This document discusses downstream processing (DSP) in bioproduct manufacturing. It defines DSP as a series of isolation and purification steps to separate biological molecules from complex mixtures. DSP is challenging due to the similar properties of products, impurities, and contaminants. The document outlines various unit operations used in low, medium, and high resolution separation. It emphasizes that DSP is a major cost in bioproduct production and recovery method selection impacts overall economics. Proper method choice requires understanding the product properties, quality needs, and ability to scale the process.

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3. Definitions:
Down Stream Processing
“Complicated series of Isolation and Purification steps,
which are usually quite costly”
“Application of Principles of Bioseparations in a manner
that will apply to bioproducts not yet invented, and
biological molecules not yet produced on a large scale, as
well as the molecules that form the basis of the current
industry”
“Application of fundamental engineering and biological
principles to the design of adsorbents, systems, and
processes for the separation of biological molecules with
a mechanistic analysis as a key phenomena at a
microscopic level”
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4. Challenge of Down Stream Processing
It is difficult to efficiently and economically
recover a high purity biochemical product
from a complex mixture of related and
functional molecules, impurities and
contaminants which have similar physical and
chemical properties.
Bioseparations Engineering
“The Systematic study of the scientific and engineering
principles utilized for large-scale purification of
biological products
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BIOLOGICAL PRODUCTS
(with different bioseparation process)
Product Nature of bioseparation required
Alcoholic beverages:
Beer, wine, spirits
Clarification, distillation
Organic acids:
Acetic acid, citric acid
Precipitation, filtration, adsorption, solvent
extraction
Vitamins:
Vitamin C, vitamin B12, riboflavin
Precipitation, filtration, adsorption, solvent
extraction
Amino acids:
Lysine, glycine, phenylalanine
Precipitation, filtration, adsorption, solvent
extraction
Antibiotics:
Penicillins, neomycin, bacitracin
Precipitation, filtration, adsorption, solvent
extraction
Carbohydrates:
Starch, sugars, dextrans
Precipitation, filtration, adsorption
Lipids:
Glycerol, fats, fatty acids
Precipitation, filtration, adsorption, solvent
extraction

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Proteins:
Food and food additives
Neutraceuticals
Industrial enzymes
Hormones
Pharmaceutical enzymes
Plasma derived products
Monoclonal antibodies
Growth factors
Clotting factors
Thrombolytes
r-DNA derived proteins
Diagnostic proteins
Vaccines
Filtration, precipitation, centrifugation,
adsorption, chromatography, membrane
based separations
DNA based products:
DNA probes, plasmids, nucleotides,
oligonucleotides
Filtration, precipitation, centrifugation,
adsorption, chromatography, membrane
based separations
BIOLOGICAL PROD UCTS
(with different bioseparation process)

9. THE DIVERSE BIOCHEMICAL PROCESS INDUSTRY
•PRODUCTS AND SERVICES FORMULTIPLE MARKETS
Food & Beverage
Health Care
Therapeutics
Diagnostics
Device
Specialty Chemical
Commodity Chemicals
Waste Treatment
•MANUFACTURING BY MULTIPLE SYNTHETIC & EXTRACTIVETECHNOLOGIES
Biosynthetic
-Microbial, Animal, Plant
Extractive
–Animal, Plant
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10. THE DIVERSE BIOCHEMICAL PROCESS INDUSTRY (Continued)
• PRODUCTS BELONG TO MULTIPLE CLASSES
– Small Molecules
– Proteins
– Nucleic Acids
– Carbohydrates
– Catabolites & Anabolites
– Cells And Viruses
• PRODUCTS & PROCESSES REGULATED
– FDA
– EPA
– OSHA
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11. POINTS TO CONSIDER IN DOWNSTREAM PROCESSING
• DSP begins with Raw Material selection
“Garbage in means garbage out”
• There are trade offs, e.g. between purity and yield
“No Free lunch”
• Mass and Energy are conserved.
“What goes in must come out somewhere and in some form.
There may be transformation in form.”
• There are Impurities and Contaminants
• You will be watched.
e.g. by customers (internal and external) and the FDA.
Define metrics and appropriate analytical methods.
• Regulation by FDA, EPA, and OSHA
• Design:
– Target –the Spec Sheet
– Path –the PFD
– Measure –Analytical
•Murphy’s Law
– Contaminants –need control
– Lost material –need robustness
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Product Bioseparation
cost (%)
Solvents e.g. ethanol, acetone 15-20
Cells, e.g. bakers yeast, brewers yeast 20-25
Crude cellular extracts, e.g. yeast extract 20-25
Organics acids, e.g. citric acid, lactic acid 30-40
Vitamins and amino acids e.g. lysine, ascorbic acid 30-40
Gums and polymers, e.g. xanthan, gellan 40-50
Antibiotics, e.g. penicillins, rifanpicin 20-60
Industrial enzymes, e.g. Amyloglucosidase, glucose isomerase 40-65
Non-recombinant therapeutic proteins, e.g. pancreatin, papain 50-70
r-DNA products, e.g. recombinant insulin, recombinant streptokinase 60-80
Monoclonal antibodies 50-70
Nucleic acid based products 60-80
Plasma proteins, human albumin, human immunoglobulins 70-80
Economic importance of bioseparation
(cost of bioseparation)

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Concentration in the broth versus final selling price.
(Reprinted by permission from J. L. Dwyer, “Scaling Up Bioproduct Separation with High Performance Liquid Chromatography,”
Bio/Technology, vol. 2, p. 957, 1984.)
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14. Low resolution-high throughput
 Cell disruption
 Precipitation
 Centrifugation
 Liquid-liquid extraction
 Leaching
 Filtration
 Supercritical fluid extraction
 Microfiltration
 Dialysis
High resolution-low throughput
 Ultracentrifugation
 Adsorption
 Packed bed chromatography
 Affinity separation
 Electrophoresis
High resolution-high throughput
o Ultrafiltration
o Fluidized bed chromatography
o Membrane chromatography
o Monolith column chromatography
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15. SUPPLY CHAIN IN BIOPROCESSING
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1. What is the product?
2. What is the value of product?
3. What is the acceptable product quality for the proposed end
use?
4. Where is the product in the complex mixture?
5. What are the physical and chemical properties of the
product and the impurities?
6. Is the product stable?
7. What are the economic of the various isolation procedure?
8. Are they any contamination / health risk?
9. Can the isolation procedure be scaled up?
HOW TO CHOOSE A GOOD
SEPARATION METHOD?

17. WHEN SELECTING UNIT OPERATIONS THERE ARE CHOICES AND DECISIONS
MUST BE MADE
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19. Role and Importance of DSP:
• A key segment of the production and marketing of a pharmaceutical
product (antibiotic, peptide or a complex protein) is the processing of
the material from its initial milieu (tissue or fermentation broth) to
a pure form suitable for its intended use.
• The knowledge of genetic basis of biological functioning creates
the challenges and opportunities of applying information derived
from genomes to the production of biological products.
• Large-scale Bioseparations represents both art and science.
• The manner in which the bioproduct is associated with the cell or
organism defines its initial recovery characteristics.
• Product recovery is usually accounts for a large portion of the product
cost and in some cases it is the major manufacturing cost.
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