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1. Industrial Biotechnology
Downstream processing
By
Ali Safaa97

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Contents
Introduction.................................................................................................................3
Stages ..........................................................................................................................3
Analytical Support of Downstream Processing..........................................................4
Cell Disruption............................................................................................................7
Traditional Chromatography Methods........................................................................9
Continuous filtration .................................................................................................10
Continuous homogenization and cell lysis ...............................................................11
Summary ...................................................................................................................11
References.................................................................................................................13

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Introduction
Downstream processing
refers to the recovery and the 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. Downstream processing is usually considered a specialized field in
biochemical engineering, itself a specialization within chemical engineering,
though many of the key technologies were developed by chemists and biologists
for laboratory-scale separation of biological products.
Downstream processing and analytical bioseparation both refer to the separation or
purification of biological products, but at different scales of operation and for
different purposes. Downstream processing implies manufacture of a purified
product fit for a specific use, generally in marketable quantities, while analytical
bioseparation refers to purification for the sole purpose of measuring a component
or components of a mixture, and may deal with sample sizes as small as a single
cell.
Stages
A widely recognized heuristic for categorizing downstream processing operations
divides them into four groups which are applied in order to bring a product from its
natural state as a component of a tissue, cell or fermentation broth through
progressive improvements in purity and concentration.
Removal of insolubles is the first step and involves the capture of the product as a
solute in a particulate-free liquid, for example the separation of cells, cell debris or
other particulate matter from fermentation broth containing an antibiotic. Typical
operations to achieve this are filtration, centrifugation, sedimentation,
precipitation, flocculation, electro-precipitation, and gravity settling. Additional
operations such as grinding, homogenization, or leaching, required to recover

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products from solid sources such as plant and animal tissues, are usually included
in this group.
Product isolation is the removal of those components whose properties vary
considerably from that of the desired product. For most products, water is the chief
impurity and isolation steps are designed to remove most of it, reducing the
volume of material to be handled and concentrating the product. Solvent
extraction, adsorption, ultrafiltration, and precipitation are some of the unit
operations involved.
Product purification is done to separate those contaminants that resemble the
product very closely in physical and chemical properties. Consequently steps in
this stage are expensive to carry out and require sensitive and sophisticated
equipment. This stage contributes a significant fraction of the entire downstream
processing expenditure. Examples of operations include affinity, size exclusion,
reversed phase chromatography, ion-exchange chromatography, crystallization and
fractional precipitation.
Product polishing describes the 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. Depending on the product and its intended use, polishing may also
include operations to sterilize the product and remove or deactivate trace
contaminants which might compromise product safety. Such operations might
include the removal of viruses or depyrogenation.
A few product recovery methods may be considered to combine two or more
stages. For example, expanded bed adsorption accomplishes removal of insolubles
and product isolation in a single step. Affinity chromatography often isolates and
purifies in a single step. [1]
Analytical Support of Downstream Processing
Downstream processing ensures control of process- and product-related
impurities. Analytics for measuring process-related impurities such as host cell
proteins and nucleic acids are still challenging the capabilities of the measurement
technology. Making reliable measurements at or near the detection limit with
immunological assays or qPCR demands a great deal of assay optimization.
Downstream processing can also impact the distribution and levels of
productrelated impurities. Accurate monitoring of product-related impurities,

