Downstream processing

Dr Saquiba Yesmine
 Downstream processing

Recovering a biological reagent from a cell culture supernatant is

one of the critical parts of the manufacturing procedure for
biotech products and purification costs typically outweigh those of
the upstream part of the production process.

Production of monoclonal antibodies, Protein A resin accounts for

some 10% of the cost while virus filtration can account for some
40% of the cost
 Down stream processing

• Separating the impurities from the product protein requires a series

of purification steps, each removing some of the impurities and
bringing the product closer to its final specification.
• In general, the starting feedstock contains cell debris and/or whole-
cell particulate material that must be removed.
• Defining the major contaminants in the starting material is helpful in
the downstream process design.
• This includes detailed information on the source of the material (e.g.,
bacterial or mammalian cell culture).
 Down stream processing
• Downstream processing serves to
(a) recover the therapeutic protein from its producer cell source upon
completion of the upstream processing phase
(b) purify the protein and
(c) formulate the protein into final product format.
Down stream
processing
• The steps of downstream processing are:
• 1-separation of biomass
• 2-cell disruption
• 3-concentration of broth
• 4-initial purification of metabolites
• 5-metabolite specific purification
• 6-de-watering
• 7-polishing of metabolites
1-separation of biomass: separating the biomass
(microbial cells) generally carried out by centrifugation or
ultra-centrifugation. If the product is biomass, then it is
recovered for processing and spent medium is discarded. If
the product is extra cellular the biomass will be discarded.
Ultra filtration is an alternative to the centrifugation.

2-cell disruption: If the desired product is intracellular the

cell biomass can be disrupted so that the product should
be released. The solid-liquid is separated by centrifugation
or filtration and cell debris are discarded.
3-concentration of broth: The spent medium is concentrated if the
product is extracellular.

4-initial purification of metabolites: According to the physicochemical

nature of the product molecule several methods for recovery of product
from the clarified fermented broth were used ( precipitation, solvent
extraction, ultra-centrifugation, ion-exchange chromatography,
adsorption and solvent extraction)

5-metabolite specific purification: specific purification methods are

used when the desired metabolite is purified to a very high degree.
• 6-de-watering: If low amount of product is found in very
large volume of spent medium, the volume is reduced by
removing water to concentrate the product. It is done by
vacuum drying or reverse osmosis.
• 7-polishing of metabolites: this is the final step of making
the product to 98 to 100% pure. The purified product is
mixed with several inert ingredients called EXCIPIENTS. the
formulated product is packed and sent to the market for the
consumers.
•
Down stream processing
Initial product recovery

• In the case of intracellular product, cell recovery is followed by

cellular disruption, in order to release the intracellular contents,
including the protein of interest.
• Cell disruption
• Disruption techniques, such as sonication or treatment with the
enzyme lysozyme, are usually confined to laboratory-scale operations,
due either to equipment limitations or on economic grounds.
• Disruption of microbial cells and, some animal/plant tissue types is
most often achieved by mechanical methods, such as homogenization
or by vigorous agitation with abrasives.
Initial product recovery: homoginization

• An efficient cooling system minimizes protein denaturation.

