
▪ NOTE- Biosensors ≠ Bioanalytical system
56
59. WORKING PRINCIPLE
▪ Analytes diffuse from the solution to
the surface of the Biosensor.
▪ Analytes react specifically &
efficiently with the Biological
Component of the Biosensor.
▪ This reaction changes the
physiochemical properties of the
Transducer surface.
▪ This leads to a change in the
optical/electronic properties of the
Transducer Surface.
▪ The change in the optical/electronic
properties is measured/converted
into electrical signal, which is
detected.
59
63. Bioreceptor
▪ In a biosensor, the bioreceptor is designed to interact with the specific analyte of
interest to produce an effect measurable by the transducer.
▪ High selectivity for the analyte among a matrix of other chemical or biological
components is a key requirement of the Bioreceptor.
▪ While the type of biomolecule used can vary widely, biosensors can be classified
according to common types Bioreceptor interactions involving: antibody/antigen,
enzymes/ligands, nucleic acids/DNA, cellular structures/cells, or biomimetic materials.
63
64. Surface attachment of biological elements
▪ An important part in a biosensor is to attach the biological
elements (small molecules/protein/cells) to the surface of the
sensor.
▪ The simplest way is to functionalize the surface in order to coat it
with the biological elements.
▪ This can be done by polylysine, aminosilane, epoxysilane or
nitrocellulose in the case of silicon chips/silica glass.
Subsequently, the bound biological agent may be for example
fixed by Layer by layer deposition of alternatively charged polymer
coatings.
64
65. Biotransducer
▪ A Biotransducer is the recognition-transduction component of a
biosensor system. It consists of two intimately coupled parts; a bio-
recognition layer and a physicochemical transducer, which acting
together converts a biochemical signal to an electronic or optical
signal.
▪ As a result of the presence and biochemical action of the analyte
(target of interest), a physico-chemical change is produced within
the biorecognition layer that is measured by the physicochemical
transducer producing a signal that is proportionate to the
concentration of the analyte.
65
66. TYPES OF BIOSENSORS
▪ Biosensors can be classified by their Biotransducer type. The most
common types of Biotransducer used in biosensors are discussed
in the next slides :
66
68. 1. Calorimetric / Thermal Detection Biosensors.
▪ Uses Absorption / Production of Heat.
▪ Many enzyme catalyzed reactions are exothermic, generating heat which may be used
as a basis for measuring the rate of reaction and, hence, the analyte concentration.
This represents the most generally applicable type of biosensor.
▪ Temp. measured by Enzyme Thermistors - The temperature changes are usually
determined by means of thermistors at the entrance and exit of small packed bed
columns containing immobilized enzymes within a constant temperature environment
▪ Uses: Detection of:
(1) Pesticides . (2) Pathogenic Bacteria
68
69. 2. Optical Biosensors
▪ Colorimetric for color - Measures change in Light Adsorption.
▪ Photometric for Light Intensity - Detects the Photon output.
▪ Raman effect
69
70. 3. Resonant Biosensors
▪ An Acoustic Wave Transducer is coupled with Bioelement.
▪ Measures the change in Resonant Frequency.
70
71. 4. Piezoelectric Biosensor
▪ The principle of Piezoelectric Biosensor is used in sound vibrations, hence it is called
acoustic Biosensors. The basics of the Biosensors are formed by the piezoelectric
crystals and the characteristic frequencies are trembling with the crystals of positive
and negative charge. By using the electronic devices we can measure the certain
molecules on the crystal surface and alters the response frequencies using these
crystals we can attaché the inhibitors.
71
72. 5. Electrochemical Biosensors
▪ Electrochemical Biosensor is a simple device. It measures the
measurement of electronic current, ionic or by conductance
changes carried by bio-electrodes.
72
73. i. Conductimetric Biosensors
▪ Measures Electrical Conductance/Resistance of the solution.
▪ Conductance Measurements have relatively Low Sensitivity.
▪ Electrical Field is generated using sinusoidal(ac) voltage, which
helps in minimizing undesirable effects like:
i. Faradaic processes.
ii. Double layer charging &
iii. Concentration polarization
73
74. ii. Blood Glucose Biosensor
▪ The Blood glucose Biosensors are used widely throughout the world for diabetic
patients. It has a single use disposable electrodes with glucose oxide and derivatives
of a mediator (Ferrocene) and the shape of the blood glucose Biosensor looks like a
watch pen. With the help of hydrophilic mesh electrodes are converted.
74
75. iii. Potentiometric Sensors.
▪ Working Principle – When ramp voltage is applied to an electrode
in solution, a current flow occurs because of electrochemical
reactions.
▪ Measured Parameter – Oxidation / reduction Potential of an
Electrochemical run.
Amit Gothe 75
77. CRYSTALLIZATION
▪ Crystallization is a separation
and purification method widely
used for final purification of
components.
▪ Crystallization consists of two
stages: formation of nuclei and
growth of crystals.
▪ For crystallization to occur the
solution should be first
supersaturated.
Amit Gothe 77
78. NUCLEATION
▪ The first step of crystallization is formation of nucleation where
crystals are formed when solute molecules dispersed in the
solvent start to gather into clusters. And became stable under the
current operating condition .
▪ These stable structures together form a nuclei . It is at the stage of
nucleation that atoms arrange in periodic manner to form crystal
structure.
▪ There are two different nucleation formations – primary and
secondary.
Amit Gothe 78
79. Crystal growth
▪ The second step of crystallization is crystal growth where nucleus
size increases after the critical cluster size is achieved.
▪ It is the Growth of nuclei to the next stage .
▪ Crystal growth rate is affected by various physical factors, such as
surface tension of solution, pressure, temperature, relative crystal
velocity in the solution, Reynolds number and other factors
▪ Polymorphism : ability to crystallize with different crystal
structures
Amit Gothe 79
80. Crystallization processes
▪ Influencing factors : temperature and concentration
A) cooling crystallization
B) evaporative crystallization - Generating crystals by evaporating a
solution at constant temperature.Most of the industrial crystallizers
are evaporative.
▪ Example: sodium chloride and sucrose
Amit Gothe 80
81. Applications
▪ Crystallization is an important downstream processing method in
bioprocess technology and in all chemical industry.
▪ Downstream processing can contribute to a large portion of end
product price. Therefore crystallization has an advantage
compared to other solid liquid separation operations such as
distillation since crystallization is a rather energy efficient unit
operation.
▪ In addition the yielded product has very high purity level and
therefore wide scale of end use opportunities.
Amit Gothe 81