BTH – 401: BIOPROCESS ENGINEERING

ASSIGNMENT ON

Downstream processing: Chromatography,

Drying Devices (Lyophilization and spray
dry technology), Crystallization, Biosensors
(construction & applications)

Presented to:
Dr. Santosh Anand
Department of Biotechnology
RCASC

Submitted by:
Melissa Simte
BT180218
th
M.Sc. Biotechnology 4 semester

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 INDEX

CONTENTS Page no.

Introduction 3-4

Chromatography & Types 4-8

Drying devices (Lyophilization & 9-11

drying devices)

Crystallization 12

Biosensors 13-14

References 15

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 ÞIntroduction
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 human growth hormone),
antibodies (e.g. infliximab and abciximab) and vaccines; antibodies and
enzymes used in diagnostics; industrial enzymes; and natural fragrance
and flavor compounds. Downstream processing is usually considered a
specialized field in biochemical engineering.
Stages in Downstream Processing:
• Stage # 1. Solid-Liquid Separation
• Stage # 2. Release of Intracellular Products
• Stage # 3. Concentration
• Stage # 4. Purification by Chromatography
• Stage # 5. Formulation

STEPS IN DOWNSTREAM PROCESSING

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 The purification of the product, the so-called downstream
process (DSP), tends to be one of the costliest aspects of modern
bioprocessing, especially in the case of proteins. In such cases,
chromatography is still the major tool on all levels of the DSP from the
first capture to the final polishing step.

ÞChromatography:

‘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. There are many types of chromatography,
they are:
1. Gel- filtration Chromatography
2. Affinity Chromatography
3. Ion- exchange Chromatography
4. Hydrophobic interaction
The principles of each types are briefly described below:

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 • 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.

• Ion-exchange chromatography:
Ion-exchange chromatography is a type of chromatography that
separates analytes based on charge. A column is used that is filled
with a charged stationary phase on a solid support, called an ion-
exchange resin. Strong cation-exchange chromatography
preferentially separates out cations using a negatively charged resin
& strong anionic- exchange separates out anions using a positively
charged resin.

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 Ion-exchange chromatography is a two-step process:
1. The sample is loaded onto the column in a loading buffer, and
the sample binds to the column according to its charges. Thus,
the samples of opposite polarity to the resin are strongly bound
and the molecules that are not charged or are of the same
charge are not bound.
2. The samples bound to the column are eluded using a salt
gradient where the amount of salt in the buffer is gradually
increased. The eluded sample is collected, and the sample of
interest is recovered.

• Affinity chromatography:

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 sample is passed

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through the column and the specific sample binds to the ligand
like lock & key.

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.

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 • Hydrophobic interaction chromatography (HIC):

This is based on the principle of weak hydrophobic interactions

between the hydrophobic ligands (alkyl, aryl side chains on
matrix column) 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.
It is more widely used as it is less denaturing, and
it preserves the biological activity of the protein. It is often used in
combination with techniques such as ion exchange or gel filtration
chromatography.

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 ÞDrying devices:
(Lyophilization & spray dry technology)

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. Based on the method of heat transfer, drying
devices may be categorized as contact, convection, radiation
dryers. These three types of dryers are commercially available.

• 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.

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There are different types of spray dryers:

1. Co-current flow dryer

2. Counter current flow dryer
3. Mixed flow dryer
4. Open cycle dryer
5. Closed cycle dryer &Semi-closed cycle dryer
6. Single stage dryer & Two stage dryer
7. Vertical dryer
8. Horizontal dryer

• Lyophilization:

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 (solid

to vapor state) 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 to stop all of the biological
activity.

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• Freezing: Decreasing the temperature of the product to
-50ºc with low vacuum.
• Primary drying (Sublimation): Introduction of heat to
the product to remove moisture.
• Secondary drying (Desorption): Increasing of
temperature to 50ºC – 60ºC and lowering of vacuum to
remove bound water.

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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.

• The first step of crystallization is formation of nucleation where

crystals are formed when particles gather into clusters. There
are two different nucleation formations – primary and
secondary.

• The second step of crystallization is crystal growth where

nucleus size increases after the critical cluster size is achieved.

Mechanism and processes:

The steps involved in crystallization are:

• Super saturation of the solution using one of the methods like
heating, cooling & salting.
• Nucleation, where the atoms, molecules or ions come closer to
each other and form aggregates called clusters, these clusters
join to form embryo and finally these embryos join to form
nuclei from which crystals are formed.
• Crystal growth occurs once nuclei formation stops.

Applications of crystallization:

1. Purification of drugs
2. Preparation of organic & inorganic API
3. Improved bioavailability of drug

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 Þ Biosensors:
(Construction & Applications)

A biosensor is an analytical device ,used for the detection of an

analyte that combines a biological component with a physiochemical
detector.

The most common types biosensors are:

• electrochemical biosensors.
• optical biosensors.
• electronic biosensors.
• piezoelectric biosensors.
• gravimetric biosensors.
• pyroelectric biosensors.

Main Components of a Biosensor

The block diagram of the biosensor includes three segments namely,
sensor, transducer, and associated electrons. In the first segment,
the sensor is a responsive biological part, the second segment is the
detector part that changes the resulting signal from the contact of the
analyte and for the results it displays in an accessible way. The final
section comprises of an amplifier which is known as signal
conditioning circuit, a display unit as well as the processor.

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Þ Applications:

1. Biological devices like glucose monitoring, pregnancy test.

2. Environmental applications, for testing river water contaminants
& pesticides.
3. Drug discovery & evaluation of new biological activities of new
compounds.
4. Remote sensing of air-borne bacteria or remote sensing of water
quality.

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 References:
• elprocus.com
• nature.com
• link.springer.com
• ncbi.nlm.nih.gov
• biologydiscussion.com

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