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including higher molecular weight and lower molecular weight species is critical
for product safety.
The analytics for downstream bioprocessing have improved considerably. With the
emergence of multiplexed techniques, approaches for more rapid sample handling,
and higher-throughput clean-up techniques it has become easier to support the
development of downstream unit operations. In addition, some of these techniques
are viable for larger scale manufacturing.
In many instances, loading and optimal operation of the first downstream unit
operation requires knowledge of the product concentration (i.e., titer) in the cell
culture media. A range of analytical technologies are available to measure titer.
The suitability of the titer method for GMP testing should be considered. With
improved analytics comes the ability to more precisely control and diagnose
purification operations, and thus, ensure consistency from batch to batch. There are
various locations where analytics can play a major role. Most often a simple titer
assay (e.g., A280 or HPLC based) is used to determine column loading. By loading
a consistent amount of protein on a column, it may be possible to ensure
consistency of the product quality further downstream. Likewise, the column
elution pooling criteria could be determined using a variety of assays. In most
cases, unless data is available to prove otherwise, pooling criteria should be
determined to increase the product purity by reducing the levels of process and
product-related impurities.
Depending on the susceptibility of the protein, significant degradation (e.g.,
oxidation, deamidation, isomerization, clipping) could occur during downstream
processing. Process holds, especially when conducted in buffers far from neutral
pH, at ambient temperature, or with light exposure, can generate degraded species.
LC-MS peptide mapping is an ideal method for detecting and quantifying these
types of degradation, but the necessary infrastructure for these experiments is
required (e.g., instrumentation, software, trained analysts for routine testing).
UVbased peptide mapping is also feasible, but additional method development is
required to ensure that the peptide peaks are well separated and appropriately
characterized. Finally, other methods, including charge heterogeneity methods or
reverse phase chromatography may be able to distinguish degraded from
nondegraded protein depending on the impact of the modification. These methods
can be identified during forced degradation studies. [4]

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Figure 1: Steps in downstream processing. [5]

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Cell Disruption
Downstream processing of fermentation broths usually begins with removal of the
cells by filtration or centrifugation. The next step depends on the location of the
desired product. For substances such as ethanol, citric acid, and antibiotics that are
excreted from cells, the product is recovered from the cell-free broth using unit
operations such as those described later in this chapter. In these cases, the biomass
separated from the liquid is discarded or sold as a by-product. For products such as
enzymes and recombinant proteins that remain in the biomass, the harvested cells
must be broken open to release the desired material. A variety of methods is
available to disrupt cells. Mechanical options include grinding with abrasives,
high-speed agitation, high-pressure pumping, and ultrasound. Nonmechanical
methods such as osmotic shock, freezing and thawing, enzymic digestion of the
cell walls, and treatment with solvents and detergents can also be applied. A
widely used technique for cell disruption is high-pressure homogenisation. The
forces generated in this treatment are sufficient to completely disrupt many types
of cell. A common apparatus for homogenisation of cells is the Gaulin
homogeniser. The homogeniser is of general applicability for disruption of all
types of cell; however the homogenising valve can become blocked when used
with highly filamentous organisms. Greater disruption is achieved by maintaining
a small gap around the valve so that the cells strike the impact ring with high
velocity. The exact mechanisms responsible for cell disruption in the Gaulin
homogeniser remain a subject of debate. Cavitation, fluid shear, impact, and
pressure shock may all play a role in the breakage process. [6]

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Figure 2: Process development for purifying high-value recombinant proteins from
bacteria and microalgae. [7]

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Figure 3: Continuous downstream processing for high value biological products.
[11]
Traditional Chromatography Methods
Traditional chromatography methods in industrial processes encompass affinity
(AC), ion exchange (IEX), and hydrophobic interaction (HIC) chromatography.
Size exclusion chromatography (SEC) is found in older and/or smaller scale
industrial processes and is not further discussed here because of its throughput N.
Singh and S. Herzer limitations and column packing challenges. Both limitations
ensure that size exclusion chromatography remains a poor choice for an industrial
process. AC, and IEX in particular, are factotums in the industry because of their
robustness, simplicity, and, of course, familiarity. With that said, poor mass
transfer and back pressure are inherent limitations of traditional chromatography.
Mass transfer in traditional chromatography media is intraparticle pore diffusion
controlled, and pore concentration is inversely proportional to bead radius and
pressure drop is inversely proportional to the bead radius squared. As improved
mass transfer is pitted against an increase in back pressure (cost consideration of
smaller bead size aside), traditional bead based chromatography always operates
within these constraints. Downstream processing is challenged with developing
robust, fast, productive processes to enable purification high titer upstream
processes. Traditional chromatography is perceived as a bottleneck and its future