Homogenizers capable of handling large quantities of cellular
suspensions are now available, many of which can efficiently process
several thousand litres per hour.
• An additional method often employed to achieve microbial cell
disruption, both at the laboratory level and on an industrial scale,
involves cellular agitation in the presence of glass beads.
• Industrial- scale bead-milling systems can process in excess of 1000 l of
cell suspension per hour. Cooling systems minimize protein inactivation
by dissipating the considerable heat generated during this process.
Removal of nucleic acid
• Effective removal of nucleic acids during protein purification may be
achieved by precipitation or by treatment with nucleases.
• A number of cationic (positively charged) molecules are effective
precipitants of DNA and RNA; they complex with, and precipitate, the
negatively charged nucleic acids.
• The most commonly employed precipitant is polyethylenimine, a
long-chain cationic polymer.
• The precipitate is then removed, together with cellular debris, by
centrifugation or filtration.
Removal of nucleic acid
• Nucleic acids may also be removed by treatment with nucleases
• Nucleases catalyse the enzymatic degradation of these biomolecules.
• Indeed, nuclease treatment is quickly becoming the most popular method
of nucleic acid removal during protein purification.
• This treatment is:
- efficient
- inexpensive
Unlike many of the chemical precipitants used, nuclease preparations
themselves are
- innocuous and do not compromise the final protein product
Initial product concentration:
Ultrafiltration
• Membranes, pore diameters normally range from 1 to 20 nm. These
pores are sufficiently small to retain proteins of low molecular mass.
• Traditionally, ultrafilters have been manufactured from cellulose
acetate or cellulose nitrate.
• Several other materials, such as polyvinyl chloride and polycarbonate,
are now also used in membrane manufacture. Such plastic-type
membranes exhibit enhanced chemical and physical stability*.
• An important prerequisite in manufacturing ultrafilters is that the
material utilized exhibits low protein adsorptive properties.
• Large-scale ultrafiltration systems
invariably employ cartridge-type filters
(Figure). This allows a large filtration
surface area to be accommodated in a
compact area.
Ultrafiltration:
Ultrafiltration has become prominent as a method of protein concentration for a
variety of reasons:
• the method is very gentle, having little adverse effect on bioactivity of the
protein molecules;
• high recovery rates are usually recorded, with some manufacturers claiming
recoveries of over 99 per cent
• processing times are rapid when compared with alternative methods of
concentration
• little ancillary equipment is required
Drawback:
• Its susceptibility to rapid membrane clogging.
• Viscous solutions also lead to rapid decreases in flow rates and prolonged
processing times.
Filtration
• Filters are divided into three main categories:
• Cake filters
• Clarifying filters
• Cross-flow filters
Filtration
• Slurries with high solid concentration are operated in cake filters
• Very dilute liquid are passed through the clarifying filters
• The cross-flow filters are used for concentrating the solution by using
filtration technique.
Chromatographic purification
• Once the protein is recovered from its producer source and concentrated it
must be purified to homogeneity. Purification is generally achieved by column
chromatography.
• In general, a combination of two to four different chromatographic
techniques is employed in a typical downstream processing procedure.
• Gel-filtration and ion-exchange chromatography are amongst the most
common.
• Affinity chromatography is employed wherever possible, as its high
biospecificity facilitates the achievement of a very high degree of purification.
• Examples include the use of immunoaffinity chromatography to purify blood
factor VIII and lysine affinity chromatography to purify tPA.
Size-exclusion chromatography
(gel filtration)
• Fractionation of proteins by size-exclusion chromatography is achieved by
percolating the protein- containing solution through a column packed with a
porous gel matrix in bead form. As the sample travels down the column, large
proteins cannot enter the gel beads and hence are quickly eluted.
• Protein molecules, therefore, are usually eluted from a gel-filtration column in
order of decreasing molecular size.
• In most cases the gel matrices utilized are prepared by chemically cross-linking
polymeric molecules such as dextran, agarose, acrylamide and vinyl polymers.
• Although size-exclusion chromatography is an effective fractionation tech-
nique, it generally results in a significant dilution of the protein solution
relative to the starting volume applied to the column.
Size-exclusion chromatography
(gel filtration)
• Although size-exclusion
chromatography is an effective
fractionation technique, it
generally results in a significant
dilution of the protein solution
relative to the starting volume
applied to the column.
 Ion-exchange
chromatography
• Ion-exchange chromatography is based upon the principle of reversible
electrostatic attraction of a charged molecule to a solid matrix that contains
covalently attached side groups of opposite charge.
• Proteins may subsequently be eluted by altering the pH or by increasing the
salt concentration of the irrigating buffer.
• Ion-exchange matrices that contain covalently attached positive groups are
termed anion exchangers i.e., species such as aminoethyl and
diethylaminoethyl groups with positively charged functional groups.
• These will adsorb anionic proteins, e.g. proteins with a net negative charge.
• Matrices to which negatively charged groups are covalently attached are
