UNIT VI

DOWNSTREAM
PROCESSING
 WHAT IS A BIOPRODUCT?
 Bioproducts—chemical substances or combinations of
chemical substances that are made by living things
 Bioproducts can be broadly classified into three categories:
small molecules, large molecules, and particulate products.

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 CLASSIFICATION OF
BIOTECHNOLOGY PRODUCTS
• BASED ON CHEMICAL NATURE: Solvents, organic
acids, amino acid, proteins, antibiotics, lipids,
nucleic acids, cells, purified proteins, crude cell
extract, sugars/carbohydrates.

• BASED ON APPLICATION: Industrial chemicals,

agrochemicals, biopharmaceuticals, food, food
additives, diagnostic products, cosmetics,
laboratory products.
 CHARACTERISTICS OF
BIOTECHNOLOGY PRODUCTS
• Present in very low concentration.
• Several impurities are present along with the
desired product.
• Stringent quality requirements.
• Susceptibility to denaturation.
• Thermolabile.
Bioseparations are elaborate and expensive
 Up to 90% of new product cost is in downstream processing.
 More than 60% of the cost of third and fourth generation
antibiotics is in purification.
 For recombinant DNA fermentation products, downstream
processing can account for 80 to 90% of the overall
processing cost.

INVERSE RELATIONSHIP
BETWEEN PRICE AND
PRODUCTION
 FORMS OF SEPARATION
FORM OF PROCESS TECHNIQUES USED
SEPARATION
Separation of cells from Filteration, membrane
culture media separation, centrifugation
Particle- Liquid
Removal of bacteria from Centrifugation,
product solution sedimentation, floatation
Separation of plasmid Density Gradient
Particle-Particle DNA from chromosomal centrifugation, Membrane
DNA separation
Removal of solvent from Centrifugation, filteration,
solute sedimentation
Removal of dissolved
Solute-Solvent impurities from liquid Evaporation, Distillation,
product dialysis, Precipitation
Replacement of solvent
with some other solvent
 FORMS OF SEPARATION
FORM OF PROCESS TECHNIQUES USED
SEPARATION
One protein from another Ultrafilteration, dialysis
Solute-Solute Eg. Serum albumin from
serum proteins
Solvents such as acetone, Distillation, phase
Liquid-Liquid ethanol from aqueous separation
media
 POINTS TO BE CONSIDERED WHILE
DESIGNING BIOSEPARATION
1. The nature of starting material: e.g. a cell suspension,
a crude protein solution.
2. The initial location of the target product: e.g.
intracellular, extracellular, embedded in solid material
such as inclusion bodies.
3. The volume or flow-rate of the starting material.
4. The relative abundance of the product in the starting
material, i.e. its concentration relative to impurities.
 POINTS TO BE CONSIDERED WHILE
DESIGNING BIOSEPARATION
5. The susceptibility to degradation e.g. its pH
stability, sensitivity to high shear rates or
exposure to organic solvents
6. The desired physical form of the final
product, e.g. lyophilized powder, sterile
solution, suspension
7. The quality requirements, e.g. percentage
purity, absence of endotoxins or aggregates
8. Process costing and economics
 PHYSIOCHEMICAL BASIS OF
BIOSEPARATION
METHOD CHARACTERISTIC TECHNIQUES USED
SIZE Filteration, membrane
separation, centrifugation
DENSITY Centrifugation,
PHYSICAL sedimentation, floatation
DIFFUSIVITY Membrane separation
SHAPE Centrifugation, filteration,
sedimentation
POLARITY Chromatography, adsorption
SOLUBILITY Precipitation, crystalization
CHEMICAL ELECTROSTATIC Adsorption, electrophoresis
CHARGE
VOLATILITY Distillation, Evaporation
 RIPP Scheme in DSP
• RIPP- Recovery, Isolation, Purification and
Polishing
• commonly used in DSP
• RECOVERY AND ISOLATION- low resolution
techniques (e.g. precipitation, filtration,
centrifugation, and crystallization). Significantly
reduce the volume and overall concentration of the
material being processed.
• PURIFICATION AND POLISHING- high
resolution techniques (e.g. affinity separations,
chromatography, and electrophoresis). To obtain
pure and polished finished products.
PURIFICATION
OF REAGENT
GRADE
ANTIBODY
 Protein: Separation
• Different proteins exists within one cell
– Many steps needed to extract protein of interest, and separate
from many contaminants
– Separation techniques – Size, charge, solubility, binding
properties
– Before purification begins, protein must be released from cell by
homogenization
• Initial Recovery of Protein
– Intracellular or
– Extracellular
 Steps Involved
• Disrupt the biological source
– Blender, homogenizer
• Remove debris
– Centrifugation and filtration
• Precipitate/concentrate
– Ammonium sulfate
• Remove salt
– Dialysis
• Purify
– Chromatography
• Analyze and Polish
– Activity, molecular weight
– Removal of solvent, Lyophilization
 Cell Disruption
• Several method to disrupt cells
• When the membrane is disrupted, two things are added
– Buffer to maintain pH and compound dissolved in it to support
cell osmolarity
– Antioxidant – dithioth eitol o β-mercaptoethanol
• Animal cells (no cell wall)
– Homogenizer – A thick walled test tube with a tight fitting
plunger
– Osmotic shock: Mainly for cells without cell wall
– Freeze-thaw cycle: Continuous freezing and thawing – Ruptures
cells
 Cell Disruption
• Plant cells (Cell wall)
– Blender: Grinding tissue in a blender with a suitable buffer
– Releases soluble proteins and various subcellular organelles
• Microbial cells (Cell wall)
– Treatment with chemicals
• Detergents (e.g. triton x100, sodium cholate)
• Solvents (e.g. toulene, acetone)
• Chaotropic agents (e.g. urea, guanidine)
– Sonication
• Sound waves to break open cells
– Homogenization
• Agitation in the presence of abrasives (usually glass beads or sand)
– Lysozyme treatment
 Removal of Whole Cells and Debris
• Centrifugation
– Sample is spun, after lysis, to separate unbroken cells, nuclei,
other organelles and particles not soluble in buffer
– Differential Centrifugation
– Density Gradient Centrifugation
– Centrifuges
• Fixed angle
• Swinging bucket

