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MODULE 5: DOWNSTREAM PROCESSING:
Objectives And Criteria,
Foam Separation,
Precipitation Methods,
Filtration,
Centrifugation,
Cell Disruption Methods,
Liquid Extraction,
Membrane Filtration,
Chromatography,
Drying Devices,
Crystallization.
Solvent Recovery.
Effluent Treatment
Quality Control Of Fermented Products,
Process Economics.
Downstream processing in biotechnology refers to the
purification and recovery of a desired product from a
biotechnological process. This process typically follows
the upstream processes like fermentation or cell culture
where the target product is produced. The objectives and
criteria of downstream processing are crucial for
ensuring the quality, purity, and yield of the final
product. Here are the key objectives and criteria:
 Product
Purification: The primary objective of
downstream processing is to purify the target product
from the complex mixture of biomass, media
components, and other impurities generated during the
upstream process. Purification methods include
separation techniques such as chromatography, filtration,
precipitation, and extraction.
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High Yield: Downstream processing aims to maximize
the yield of the target product while minimizing losses.
High yield ensures that the process is economically
viable and that sufficient quantities of the product are
obtained for commercialization.
 Product Quality: Downstream processing must ensure
that the final product meets predefined quality
standards. This includes criteria such as purity,
potency, stability, and safety. Quality control measures
are implemented throughout the process to monitor
and maintain product quality.
 Efficiency and Cost-effectiveness: The process should
be efficient in terms of both time and resources.
Efficient downstream processing minimizes processing
time, reduces the consumption of reagents and
consumables, and lowers overall production costs.
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Scalability: Downstream processing should be scalable to
accommodate variations in production scale. The process must be
adaptable to different batch sizes without compromising product
quality or yield.
Process Robustness: The downstream process should be robust
and capable of handling variations in raw materials, operating
conditions, and environmental factors. Robust processes are less
susceptible to fluctuations and are more reliable for consistent
product quality and yield.
Regulatory Compliance: Downstream processing must comply
with regulatory requirements and standards set by regulatory
authorities such as the FDA (Food and Drug Administration) or
EMA (European Medicines Agency). Compliance ensures that the
final product is safe, efficacious, and meets all necessary regulatory
guidelines.
Environmental Impact: Downstream processing should minimize
its environmental footprint by optimizing resource utilization,
reducing waste generation, and implementing sustainable practices
wherever possible.
The objectives and criteria of downstream processing aim to ensure
the efficient and effective purification of biotechnological products
while maintaining high quality, yield, and compliance with
regulatory standards.
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Foam separation, also known as foam fractionation or
foam flotation, is a technique used in downstream
processing in industries such as pharmaceuticals,
biotechnology, food, and environmental engineering. It is
employed to separate and purify proteins, enzymes, and
other biomolecules from complex mixtures. Foam
separation exploits the differences in surface properties
and interfacial tensions between components in a
solution to selectively concentrate or remove target
molecules.
Here's how foam separation typically works:
1. Formation of Foam: The process begins with the
generation of foam by introducing gas (usually air or
nitrogen) into the liquid mixture. This can be achieved
through various means such as sparging, agitation, or
bubbling.
2. Adsorption to Bubbles: Components in the liquid
mixture partition between the liquid phase and the gas
phase (bubbles). Surface-active molecules, such as
proteins or surfactants, tend to adsorb onto the bubble
surface due to their amphiphilic nature, while other
components may remain predominantly in the liquid
phase.
3. Foam Rise: The foam, containing the adsorbed molecules,
rises to the top of the liquid phase due to buoyancy.
4. Foam Collection: The foam is collected at the top of the
vessel using collection devices such as foam traps or
skimmers.
5. Decomposition or Desorption: The collected foam is then
subjected to conditions that cause the foam to break
down, releasing the adsorbed molecules. This can
involve methods such as mechanical agitation,
temperature changes, or the addition of antifoaming
agents.
6. Recovery: The target molecules are separated from the
foam and recovered in a concentrated form.
Foam separation offers several advantages in
downstream processing:
 Selective separation based on surface properties:
Surface-active molecules can be selectively
concentrated in the foam phase.
 Scalability: Foam separation processes can be
easily scaled up for industrial applications.
 Reduced processing time: Foam separation can
often be performed in a single step, reducing the
overall processing time and complexity.
 Potential
for continuous operation: Foam
separation processes can be designed for
continuous
operation,
further
increasing
efficiency.
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There are also challenges associated with foam
separation, such as the potential for foam
instability, loss of valuable components, and
difficulties in scaling up certain processes.
Optimization of operating conditions and careful
selection of equipment and surfactants are
important considerations to overcome these
challenges and maximize the efficiency of foam
separation in downstream processing.
Each precipitation method has its advantages and limitations, and
the choice of method depends on the specific characteristics of the
target biomolecule and the desired purity of the final product.
Additionally, optimization of parameters such as pH,
temperature, and concentration is crucial to achieving high yields
and purity during precipitation in downstream processing.
Cell Harvesting: In biotechnology and pharmaceutical
industries, cells expressing desired products (such as
proteins or antibiotics) are typically grown in
fermentation tanks. Once the fermentation process is
complete, cells need to be separated from the
fermentation broth. Centrifugation is often used for cell
harvesting, where cells are separated from the broth
based on their density. The centrifugation process
concentrates the cells into a pellet at the bottom of the
centrifuge tube or chamber, while the supernatant
containing unwanted components can be discarded.
 Clarification of Broth: Fermentation broths often contain
a mixture of cells, cell debris, media components, and
desired products. Centrifugation can be employed to
clarify the broth by separating insoluble particles and cell
debris from the desired product and the liquid medium.
This step helps in reducing the burden on subsequent
purification steps and improving the purity of the final
product.
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Separation of Biomolecules: Centrifugation can
be used for the separation of biomolecules based
on their size, density, and sedimentation rates. For
example, it can be used to separate different cell
components such as organelles, proteins, and
nucleic acids. Ultracentrifugation, a high-speed
centrifugation technique, is often employed for
such separations.
 Concentration: Centrifugation can also be used to
concentrate dilute solutions of desired products.
By centrifuging the solution at high speeds, water
or solvent can be removed, leading to a
concentrated product.
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Purification: Centrifugation is often a preliminary step in the
purification process. While it may not achieve high levels of
purity on its own, it helps in separating the crude mixture
into fractions enriched in the desired product, which can
then undergo further purification steps such as
chromatography.
Density Gradient Centrifugation: This technique involves
layering a sample on top of a density gradient medium (such
as sucrose or cesium chloride) and subjecting it to
centrifugation. Different components of the sample migrate
through the gradient until they reach a point where their
density matches that of the surrounding medium, leading to
separation based on density.
Overall, centrifugation is a versatile and widely used
technique in downstream processing, enabling efficient
separation and purification of desired products from
complex mixtures.