downstream processing

1. DOWNSTREAM PROCESSING
Objective:
An opportunity to understand different steps of
downstream processing used in the purification of
biological products from the fermentation broth and
complex mixtures, its economics and process design
criteria for various classes of bioproducts.
Module1:
Role and importance of downstream processing in
biotechnological processes. Characteristics of products,
Economics, process design criteria for various classes of
byproducts (high volume, low value products and low
volume, high value products), physico-chemical basis of
different bio-separation processes.

2. Bioprocesing/Biochemical engineering
The efficient utilization of physical, chemical and
biological processes to convert raw biological materials
into useful products, at minimal cost, with minimal
energy consumption, and with minimal impact on the
environment for research and large scale industry.

3. Why it is important?
It is required for:
1) Purification and recovery of biosynthetic products,
mostly pharmaceutics from sources which are
natural. For example, plant and animal tissues or
fermentation froth.
2) Recycling of components that can be salvaged form
waste.
3) Proper waste disposal and treatment

4. General Uses of recombinant DNA technology

5. Expression of desired proteins in Bacteria and Animals

6. Development of Transgenic plants with desired traits

7. • Upstream processing refers to the culturing of
cells and microorganisms to create the bulk
bio-product. This processing is typically done
through cell culture or fermentation
• Downstream processing includes isolation and
purification of the desired product from
biological cells.
Upstream- Downstream

8. Fermentation (upstream processing)
Bioprocessing
+
Product recovery (downstream processing)
e.g.

9. • Because bioprocesses use living material,
they offer several advantages over
conventional chemical methods of
production:
 They usually require lower temperature, pressure, and
pH.
 They can use renewable resources as raw materials;
and greater quantities can be produced with less
energy consumption.

10. Bioprocessing Schematic

11. Bioprocessing Schematic Upstream and Downstream

12. Steps of downstream processing are:
1) Solid-Liquid Separation: It is isolation of whole cells and other
ingredients that are insoluble from the culture media. Several
methods used for solid-liquid separation are floatation,
filtration, flocculation and centrifugation.
2) Release of Intracellular Products: Sometimes, the required
product like vitamins, enzymes are located within the cells. So,
they should be released maximally and in an active form for
their isolation and processing. Yeasts, bacteria, protozoa and
other cells can be disrupted by chemical, physical or enzymatic
methods.

13. 3) Concentration: Filtrate free from suspended particles usually has 80-98% of
water. The required constituent is in very small quantity minor and water should
be extracted to achieve the product concentration. Most commonly used
methods for concentrating such products are evaporation, membrane
filtration, liquid-liquid extraction, adsorption and precipitation.
4) Purification by Chromatography: The products of fermentation are very
effectively purified by chromatography. Different types of chromatography used
in downstream processing are Ion-exchange chromatography, affinity
chromatography, Gel-filtration chromatography, hydrophobic interaction and
immobilized metal-ion affinity etc.
5) Formulation: It means the maintenance of stability and activity of desired
products during storage and distribution. The formulation can be done by
concentrating them with removal water. For certain small molecules, it can be
done by crystallization by adding salts. Proteins usually require certain
stabilizing additives to increase their shelf life.

14. Fermentor

15. Bioreactor and fermentor are two words for basically the same
thing. Scientists who cultivate bacteria, yeast, or fungi often use the
term fermentor. The term bioreactor often relates to the cultivation of
mammalian cells but is also generically used.
•The major types are:
•(1) Continuous Stirred Tank Bioreactors.
•(2) Bubble Column Bioreactors.
•(3) Airlift Bioreactors.
•(4) Fluidized Bed Bioreactors.
•(5) Packed Bed Bioreactors.
The word fermentation originally came from a Latin word “fervere” that
means to boil. Fermentation is a process used for the production of a
product by the aid of microorganisms. Louis Pasteur defined the
fermentation process as ‘la vie sans l'air,’ which means ‘life without air.’