• Filtration
– Cheese cloth: mainly removes materials that are not
homogenized
– Problem – plugging reduces the flow and ultimately clogging
– Removal of whole cells and cell debris after cell disruption
Centrifugation
Concentration and Primary Purification
• Large volumes of dilute broth are to be brought down to
Manageable amount
• In laboratory scale:
– Ultrafiltration
– Precipitation
– Dialysis
– Ion-exchange
• Solubility of Proteins in water
– Due to hydrophilic amino acids on their surfaces, they attract or
interact with water, proteins are soluble in water solutions
 Concentration by Ultrafiltration
• Most widely applied method both in laboratory and
industrial scale
• Ultrafiltration membranes (pore diameters: 1 – 20 nm)
• Traditional materials: cellulose acetate and cellulose
nitrate
• Nowadays: PVC and polycarbonate
• It’s a uick p ocess and has high ecove y ate
• But is susceptible to rapid membrane clogging
 Concentration by Precipitation
• One of the oldest and straight forward methods
• Addition of neutral salts, e.g. Ammonium sulfate (NH4)2SO4
– Solubilized proteins can be purified based on solubility (usually
dependent on overall charge, ionic strength, polarity)

• Addition of organic solvents (e.g. Ethanol and acetone)

• Addition of organic polymers (e.g. Polyethylene glycol-PEG)
• Affinity precipitation
– Addition of ligand (antibody), precipitates target protein by
biospecific-molecular interactions
 Salting Out
• Relies on the fact that proteins loose solubility as
concentration of salt is increased
– Results in a partial purification of all proteins with similar solubility
characteristics
– Salt takes away water by interacting with it, makes protein less
soluble (increases hydrophobic interactions)
• 1st step in research laboratories because it provides some
crude purification of proteins separating non-proteins and
some unwanted proteins out
• Different aliquots taken as function of salt concentration to
get closer to desired protein sample of interest (30, 40, 50,
75% increments)
– One fraction has protein of interest
 Dialysis
• Passage of solutes through a semi-permeable membrane.
• Pores in the dialysis membrane are of a certain size.
• Desired protein stays in, while water, salts, protein
fragments, and other molecules smaller than the pore
size pass through
 Separation by Column Chromatography
• Separation is based on size and shape, overall charge,
presence of surface hydrophobic groups, and ability to
bind various ligands
• Separation of different proteins is based on according to
their differential partitioning between two phases:
– Solid stationary phase:
Samples interacts with
this phase
– A liquid mobile phase:
Flows over the
stationary phase and
carries along with it the
sample to be separated
 Important Steps in Chromatography
• Pack column - Column is packed with material (resin) that
can absorb molecules based on some property (charge,
size, binding affinity, etc.)
• Wash column - Molecules washed
through the column with buffer

