DR. N. BANUASSOCIATE PROFESSOR

VELS UNIVERSITY

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

Bioseparation methods (contd..)

High resolution-high throughput Ultrafiltration Fluidized bed chromatography Membrane chromatography Monolith column chromatography