16. For certain applications, biological products can be used as crude extracts
with little or no purification. However, biopharmaceuticals typically require
exceptional purity, making downstream processing a critical component of
the overall process.
Proteins are the most important biopharmaceuticals.
Enzymes / Antibodies / Plasma / Blood components
Blood plasma fractionation was the first full - scale biopharmaceutical
industry with a current annual production in the 100 - ton scale. Mostly
physical in nature using centrifugation and filteration.
Anti - venom antibodies and other anti - toxins extracted from animal
sources are additional examples of early biopharmaceuticals, also purified by
a combination of precipitation, filtration and chromatography.
Earlier forms of Bioprocessing /Physico-mechanichal

18. Plasmapheresis equipment

19. Roles of downstream processing:
1) Production of antibiotics : Downstream processing is very important in the
manufacture of antibiotics such as penicillin. The process is applied in the
purification and separation of antibiotics from a number of mediums.
2) Large scale manufacture of monoclonal antibodies(mAbs). Since
antibodytherapies are characterized by long-term administration of large
antibody does, biopharmaceutical companies greatly appreciate down processing
for facilitating industrial manufacture of antibodies.
3) Hormones are also resultant products of downstream processing. Insulin and
FSH are major hormones utilizing Downstream processing.
4) Vaccine: The application of downstream processing in the manufacture of
important vaccines like influenza and small pox.
5) Industrial production of enzymes : Enzymes are normally synthesized by
living cells and are responsible for triggering chemical reactions. is therefore very
important since they are applied in processes such as food preservation and
processing, manufacture of textiles, paper industry, scientific research, etc.

20. Current biopharmaceuticals are almost
exclusively produced by recombinant DNA
technology.
Chromatography and membrane filtration
serve as the main tools for purification for
these products.

21. Economics of bioprocess:
The Bioprocessing Market share is expected to reach US$ 72.55 billion by 2031 from US$
25.35 billion in 2023 to record a CAGR of 14.0% from 2023 to 2031.
Key Players: A few of the major companies operating in the bioprocessing market include
Cytiva (Danaher Corporation), Thermo Fisher Scientific Inc., Sartorius AG, Merck
KgaA, 3M Company, Getinge AB, Eppendorf SE, Corning Incorporated, Agilent Technologies,
and Bio-Rad Laboratories etc.
Geographic Insights: In 2023, North America led the bioprocessing market with
a substantial revenue share, followed by Europe and APAC, respectively. Asia
Pacific is expected to register the highest CAGR during the forecast period.By
scale of operation, the bioprocessing market is segmented into commercial operations and
clinical operations. The commercial operations segment held a larger market share in
2023.In terms of process, the bioprocessing market is bifurcated into downstream
bioprocess and upstream bioprocess. The downstream bioprocess segment held a larger
market share in 2023.
In terms of application, the bioprocessing market is categorized into monoclonal antibodies,
vaccines, recombinant protein, cell and gene therapy, and others. The monoclonal
antibodies segment dominated the bioprocessing market with the largest share in 2023.

22. By Application
• Biopharmaceuticals
• Speciality Industrial Chemicals
• Environmental Aids
By Scale
• Industrial Scale (Over 50,000 Litres)
• Small Scale (Less Than 50,000 Litres)

23. Biopharmaceuticals: Drugs classified as biopharmaceuticals (ca.130 +
vaccines) had reported sales of $197bn in 2016, with above average growth
of +7.6%. These drugs now represent 24% of the global drug market. Within
this segment, the 61 approved drugs derived from antibodies had sales of
$82bn (+15.4%).
Top drugs: The biggest drug in 2016 was Humira (AbbVie) with ex-factory
sales of $16.1bn (+14%). The top 10 drugs in the world represented 25% of
the entire market. On a cumulative basis, the best-selling drug ever remains
Lipitor ($152bn).
R&D investment: Investment in R&D is key to future success. The top 15
companies invested $86.0bn in pharma R&D in 2016, representing 20.5% of
Rx drug sales. The highest spender was Roche at $10.3bn (22.6% of Rx sales).
Economics of pharmaceutical products:
Global Pharmaceuticals statistics