termed cation exchangers, adsorbing cationic proteins, e.g. positively charged
proteins.
• Negatively charged groups attached to suitable matrices forming cation
 Ion-exchange
chromatography
• During the cation-exchange process, positively
charged proteins bind to the negatively charged ion-
exchange matrix by displacing the counter ion (often
H+), which is initially bound to the resin by
electrostatic attraction.
• Elution may be achieved using a salt-containing
irrigation buffer. The salt cation, often Na+ of NaCl, in
turn displaces the protein from the ion-exchange
matrix.
• In the case of negatively charged proteins, an anion
exchanger is obviously employed, with the protein
adsorbing to the column by replacing a negatively
charged counter ion.
Principle of ion-exchange chromatography, in this case anion exchange chromatography. The chromatographic beads exhibit
an overall positive charge. Proteins displaying a nett negative charge at the pH selected for the chromatography will bind to
the beads due to electrostatic interactions
Ion-exchange
chromatography
• The vast majority of purification procedures employ at least one ion-
exchange step
• It represents the single most popular chromatographic technique in
the context of protein purification. Its popularity is based upon
- the high level of resolution achievable
- its straightforward scale-up (for industrial application)
- ease of use and ease of column regeneration
- it leads to a concentration of the protein of interest. It is also one of
the least expensive chromatographic methods available.
- Anion-exchange chromatography, therefore, is most commonly used.
Affinity chromatography
• Affinity chromatography is often described as the most powerful highly
selective method of protein purification available.
• This technique relies on the ability of most proteins to bind specifically and
reversibly to other compounds, often termed ligands.
• Affinity chromatography offers many advantages over conventional
chromatographic techniques. The specificity and selectivity of biospecific
affinity chromatography cannot be matched by other chromatographic
procedures.
• Increases in purity of over 1000-fold, with almost 100 per cent yields, are often
reported, at least on a laboratory scale. Incorporation of an affinity step could
thus drastically reduce the number of subsequent steps required to achieve
protein purification. This, in turn, could result in dramatic time and cost savings.
• A wide variety of ligands may be covalently attached to an inert
support matrix, and subsequently packed into a chromato- graphic
column.
• In such a system, only the protein molecules that selectively bind to
the immobilized ligand will be retained on the column.
• Washing the column with a suitable buffer will flush out all unbound
molecules.
• An appropriate change in buffer composition, such as inclusion of a
competing ligand, will result in desorption of the retained proteins.
• Elution of bound protein from an affinity column is achieved by
altering the composition of the elution buffer, such that the affinity of
the protein for the immobilized ligand is greatly reduced. A variety of
non-covalent interactions contribute to protein–ligand interaction. In
many cases, changes in buffer pH, ionic strength, inclusion of a
detergent or agents such as ethylene glycol (which reduce solution
polarity) may suffice to elute the protein.
Affinity Chromatography
• Despite such promise, bio-specific affinity chromatography does display
some practical limitations:
• many bio-specific ligands are extremely expensive and often exhibit poor
stability;
• many of the ligand coupling techniques are chemically complex, hazardous,
time consuming
• costly;
• any leaching of coupled ligands from the matrix also gives cause for
concern for two reasons, as (a) it effectively reduces the capacity of the
system and (b) leaching of what are often noxious chemicals into the
protein products is undesirable.
Hydrophobic interaction
chromatography
• Of the 20 amino acids commonly found in proteins, eight are classified
as hydrophobic, due to the non-polar nature of their side chains
• Hydrophobic interaction chromatography fractionates proteins by
exploiting their differing degrees of surface hydrophobicity. It depends
on the occurrence of hydrophobic interactions between the
hydrophobic patches on the protein surface and hydrophobic groups
covalently attached to a suitable matrix.
• The most popular hydrophobic interaction chromatographic beads
(resins) are cross-linked agarose gels to which hydrophobic groups have
been covalently linked
Hydrophobic interaction
chromatography
• Protein separation by hydrophobic interaction chromatography is dependent upon
interactions between the protein itself, the gel matrix and the surrounding aqueous
solvent.
• Increasing the ionic strength of a solution by the addition of a neutral salt (e.g.
ammonium sulfate or sodium chloride) increases the hydrophobicity of protein
molecules.
• Reversed phase chromatography may also be used to separate proteins on the basis of
differential hydrophobicity.
• This technique involves applying the protein sample to a highly hydrophobic column to
which most proteins will bind.
• Elution is promoted by decreasing the polarity of the mobile phase. This is normally
achieved by the introduction of an organic solvent. Elution condi- tions are harsh and
generally result in denaturation of many proteins.
Reversed phase chromatography
• Mobile phase is significantly more
polar tha the stationary phase.
Challenge of Downstream
processing
• Low product concentration
• Large number of impurities
• Thermolabile bioproducts

An ideal bioseperation process should include high selectivity as well as

stability of the products.