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has been called into question for over a decade. Juxtaposed to this expectation of
the imminent demise of traditional chromatography is the entrenched platform
process for antibodies. As an antibody platform process shortens process
development, simplifies manufacturing, and even enables platform analytics
without jeopardizing commercial success, finding an equally robust and easy to use
process can be hard to envision. [9]
Continuous filtration
Filtration, in the form of microfiltration and ultrafiltration, is a key unit operation
in downstream processing of recombinant proteins. Microfiltration is mainly used
for cell harvesting. Ultrafiltration is used for concentration of proteins and often in
the form of diafiltration for buffer exchange. Ultrafiltration is also used to clear
viruses from recombinant DNA produced in mammalian cells. This step is also
named nanofiltration, although it is an ultrafiltration with a membrane with a rather
low pore size. For cell harvesting filtration is either used in combination with
centrifugation or as an alternative unit operation. Moreover, it has been suggested
that dynamic filtration systems combined with affinity technologies or
electrofiltration intensify processes. Operations would save on costs and could
streamline production with such a combination. Continuous filtration, also termed
cascade filtration, is used in conventional pharmaceutical manufacturing.
Continuous filtration is achieved by connecting several filtration units, where the
permeate of one filtration unit is fed onto a subsequent unit. The size of each unit
may change from step to step. In the biopharmaceutical industry, continuous
filtration may be used to (i) remove solids by microfiltration to (ii) concentrate
proteins by ultrafiltration, (iii) perform buffer exchange or conditioning of process
solutions by diafiltration, and (iv) act as a virus removal step by nanofiltration
(ultrafiltration utilizing small pores). Ultra-scaled-down models of filtration to
mimic energy dissipation and flow patterns are available. Laboratory equipmentis
also readily available and can be easily adjusted for continuous filtration.
Continuous filtration can be performed in several ways: ‘batch topped off’,
singlepass tangential-flow filtration (SPTFF), diafiltration, and membrane
cascades.

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Continuous homogenization and cell lysis
High-pressure homogenizers are the industrial way to lyse cells in pilot and full
scale. These machines consist of two main elements: a piston pump usually in the
form of a triplex pump and a valve with a slit of around 100 mm to generate
pressure up to 1500 bar. Cell lysis is caused by cavitation, which is generated at
the valve and immediately behind the valve when the pressure is suddenly
released. Modern high-pressure homogenizers can be operated continuously,
because the cells are disrupted in a single pass, which was not possible with older
homogenizers. However, when homogenizers are used in continuous mode, cell
lysis must be completed in a single pass. This is achieved by installing two
homogenization valves in series. Continuous rates must be strictly avoided.
Highpressure homogenization would destroy the plasmids. [10]
Summary
1-Downstream processing involves all unit operations after fermentation that
improve the purity of the final ethanol product. Ethanol is typically purified using
combinations of distillation and molecular sieving. The fermented broth from the
beer well is sent to a traditional distillation column to obtain 95% ethanol. Since
this ethanol contains 5% moisture, it is dehydrated using azeotropic distillation.
The presence of water increases the molecular polarity of ethanol, making it
separate when mixed with gasoline. In azeotropic distillation, a third chemical
called an entrainer (e.g., benzene or cyclohexane) is added. Since azeotropic
distillation is complicated and expensive, molecular sieving methods have been
implemented recently in large-scale ethanol purification. The molecular sieves let
the smaller water molecules (0.28 nm) pass through while retaining dehydrated
ethanol (0.44 nm). It is possible to regenerate molecular sieves by heating or
applying vacuum. As indicated in Table 5.2, many food waste feedstocks have
ethanol yields lower than 5% w/v, illustrating the need to optimize more
economical downstream processing technologies. [2]
2-Downstream processing equipment covers a vast range of systems varying in
size and complexity. The scales covered include laboratory systems, pilot-scale
equipment, and production scale. The complexity of equipment decreases with the
scale, but in general, even larger-scale equipment needs to offer a certain degree of