24. India Bioprocessing industry statistics
India is well known as a key destination for bioprocess outsourcing and a
major supplier of active pharmaceutical ingredients and raw materials.
India has the potential to capture 8–10% of this industry and become one
of the top 10 global markets by value.
India’s biotechnology industry (consisting of biopharmaceuticals as well
as other sectors) has gained global attention only within the past five to
six years.
Although reports indicate that a majority of India’s industry in the next
decade will continue consist of small-molecule drugs, the
biopharmaceuticals market (~62% of biotech industry) will grow .

25. LEADING COMPANIES IN INDIA
Pharmaceutical/ Biopharmaceutical:
Ranbaxy; Dr Reddy’s Laboratories; Cipla; Sun Pharma Industries, Lupin Labs, Aurobindo Pharma,
GlaxoSmithKline Pharma, Cadila Healthcare, Aventis Pharma, Ipca Laboratories.
Biocon, Serum Institute of India, Panacea Biotec, Reliance Life Sciences, Novo Nordisk, Shantha
Biotech, Indian Immunologicals, Bharat Biotech, Eli Lilly, Bharat Serums, Hafkine Biopharma, Cadila
Healthcare, GSK, Intervet India, Intas Biopharma

26. The global pharmaceutical manufacturing market size was valued at USD
405.52 billion in 2020 and is expected to grow at a compound annual
growth rate (CAGR) of 11.34% from 2021 to 2028. The pharmaceutical
landscape has undergone a massive transformation with the emergence of
new technologies, cost-effective, and more efficient manufacturing
approaches. In 2020, the conventional drugs
(small molecules) segment
accounted for the highest
revenue share of over 65%.
According to an article
published in August 2019, in the
pharmaceutical market, small
molecule drugs account for up
to 90% of the total global drug
sales.
The COVID-19 pandemic is anticipated to serve as the key driving force for R&D
expenditure in this segment. Moreover, growing awareness about women’s health
has driven significant attention of operating players towards the development of
therapies to address key conditions in women. On the other hand, cancer
therapies are anticipated to register the fastest CAGR from 2021 to 2028. This is
owing to the high sales of oncology drugs, especially KEYTRUDA of Merck and
HUMIRA of AbbVie, Inc. Several studies have reported that healthcare spending
on cancer treatments has doubled in the last few years. Moreover, a huge number
of clinical tests in immuno-oncology globally are driving the segment growth.

27. The global industrial enzymes market size was valued at USD 5.6 billion in 2019
and is expected to grow at a compound annual growth rate (CAGR) of 6.4%
from 2020 to 2027. Rising product demand from the end-use industries, such
as biofuel, home cleaning, animal feed, and food and beverage, is projected to
fuel the industry growth over the forecast period.
Carbohydrases are used predominantly as a catalyst for the conversion of
carbohydrates into sugar syrup, including fructose and glucose, which are
further utilized in the pharmaceutical and food and beverage industries.
Protease, another prominent product, is majorly used for protein breakdown,
catalytic hydrolysis of protein peptides to amino acids, and in various
applications, such as food, pharmaceutical, detergents, animal feed, chemicals,
and photography. Serine, threonine, cysteine, aspartate, papain, glutamic acid,
The market is anticipated to be
driven by ascending demand
for carbohydrase and proteases in
the food and beverage
applications, especially in the
emerging economies of Asia
Pacific, such as China, India, and
Japan. Moreover, growth in the
developed economies can be
attributed to increasing
industrialization, along with
advancements in the nutraceutical
sector.