• Collect fractions - Fractions are taken,

at some point your molecule will elute
 Different Chromatographic Techniques
• Ion Exchange Chromatography
– Differences in protein surface charge at a given pH
• Gel Filtration Chromatography
– Differences in mass or shape of different proteins
• Affinity Chromatography
– Based on biospecific interaction between protein and ligand
 Ion Exchange Chromatography
• Proteins possess both acidic and basic
amino acids
– Interaction based on overall charge (less
specific than affinity)
• Cation exchanger
– Negatively charged resin – bound to Na+ or K+ ions
• Anion exchanger
– Positively charged resin – bound to Cl- ions
• Principle
– Reversible electrostatic attraction of a charged
molecule to a solid matrix possessing opposite charge
(separate by charge)
– Elution is done by increasing salt concentration or
changing pH
 Ion Exchange Chromatography
Carboxymethyl (CM)
Negatively charged resin
O
Column- CH2-C
O-

Diethylaminoethyl (DEAE)
Positively charged resin
+ C2H5
Column- CH2-CH2-NH
C2H5
 Ion Exchange Chromatography
• pH of medium vs. pI of protein
• Column is equilibrated with buffer of suitable pH and ionic
strength
• Proteins – net charge opposite to that of exchanger stick
to column
• No net charge or same charge elute - first
 Ion Exchange Chromatography
• If the column is negatively charged i.e. carboxymethyl then
– If the pH of the environment is below the pI (more acidic, >[H+]),
protein will attain +ve charge and will bind to the column
– If the pH of the environment is above the pI (more basic, >[OH-]),
protein will attain –ve charge and not bind to column but elute

• If the column is positively charged i.e. DEAE then

– If the pH of the environment is below the pI (more acidic, >[H+]),
protein will attain +ve charge and not bind to column but elute
– If the pH of the environment is above the pI (more basic, >[OH-]),
protein will attain –ve charge and will bind to the column
 Gel Filtration Chromatography
• Separation based on size and shape, also known as
Size Exclusion Chromatography
• Porous gel matrix in bead form is used as stationary
phase:
– e.g. Carbohydrate polymer such as
cross-linked dextran (Sephadex) or
agarose (Sepharose)
– Polyacrylamide (Bio-Gel)
• Extent of cross-linking can be
controlled to determine pore size
• Estimate molecular weight by
comparing sample with a set of
standards
 Gel Filtration Chromatography
• Smaller molecules enter the pores
and are delayed in elution time.
• Larger molecules do not enter and
elute from column before smaller
ones
 Affinity Chromatography
• It relies on the ability of most proteins to bind specifically
and reversibly to their ligands
• Most powerful and highly selective method
• Generally used in late purification steps
• Biospecific affinity chromatography
– General ligand approach: Cofactors (NAD+) or lectins
– Specific ligand approach: enzyme substrates, substrate
analogues or inhibitors, antibodies
• Increase in purity of over 1000-fold, with almost 100 %
yields are reported (at least in lab scale)
 Affinity Chromatography
• Stationary phase has a polymer that can be covalently
linked to a compound called a ligand that specifically
binds to protein
• Bound protein can be eluted from
column by adding high
concentrations of ligand in
soluble/mobile form.
• Competition –
proteins bound to
ligand in column will
bind to mobile
ligands
 Affinity Chromatography
• General ligand approach: Cofactors (NAD+)
– Use bound Adenosine -5’-monophosphate (AMP-Sepharose),
this is part Of NAD+.
– LDH will binds to AMP
– Release LDH by adding NADH, which binds tighter to the LDH
• Immunoaffinity purification
– Polyclonal antibodies: low binding capacity, some other proteins
can also bind
– Monoclonal antibodies: monospecific
– Relatively high cost technique
 Electrophoresis
• Charged particles migrate in electric
field toward opposite charge
• Proteins have different mobility:
– Charge
– Size and shape

• Polyacrylamide used mostly for proteins

 SDS- Polyacrylamide Gel Electrophoresis
• Protein is treated with detergent (SDS) sodium dodecyl
sulfate
– SDS breaks all tertiary and quaternary structures.
– Separates subunit by molecular weight
– Once denatured all proteins attain the form of random coil
– SDS form micelles around proteins thereby making proteins
negatively charged
– Proteins now separate by molecular weight
• Polyacrylamide has more resistance towards larger
molecules than smaller (SDS-PAGE)
– Smaller proteins move through faster
• Molecular weight of DNA and protein can be determined
SDS- PAGE
Extracellular
Product

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 Process for ethanol production from fermentation:

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 Manufacture of citric acid:

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 Penicillin production:

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 Production of an intracellular enzyme:

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 Processing scheme for recombinant insulin:

(Inclusion Body)

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 Processing scheme for recombinant insulin (2/2):

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