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flexibility to be able to address various production scenarios. Downstream
equipment by itself does not contribute to the actual purification event, but
wrongly designed/chosen equipment can have a deterioration effect on the process
yield and economy. For instance, in the case of a chromatography system, system
contribution to zone broadening because of additional mixing and/or no-flow
zones will result in high buffer consumption and lower yield or lower purity for a
predefined set of operating conditions. [3]
3-Separation science continues to occupy the central position in the overall
strategy for the downstream processing and purification of therapeutic protein
products for human use. Increasing product titers from mammalian cell culture and
new emerging classes of biopharmaceuticals has presented a challenge to the
industry to identify ways of improving the robustness and economics of
chromatography processes. In commercial manufacturing, there is always a need to
increase the scale of the chromatography operations which are typically developed
and optimized in small-scale laboratory experiments. [8]
4-Downstream processing (DSP) is engaged in the separation and refinement of
mixtures of components. In its simplest definition, DSP encompasses a tool box of
separation techniques designed to achieve mass transfer phenomena, converting
mixtures of substances into subsets of mixtures or fractions. At its onset, industrial
DSP of proteins considered many of the traditional chemical unit operations, such
as aqueous two phase systems (ATPS), precipitation, crystallization, and
extraction. Although these chemical techniques still have a stronghold in sister
industries such as plasma fractionation and vaccine manufacture, the lack of
general utility, emergence of higher yielding, less harsh techniques, and scale-up
limitations in cell culture-based protein DSP stifled popularity of these techniques.
Novelty in the context of these unit operations therefore mostly derives from
improvements in experimental design, high-throughput screening (HTS),
equipment, and systems. A deepened understanding of protein surface mapping
going hand-in-hand with improved protein engineering contributes as well.
Advances in separation sciences over the last five decades have enabled DSP as it
stands today, where process chromatography and filtration have evolved into the
pillars of downstream processing of protein biologics for the last three decades. A
brief review of their development and capabilities serves here to lay the framework
as to what targets need to be surpassed to achieve similar success in DSP for
alternate unit operations. [9]

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References
1- https://en.wikipedia.org/wiki/Downstream_processing#:~:text=Downstream%2
0pr
ocessing%20refers%20to%20the,treatment%20and%20disposal%20of%20was
te.
2- Swati Hegde, Thomas A. Trabold, in Sustainable Food Waste-To-energy
Systems, 2018 sciencedirect.com/topics/engineering/downstream-processing
3- Mikael I. Johansson, ... Günter Jagschies, in Biopharmaceutical Processing,
2018
4- Matthew J. Traylor, ... Johnson Varghese, in Biopharmaceutical Processing,
2018
5- Islam, Tuhidul & Eldamrawy, Saad. (2013). Novel strategies for the purication
of biomolecules by anity chromatography: Generation and use of ceramic
uorapatite binding peptides for the development of self-assembled systems and
ligand-less adsorbents.
6- Pauline M. Doran, in Bioprocess Engineering Principles (Second Edition),
2013
7- https://bioseparations.tamu.edu/research/
8- Milne JJ. Scale-Up of Protein Purification: Downstream Processing
Issues. Methods Mol Biol. 2017;1485:71‐84. doi:10.1007/978-1-4939-6412-3_5
9- Singh N, Herzer S. Downstream Processing Technologies/Capturing and Final
Purification : Opportunities for Innovation, Change, and Improvement. A Review
of Downstream Processing Developments in Protein Purification. Adv Biochem
Eng Biotechnol. 2018;165:115‐178. doi:10.1007/10_2017_12
10- Jungbauer A. Continuous downstream processing of biopharmaceuticals.
Trends Biotechnol. 2013;31(8):479‐492. doi:10.1016/j.tibtech.2013.05.011

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11-Zydney, Andrew L. “Continuous downstream processing for high value
biological products: A Review.” Biotechnology and bioengineering 113 3 (2016):
465-75 .