28. Therapeutic Uses Of Enzymes
Enzymes are used for this purpose where some inborn errors of metabolism occur
due to missing of an enzyme where specific genes are introduced to encode
specific missing enzymes. However, in most cases, certain diseases are treated by
administering the appropriate enzyme.
For example, virilisation of disease developed due to loss of hydroxylase enzyme
from adrenal cortex and introduction of the hydroxyl group (-OH) on 21-carbon of
the ring structure of steroid hormone. Steroids are compounds having a common
skeleton in the form of perhydro-1, 2-cyclo-pentano-phenanthrene. The missing
enzyme synthesises aldosterone (male hormone) in excess leading to
masculinization of female baby and precocious sexual activity in males in about 5-
7 years.
Similarly, treatment of leukaemia (a disease in which leukemic cells require
exogenous asparagine for their growth) could be done by administering
asparaginase of bacterial origin.

29. Analytical Uses Of Enzymes
Use of enzymes for analytical purposes is also important. Generally,
endpoint and kinetic analysis are possible. Endpoint analysis refers to
the total conversion of substrates into products in the presence of
enzymes in a few minutes while kinetic analysis involves the rate of
reaction and substrate/product concentration. Moreover, the
analysis of antibodies, immunoglobins, necessary for human use
poses a great promise. The usable enzymes are alkaline phosphatase,
b-galactosidase, b-lactamase, etc.
Another use of enzyme is in biosensor, device of biologically active
material displaying characteristic specificity with chemical or
electronic sensor to convert a biological compound into an electronic
signal. It is constructed to measure almost anything from blood
glucose. A simple carbon electrode, an ion-sensitive electrode,
oxygen electrode or a photocell, maybe a sensor.

30. In Brewing Industry
Enzymes used in the brewing industry are a-amylase, b-glucanase
and protease which are required for malt in substitution of barley.
Source of these enzymes is B. amyloliquefaciens. a-amylase is not
required for liquefaction or brewing adjuncts and b-glucanase
alleviates filtration problems due to poor malt quality and neutral
protease helps in the inhibition of alkaline protease by an inhibitor.
In Wine Industry
Pectic enzymes are utilised in the wine business for top yield of
product of improved quality. The pectic enzymes are pectin
transeliminase (PTE), polymethyl galacturonase (PMG),
polygalacturonases (PG), pectin esterase (PE), etc. However,
cellulose enzymes provide a smart result once combined with
different enzymes e.g. protease glucoamylase, etc.

31. In Starch Industry
It has been mentioned earlier that hydrolysis of starch began in the early 1960s
to prepare dextrose and glucose syrups. Furthermore, for complete acid
hydrolysis of starch to dextrose glucoamylase was coupled with bacterial a-
amylase. Currently, various enzymatic processes are applied to various products.
Glucose isomerase is an important enzyme used commercially in the conversion
of glucose to fructose via isomerisation. Fructose is used in the preparation of
fructose syrup. The reaction mixture at the end contains 42% fructose, 52%
glucose, and 6% dextrins. The mixture is sweeter than glucose and as sweet as
sucrose. Now, techniques have been developed to obtain 55% fructose
concentration in syrup.
In Detergent Industry
During normal washing proteinaceous dirt often precipitates on solid cloths and
proteins facilitate to adhere the dirt on textile fibres and make stains on cloths.
These stains are difficult to remove from clothes. Nevertheless, it can be easily
removed by adding proteolytic enzymes to the detergent. It attacks peptide
bonds and therefore, dissolves protein. The alkaline serine protease obtained
from B. licheniformis is most widely preferred to use in detergent. In addition, the
serine protease of B. amyloliquefaciens is also used for this purpose. It contains a-
amylase, hence to some extent it may be advantageous.

