This document discusses downstream processing in biotechnology. It defines downstream processing as the steps occurring after fermentation to recover and purify products. The key unit operations in downstream processing include cell removal, concentration, and purification techniques like chromatography. The level of purification required depends on the intended use and market for the product. Common downstream processing techniques are outlined along with considerations for designing efficient bioseparation processes.

1. DR. N. BANU
ASSOCIATE PROFESSOR
VELS UNIVERSITY

2. What is Downstream Processing
 Downstream – ‘after the fermentation process’
 Primary ‘unit operations’ of Downstream
Processing
 Cell recovery/removal
 Centrifugation
 Dewatering
 Ultrafiltration
 Precipitation
 Spray drying

3. Downstream Processing
 Secondary ‘unit operations’
 Protein purification
 Adsorption chromatography
 Gel permeation chromatography
 Protein processing
 Immobilisation
 Beading/Prilling
 Protein packaging
Sterilisation
 Bottling etc

6. Bioprocess Overview
Solid-liquid
Separation
Concentration
Purification
Formulation
Intra-Cellular
Product
Final Product
Extra-Cellular
Product
Cell Disruption
Upstream Processing
Centrifugation/Sedimentation,
Extraction, Filtration
Evaporation, Ultrafiltration,
Adsorption, Precipitation
Chromatography
Crystallisation, freeze drying,
Spray drying, sterile filtration
Chemical/Enzymatic/
Mechanical/Physical
Basic Biotechnology, 2nd Ed, Ch 9

7. Downstream processing depends on
product use
1. Enzyme preparations for animal feed
supplementation (e.g., phytase) are not
purified
2. Enzymes for industrial use may be partially
purified (e.g., amylase for starch industry)
3. Enzymes for analytical use (e.g., glucose
oxidase) and pharmaceutical proteins (e.g.,
TPA) are very highly purified

8. Fermentation
Culture supernatant
Centrifugation
to remove cells
Liquid preparation
to animal feed
market
Fermentation
Culture supernatant
Fermentation
Cell pellet
Intracellular fraction
Animal feed enzyme Analytical enzyme Therapeutic protein
Centrifugation
to remove cells
Centrifugation
to remove
medium
Protein
precipitation
Cell
lysis Centrifugation
Protein fraction
Protein
precipitation
Protein fraction
1 or 2 purification
steps
Semi-purified
protein 3-4 purification
steps
Homogeneous
protein
Sterile
bottling
To pharmaceuticals market
Lyophilisation
Bottling
To chemicals market

9. Operational diagram of large-scale fungal batch
fermentation system
Preculture Preparation of Fermentation Recovery of enzyme-
inoculum containing medium

10. Introduction to Bioproducts and
Bioseparations
• Bioproducts: They are produced by living cells or are
localized in cells from which they must be isolated.
• Bioseparation: Recovery, isolation, purification and
polishing of products synthesized by biotechnological
processes. Extended definition: Final polishing steps of
processes such as biotechnology based effluent
treatment and water purification
ream
processing
Bioreaction
Downstream
processing
Bioproduct/s
Impurities

11. Why do we need bioseparation?
 Enrichment of target product
 Reduction in bulk
 Removal of specific impurities
 Enhancement of product stability
 Achievement of product specifications
 Prevention of product degradation
 Prevention of catalysis other than the type desired
 Prevention of catalyst poisoning

12. Challenges in bioseparations
engineering
• Low product concentration concentrations
• Large number of impurities,
• Thermolabile bioproducts.
• Narrow operating pH and ionic strength window
• Shear sensitivity of bioproducts
• Low solubility of bioproducts in organic solvents
• Instability of bioproducts in organic solvents
• Stringent quality requirements
• Percentage purity
• Absence of specific impurities
An ideal bioseparation process should combine high
throughput with high selectivity, and should ensure
stability of product.

