Downstream processing
Transcript of Downstream processing
What is Downstream Processing
Downstream – ‘after the fermentation process’
Primary ‘unit operations’ of Downstream Processing Cell recovery/removal
Centrifugation Dewatering
UltrafiltrationPrecipitationSpray drying
Downstream Processing
Secondary ‘unit operations’ Protein purification
Adsorption chromatography Gel permeation chromatography
Protein processing Immobilisation Beading/Prilling
Protein packagingSterilisation Bottling etc
Bioprocess Overview
Solid-liquidSeparation
Concentration
Purification
Formulation
Intra-CellularProduct
Final Product
Extra-CellularProduct
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
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
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
Proteinprecipitation
Celllysis Centrifugation
Protein fractionProtein
precipitation
Protein fraction
1 or 2 purificationsteps
Semi-purifiedprotein 3-4 purification
steps
Homogeneousprotein
Sterile bottling
To pharmaceuticals market
LyophilisationBottling
To chemicals market
Operational diagram of large-scale fungal batch fermentation system
Preculture Preparation of Fermentation Recovery of enzyme- inoculum containing medium
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
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
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.
Classification of Bioproducts Small molecules Macromolecules
Proteins Nucleic acids and nucleotide Polysaccharides
Engineering analysis
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)
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
Unit Operation Properties Used in separation ApplicationFiltration Size Solid RemovlCentrifugation Size, density Solid RemovlMicrofiltration Size Solid Removl Extraction Solvent Extraction Partition IsolationAqueous Two -Phase Extraction Partition Isolation Adsorption Ion Exchange Chromatography Charge Isolation, PurificationAffinity 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 PurificationReverse Phase Chromatography Size, molecular interaction PurificationChromatofocusing Charge, Mobility PurificationDisplacement Chromatography Molecular interactions Purification Electrophoresis Charge, Mobility PurificationUltrafiltration Size Isolation, PurificationReverse Osmosis Size, Molecular diffusivity Isolation, PurificationPrecipitation Solublity IsolationCrystalization Solublity, Molecular interactions Purification, Polishing
A good bioseparation process: Ensures desired purity of
product Ensures stability of product Keeps cost low Is reproducible Is scalable Meets regulatory guidelines
Common Stages of Bioseparation
• Removal of solids
• Isolation (volume reduction)
• Purification
• Polishing
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,
solventrecovery, water preparation
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
Biological products (contd..)Proteins:Food and food additivesNutraceuticalsIndustrial enzymesHormonesPharmaceutical enzymesPlasma derived productsMonoclonal antibodiesGrowth factorsClotting factorsThrombolyticsr-DNA derived proteinsDiagnostic proteinsVaccines
Filtration, precipitation, centrifugation, adsorption, chromatography, membrane based separations
DNA based products:DNA probes, plasmids, nucleotides, oligonucleotides
Filtration, precipitation, centrifugation, adsorption, chromatography, membrane based separations
Economic importance of bioseparation engineeringCost of bioseparation
Product Approximate relative price
Downstream processing cost
(%)Ethanol 1 15
SCP 0.8 20Yeast biomass 2 20
Citric acid 3.2 30-40Monosodium glutamate
5 30-40
Xanthan 20 50Penicillin G 60 20-30
Bulk enzymes 100 40-65
Therapeutic proteins/DNA
>500 60-80
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
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
Bioseparation methodsLow resolution-high throughput Cell disruption Precipitation Centrifugation Liquid-liquid extraction Leaching Filtration Supercritical fluid extraction Microfiltration DialysisHigh resolution-low throughput Ultracentrifugation Adsorption Packed bed chromatography Affinity separation Electrophoresis