32. Enzyme based cell lysis methods

36. Identify Strengths and Opportunities
Scientific understanding of the biological drivers of disease, combined with the
ability to influence human biology, has never been greater.
Momentum will be fueled by a surge in the identification of more compelling
biological targets and the expansion of therapeutic modalities that enable
modulation of both newly discovered and historically undruggable targets.
The biopharma industry is currently experiencing a surge in innovation and
strategic shifts, with several key focus areas driving its evolution. These
include personalized medicine, cell and gene therapies, biosimilars, sustainability
initiatives, and the integration of data analytics and AI.
Additionally, therapeutic areas like oncology, immunology, and neuroscience are
receiving significant attention, alongside the development of vaccines and
therapies for rare and chronic diseases.
Examples of current new treatments include gene therapies for spinal muscular
atrophy, hemophilia A, sickle cell disease, and CAR-T therapies for multiple
myeloma. Already, the market share of first-in-class products has risen from 20%
in 2000 to 50% today. We estimate that some 15% of the market in 2030 will
consist of novel modalities versus only about 5% in 2020.

37. Investing in new tools:
1.AI's Transformative Potential: AI can analyze large datasets, screen compounds,
and design drug candidates, potentially reducing preclinical timelines by 30% to
50% and costs by 25% to 50%.
2.High AI Success Rates: AI-driven approaches have achieved Phase 1 success rates
exceeding 85% in some cases, showcasing their early promise.
3.Slow Adoption in Traditional Pharma: Over 40% of traditional pharma and
biotech companies have not yet integrated AI significantly into their drug discovery
processes.
4.AI-First Startups Advantage: AI-focused biotech startups are building therapeutic
pipelines faster, particularly in data-rich areas like oncology, though late-stage
success is still unproven.
5.Need for AI Strategy Refinement: To fully leverage AI, companies must refine
strategies, adapt operating models, and upskill their workforce for long-term
impact.

38. New Translational models
1.Translational Models as Game Changers: Effective translational models,
like the replicon model for hepatitis C, have historically driven major
breakthroughs, enabling high cure rates (e.g., 90% for hepatitis C).
2.Limitations of Traditional Models: Animal and biochemical assay
models often have low concordance with human disease biology,
especially in neuroscience, leading to high failure rates.
3.Advances in Translational Models: Organoids and organ-on-a-chip
technologies offer more human-relevant disease models, improving
predictive power and reducing late-stage failures.
4.Growing Research in Translational Models: Publications on organoid-
adjacent models have increased 11-fold from 2014 to 2024, indicating
significant research momentum.
5.Need for Collaboration and Investment: Sectorwide collaboration
among pharma, regulators, investors, and academia is critical to integrate
translational models into R&D and unlock their potential.

39. Key Points on Navigating Revenue and Cost Pressures in Pharma
1.Patent Cliff Impact: Drugs worth $350 billion in annual global
revenues face patent expiration by 2030.
2.Top Companies Affected: The top 20 pharma companies account
for 80% of this revenue loss.
3.Revenue at Risk: 8% of branded drug revenue is at risk from 2026–
2030, compared to 12% from 2010–2015.
4.Unique Challenges: The current patent cliff differs significantly
from past waves, with at least two major distinct factors

41. India Bioprocessing industry statistics
India is well known as a key destination for bioprocess outsourcing and a
major supplier of active pharmaceutical ingredients and raw materials.
India has the potential to capture 8–10% of this industry and become one
of the top 10 global markets by value.
India’s biotechnology industry (consisting of biopharmaceuticals as well
as other sectors) has gained global attention only within the past five to
six years.
Although reports indicate that a majority of India’s industry in the next
decade will continue consist of small-molecule drugs, the
biopharmaceuticals market (~62% of biotech industry) will grow .