13. Classification of Bioproducts
 Small molecules
 Macromolecules
 Proteins
 Nucleic acids and nucleotide
 Polysaccharides
 Engineering analysis

14.  Three factors of designing bioseparation processes
 (1) purity
 (2) cost
 (3) market
 Material state and choice of separation methods
 Material secreted ( ultrafiltration, centrifugation)
Not secreted material (cell disruption, solid-liquid
separation)
 Material in liquid ( ultrafiltration, adsorption)
 Material in solid (extraction into aqueous solution)

15. Characteristics of Bioseparations
• Starting Materials
– Fermentation broth (bacteria and yeasts, mycelial fungi and
streptomycetes, mammalian or insect cell cultures) or defined
media and complex media
– Biological materials (blood, plant and animal tissues or
organs)
– Product concentration is usually dilute
• Properties utilized in bioseparations
– size, density, solubility, partitioning, mobility, charge,
hydrophobic interactions, biological molecular interactions, etc.
• Quality of products
– activity, purity, contaminants
– For biologics, consistency of products
• The “structure” or “composition” of the product is not very well
defined,
e.g. virus, glycoprotein.The sugar of protein has a lot of heterogeneity

16. Unit Operation Properties Used in separation Application
Filtration Size Solid Removl
Centrifugation Size, density Solid Removl
Microfiltration Size Solid Removl
Extraction
Solvent Extraction Partition Isolation
Aqueous Two -Phase Extraction Partition Isolation
Adsorption
Ion Exchange Chromatography Charge Isolation, Purification
Affinity Chromatography Molecular interaction Purification, Isolation
Hydrophobic Interaction Chromatography Prtein-ligand interaction Purification, Isolation
Metal Ion Chromatography Sequence-specific tag-metal interaction Purification, Isolation
Elution Chromatography
(Liquid Cromatography, HPCL)
Gel permeation Chromatography Size and Shape ofmolecules Purification
Reverse Phase Chromatography Size, molecular interaction Purification
Chromatofocusing Charge, Mobility Purification
Displacement Chromatography Molecular interactions Purification
Electrophoresis Charge, Mobility Purification
Ultrafiltration Size Isolation, Purification
Reverse Osmosis Size, Molecular diffusivity Isolation, Purification
Precipitation Solublity Isolation
Crystalization Solublity, Molecular interactions Purification, Polishing

17. A good bioseparation
process:
 Ensures desired purity of product
 Ensures stability of product
 Keeps cost low
 Is reproducible
 Is scalable
 Meets regulatory guidelines

18. Common Stages of Bioseparation
• Removal of solids
• Isolation (volume reduction)
• Purification
• Polishing

19. Typical Operations of Bioseparation
• Removal of solids
– Filtration, centrifugation, microfiltration
• Isolation of product (volume reduction)
– Cell disruption, extraction,
adsorption,ultrafiltration,
precipitation
• Purification
– Adsorption, elution chromatography, ultrafiltration,
electrophoresis, precipitation, crystallization
• Polishing
– Crystallization, drying, auxiliary process, solvent
recovery, water preparation

20. Biological products
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

21. Biological products (contd..)
Proteins:
Food and food additives
Nutraceuticals
Industrial enzymes
Hormones
Pharmaceutical enzymes
Plasma derived products
Monoclonal antibodies
Growth factors
Clotting factors
Thrombolytics
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

22. Economic importance of bioseparation engineering
Cost of bioseparation
Product Approximate relative
price
Downstream
processing cost (%)
Ethanol 1 15
SCP 0.8 20
Yeast biomass 2 20
Citric acid 3.2 30-40
Monosodium
glutamate
5 30-40
Xanthan 20 50
Penicillin G 60 20-30
Bulk enzymes 100 40-65
Therapeutic
proteins/DNA
>500 60-80

23. Strategies for bioseparation
A large number of bioseparation methods are available
The strategy is based on how best these can be utilized for
a given separation
The following need to be taken into account:
 The volume of process stream
 The relative abundance of the product in this process
stream
 The intended use of the product, i.e. purity
requirements
 The cost of the product
 Stability requirements

24. Conventional strategy:
The RIPP scheme
 Recovery, isolation, purification and polishing
 Based on a logical arrangement of bioseparation
methods
 Low-resolution, high-throughput techniques (e.g.
precipitation, filtration, centrifugation,
crystallization) are first used for recovery and
isolation
 High-resolution techniques (e.g. adsorption,
chromatography, electrophoresis) are then used for
purification and polishing
It is now possible to avoid this RIPP scheme

25. Bioseparation methods
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

26. Bioseparation methods
(contd..)
High resolution-high throughput
 Ultrafiltration
 Fluidized bed chromatography
 Membrane chromatography
 Monolith column chromatography