42. Over 2020, the Indian BioEconomy sector registered an impressive
growth of 14.1%, which accounts for about 2.6% of India’s GDP.
The sector was valued at $80.12 billion in 2021, increasing from
$70.2 billion in 2020[2]
.
As one of the largest suppliers of low-cost drugs and vaccines
worldwide and home to over 5,000 startups, the Indian BioEconomy
sector is poised to reach a value of $300 billion by 2030[3]
. India is
among the top 12 hubs of biotechnology worldwide, and the third
largest destination for biotechnology in the Asia Pacific region.
India's biotechnology industry covers several areas, such as
biopharma, bio-services, bio-agriculture, bio-industry, and
bioinformatics.
According to the India BioEconomy Report of 2022, the biopharma
segment is the largest contributor to the biotechnology industry in
India, with a market share of 49% and total economic contribution
of approximately $39.4 billion in 2021.

43. Lacunae in Bioprocessing Industries

44. Steps of downstream processing are:
1) Solid-Liquid Separation: It is isolation of whole cells and other
ingredients that are insoluble from the culture media. Several
methods used for solid-liquid separation are floatation,
filtration, flocculation and centrifugation.
2) Release of Intracellular Products: Sometimes, the required
product like vitamins, enzymes are located within the cells. So,
they should be released maximally and in an active form for
their isolation and processing. Yeasts, bacteria, protozoa and
other cells can be disrupted by chemical, physical or enzymatic
methods.

45. 3) Concentration: Filtrate free from suspended particles usually has 80-98% of
water. The required constituent is in very small quantity minor and water should
be extracted to achieve the product concentration. Most commonly used
methods for concentrating such products are evaporation, membrane
filtration, liquid-liquid extraction, adsorption and precipitation.
4) Purification by Chromatography: The products of fermentation are very
effectively purified by chromatography. Different types of chromatography used
in downstream processing are Ion-exchange chromatography, affinity
chromatography, Gel-filtration chromatography, hydrophobic interaction and
immobilized metal-ion affinity etc.
5) Formulation: It means the maintenance of stability and activity of desired
products during storage and distribution. The formulation can be done by
concentrating them with removal water. For certain small molecules, it can be
done by crystallization by adding salts. Proteins usually require certain
stabilizing additives to increase their shelf life.

46. Process Development in Downstream Processing (DSP)
DSP development focuses on yield and productivity as well as on
purity and process capacities. An increase in separation efficiency
of single unit operations is achieved by expansion of existing
facilities and by optimization of existing and alternative processes.
New methods for process development are under investigation.
These include the establishment of platform technologies, high-
through-put methods with approaches based on QbD and DoE-
based experimental optimizations .
Additionally, an integration of modeling and simulation of unit
operations as well as the use of mini-plant facilities is applied in
process development.

47. In batch processing, it is known that protein concentration, pH, conductivity, buffer type,
viscosity, additives, operating pressure, and pressure release times can affect virus filter
performance. The question is: which of these process variabilities are relevant for
continuous virus filtration? To begin answering this question, a design-of-experiment
(DoE) study was conducted to define the design space for continuous virus filtration.
DoE. A full factorial DoE a DoE type of validation by identifying the critical
parameters (e.g., concentration, flow, pH, conductivity) was performed including a
total of 10 experiments that varied the length of the run, the operating pressure, and
either a monoclonal antibody (mAb) or buffer feed. Depending on the total length of each
run, pressure was applied for 24 or 48 hours twice with a 30-minute pressure release after
each filtration period as shown in Figure. For the 48-hour runs, an additional pressure
release of 60-minutes was conducted. Fractions were collected in the beginning of each
filtration and before and after each filtration period and pressure release to evaluate any
impacts of the pressure profile.

48. Possible process implementation of continuous virus filtration
showing operation mode (left) and preparation mode with integrity
testing (IT) (right).

49. Process design criteria for various classes of byproducts
Fundamentals of Process design with emphasis on bioseperation process
1-Design: design is a creative activity and is defined as the synthesis, the putting
together of ideas to achieve a desired purpose. Also it can be defined as the creation
of manufacturing process to fulfill a particular need. The need may be public need or
commercial opportunity.
2-Process Design: process design establishes the sequence of chemical and physical
operations; operating conditions; the duties, major specifications, and materials of
construction (where critical) of all process equipment (as distinguished from utilities
and building auxiliaries); the general arrangement of equipment needed to ensure
proper functioning of the plant; line sizes; and principal instrumentation.
3-Plant Design: includes items related directly to the complete plant, such as plant
layout, general service facilities, and plant location

50. Process Development in Upstream Processing Process
development and optimization in USP includes various parts:
Cell line development and engineering, cell clone selection,
media and feed development, bioprocess development and
scale up.
Reactor design, cell harvesting, process control and the
corresponding analytics can be part of the optimization
process as well.
These areas are optimized individually and focus on a robust
generation of a high product titer, high productivity and
defined quality

51. Optimization fields in Upstream processing

52. Due to the intense time pressure in process development,
high-throughput (HTP) methods are employed in early
process development.
They permit running a large number of screening
experiments in a very small scale and can be performed
with minimal amounts of material. Large amounts of data
are provided in a short period of time.
HTP methods are often combined with statistically
planned experiments, Design of Experiments, (DoE). In
statistically planned experimental designs, several factors
can be changed within one set of experiments.

53. HTP methods and DoE are both applied in the
development of upstream as well as downstream
processing.
Furthermore, concepts of Quality by Design (QbD) in
combination with HTP methods or DoE
QbD is a manufacturing principle in which product
quality is built into the manufacturing process by
understanding the associated risks and including
strategies to mitigate those risks during manufacture.
QbD-based development of analytical methods for
antibody aggregates and size heterogeneity have been
successfully reported

54. Optimization fields in downstream processing

55. Bioprocess design of various Industrial manufacturers

56. Physico- Chemical processes in Bioseperation

57. Traditionally, monoclonal antibodies were purified by a
sequence of different chromatographic and membrane-based
operations
A virus-inactivating operation, a filtration-based virus-reducing
step and a final diafiltration have to be included .
Downstream Processing steps in production of mABs
1 Chromatographic Separations
2 Non-Chromatographic Separations

58. Non-Chromatographic Separations
Further trends in DSP address the development of non-
chromatographic operations:
Aqueous two-phase extraction (ATPE) .
Precipitation
Crystallization
Affinity alternatives are other methods.
Non-chromatographic separations are in many cases proven in
non-pharmaceutical processes with much higher feed volumes.
They are particularly useful for DSP now that higher titers are
involved and greater amounts of buffer are required. These
“low-tech” separation methods are good for high-volume feeds
and rapidly remove a lot of liquid. This development may allow
a reduction of costs, process time and yield losses.

59. In DSP they can be used as initial harvest operation for removal
of biomass.
The former function of membranes as selective barrier for
filtration is extended towards a selective adsorption of molecules
to separate them according to their chemical behavior. This
relatively new development in membrane technology is called
membrane chromatography.
Membrane processes are one of the most important unit
operations in biopharmaceutical processing
Membrane adsorbers are used for polishing applications to
remove contaminants.
Viruses, endotoxins DNA, HCP and leached Protein A binds to the
membrane at neutral to slightly basic pH and low conductivity.

60. Aqueous Two Phase Partioning Aqueous two-phase
extraction (ATPE)
It shows potential
applications of this
process for separation of
cells and undissolved
components, of
impurities and product.
ATPE is considered a
simple and low-cost
technology compared to
Protein A chromatography.
It has advantages in
scalability, can be applied
in continuous processes
and has a high capacity
Precipitation
It can also be used for protein purification in industrial scale.

61. Other technologies for process development and integration include,
flocculation, coagulation, precipitation and magnetic separations among
others.
Coagulation and flocculation are involved in solid liquid separation,
precipitation is more often used later in purification.
Coagulation refers to the destabilization of a colloidal system by the action of
additives (coagulants.) In the case of proteins, heat or mechanical stress cause
a change in the charge of the system or the dispersed particles, enhancing their
approximation and generation of micro-flogs.
Two cases of coagulation can be distinguished:
The chemical coagulation and the bio-coagulation (or denaturation) of
proteins. The chemical coagulation usually refers to a change in the surface
charge of the particles allowing particles to come closer. On the other hand,
bio-coagulation refers to a change in the structure of, or the unfolding of, the
protein followed by aggregation of proteins caused by coagulants, heat or
vigorous mixing.

62. Chemical coagulation is widely used in waste water treatment. It
allows the removal of colloidal particles and soluble substances
which have length of 10nm to 10 μm. The most common
coagulants for waste water treatment that neutralize the negative
charge of colloids are: inorganics such as aluminum sulfate,
aluminum chloride, polyamine chloride, ferric sulfate, ferrous
sulfate and ferric chloride or organicslike polyamines and
PolyDADMAC.
Coagulation of proteins is usually referred as denaturation. This
process is widely used in downstream processing for removing
proteins and tannins from beer and to create new products such as
cheese or desserts like meringues.
Coagulation is also used in cheese making. The casein milk proteins
are unfolded due to the addition of lime, lactic acid or bacteria
(remit), before the casein coagulates and traps fat between the new
caseins bonds, producing cheese.

63. Example:
Beer is produced from barley which contains proteins and tannins. Tannins give a bitter
taste to beer and are usually attached to proteins, therefore they must be removed in
order to achieve good taste and decrease the turbidity of the beer. When the beer broth
is heated, the proteins unfold exposing their internal hydrophilic part, allowing the
generation of new hydrogen bonds between proteins and so the generation of clogs. This
process is called “Hot break” and is one of the main downstream processes for beer
purification.

64. Flocculation
In a lot of ways, flocculation is very similar to chemical coagulation. Like coagulation, the
chemical basis is that particles, generally cells, are kept apart in a suspension via
electrostatic repulsion, due to negative charges on their surface. Flocculation can be
thought of as aggregating the micro-flocs generated into much larger flocs that then
settle on the base of the vessel. Hence both are generally involved in the same stage of
the downstream processing – the initial solid-liquid separation.
One key area for flocculation is within centrifugations, which are commonly performed
within downstream processing to separate the solids from the liquid via density
differences. The settling rate of the cells is increased greatly with diameter, hence
meaning that the larger the aggregate, the faster and more well defined the separation
of it from the liquid.
Within downstream processing, an example of used flocculation agents are chitosan and
synthetic cationic polyacrylamide within the recovery of 1,3-propanediol from
fermentation broth.
A different case of the use of flocculation is in the brewing of beer and yeast
Champagne. Here, flocculation of the yeast cells occurs naturally anyway within the
fermentation broth, thus allowing the cells to be easily separated.

65. The main additional use of flocculation is
waste-water management; removing small
particles contaminants, as well as pathogens

66. Precipitation
Precipitation is a process that is used for product purification
or recovery, especially in the primary purification. Figure
shows the basic mechanism of precipitation in a simple way
(Diana Romanini, 2013). It can be attached to different stages
for the product recovery process (P.F. Stanbury, 2017).
Meanwhile, precipitation can also be used as a partial process
for enrichment and concentration. Thus, the operating
volume of the following process is lower than it was
previously. Through the precipitation stage, products can be
obtained directly or by further technique after a cell lysate
(P.F. Stanbury, 2017). The mechanism of precipitation is that
soluble compounds in solution become insoluble due to
different chemical reaction parameters, e.g. pH. For instance,
ethanol precipitation is an approach for protein precipitation
by adding ethanol as an anti-solvent.

67. Crystallization
It is mostly applied in protein
structure analysis and is already
used as a cost effective and scalable
purification procedure for small
molecules.
Examples are the purification of low
molecular weight substances like
amino acids or industrial enzymes,
like industrial lipase, or ovalbumin.
In insulin purification, crystallization
is applied as polishing step
benefitting formulation aspects of
higher stability .