Downstream Processing

Pharmaceutical Biotechnology
M. Mujtaba Khan
 Downstream processing
Downstream processing is used 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.
 Initial product recovery
• The complexity of this step depends largely
upon whether the product is intracellular or
extracellular.
– Animal cell-culture-derived biopharmaceuticals
are secreted into the media
– In many recombinant prokaryotic producer cell
types the product accumulates intra cellularly.
• In both cases, upstream processing is followed
by initial collection (harvest) of the cells.
 Extraction
• Normally achieved by centrifugation, or
sometimes micro-filtration.
• If the product is an extracellular one, then the cell
paste is discarded.
• Whereas the product-containing extracellular
fluid is subject to further processing.
• 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
• A number of very efficient systems exist that
are capable of disrupting large quantities of
microbial biomass.
 Cell Disruption
• Disruption techniques, such as sonication are
usually confined to laboratory-scale
operations, due either to equipment
limitations or on economic grounds.
 Chemical Disruption
• Chemicals such as detergents are effective in
many cases of extraction, but they suffer from a
number of drawbacks;
• they often induce protein denaturation and
precipitation.
• may adversely affect a subsequent purification
step
– (e.g. the presence of detergent can prevent proteins
from binding to a hydrophobic interaction column).
• The presence chemicals in the final preparation.
– unacceptable for medical reasons.
 Mechanical Disruption
(Homogenization)
• This process is the most often used.
• Achieved by methods, such as
homogenization or by vigorous agitation with
abrasives.
• A cell suspension is forced through an orifice
of very narrow internal diameter at extremely
high pressures generating extremely high
shear forces.
 Homogenization
• As the microbial suspension passes through the outlet
point, it experiences an almost instantaneous drop in
pressure to normal atmospheric pressure.
• These act as very effective cellular disruption forces,
and result in the rupture of most microbial cell types.
• In most cases a single pass through the homogenizer
results in adequate cell breakage, but some cases may
require a second or third pass.
• An efficient cooling system minimizes protein
denaturation (due to the considerable amount of heat
generated during the homogenization process).
• Diagrammatic representation of a cell homogenizer.
• This represents one of a number of instruments
routinely used to rupture microbial cells, and in some
cases animal/plant tissue
 Homogenization
• Upon completion of the homogenization step,
cellular debris and any remaining intact cells
can be removed by centrifugation or by
microfiltration.
 Removal of nucleic acid
• In some cases it is desirable/necessary to
remove/destroy the nucleic acid content of a cell
homogenate prior to subsequent purification of
the released intracellular protein.
• large amounts of nucleic acids often significantly
increases the viscosity of the cellular
homogenate.
• Difficult to process, particularly on an industrial
scale.
 Removal of nucleic acid
• Significant increases in viscosity place additional
demands upon the method of cell debris removal
employed.
• Increased centrifugal forces for longer time
periods may be required to collect (pellet) cell
debris in such solutions efficiently.
• If a filtration system is employed to remove
cellular debris, then the increased viscosity will
also adversely affect flow rate and filter
performance.
 Removal of nucleic acid
• Effective nucleic acid removal is particularly
important when purifying a protein destined
for therapeutic use.
– a few picograms per therapeutic dose.
 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 precipitate is then removed, together with
cellular debris, by centrifugation or fi ltration.
 Removal of nucleic acid
• Nucleic acids may also be removed by treatment
with nucleases, which catalyse the enzymatic
degradation of these biomolecules.
• Nuclease treatment is quickly becoming the most
popular method of nucleic acid removal during
protein purification.
• This treatment is efficient, inexpensive and,
unlike many of the chemical precipitants used,
nuclease preparations themselves are innocuous
and do not compromise the final protein product.
 Initial product concentration
• Concentration of the crude protein product.
• This yields smaller product volumes, which are
more convenient to work with and can be
subsequently processed with greater speed.
• Product is precipitated using salts, such as
ammonium sulfate, or solvents, such as ethanol.
• The precipitate is then collected (usually by
centrifugation, but potentially also by filtration)
and the precipitate is re-dissolved in a small
volume of processing buffer.
• Ion-exchange chromatography can also
potentially be used to concentrate protein
solutions.
• Ultrafiltration, however, is by far the most
common method now used to achieve initial
product concentration.
 Ultrafiltration
• Utilized to remove whole cells or cell debris
from solution.
• Membrane filters employed in microfiltration
generally have pore diameters ranging from 0.1
to 10 μm.
• Ultrafiltration membranes’ pore diameters
normally range from 1 to 20 nm.
• Membranes with molecular mass cut-off points
of 3, 10, 30, 50, and 100 kDa are most
commonly used and commercially available.
 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 support equipment is required.
 Ultrafiltration
• The main drawback is its susceptibility to rapid
membrane clogging.
• Viscous solutions also lead to rapid decreases
in flow rates and prolonged processing times.
• Diafiltration is generally preceded by an
ultrafiltration step to reduce process volumes
initially.
• The actual diafiltration process is identical to that
of ultrafiltration, except for the fact that the level
of reservoir is maintained at a constant volume.
• This is achieved by the continual addition of
solvent lacking the low molecular mass molecules
that are to be removed.
Chromatographic Purification

High-resolution chromatography normally yields a protein that is

98–99 per cent pure.
 Final product formulation
• This generally involves:
• Addition of various excipients.
• Filtration of the final product through a 0.22
μm absolute filter in order to generate sterile
product, followed by its aseptic filling into
final product containers.
• Freeze-drying (lyophilization) if the product is
to be marketed in a powdered format.
 Disruptive influences
• can be chemical (e.g. oxidizing agents,
detergents, etc.)
• physical (e.g. extremes of pH, elevated
temperature, vigorous agitation)
• or biological (e.g. proteolytic degradation).
 Minimization of inactivation
• can be achieved by minimizing the
exposure of the product stream to such
influences, and undertaking downstream
processing in as short a time as possible.
• it is possible to protect the protein from
many of these influences by the addition of
suitable stabilizing agents.
• Proteolytic degradation and alteration of sugar
side-chains
– Degradation of a protein is characterized by hydrolysis
of one or more peptide (amide) bonds in the protein
backbone, generally resulting in loss of biological
activity.
• Can be avoided by:
– minimizing processing times;
– processing at low temperature;
– use of specifi c protease inhibitors.
• Protein deamidation
• hydrolysis of the side chain amide group of
asparagine and/or glutamine, yielding aspartic
acid and glutamic acid respectively.
• Oxidation and disulfide exchange
• The side chains of a number of amino acids are
susceptible to oxidation by air. Although the side chains
of tyrosine, tryptophan and histidine can be oxidized,
the sulfur atoms present in methionine or cysteine are
by far the most susceptible.
• Oxidation by air normally results only in disulfide bond
formation.
• The oxidation of any constituent amino acid residue
can (potentially) drastically reduce the biological
activity of a polypeptide.
• Oxidation can be best minimized by replacing
the air in the headspace of the final product
container with an inert gas such as nitrogen,
and/or the addition of antioxidants to the final
product.
Stabilizing excipients used in final
product formulations
 Final product fill
• The bulk final product first undergoes QC testing to ensure its
compliance with bulk product specifications.
• Good practices during manufacturing will ensure that the product
carries a low microbial load but it will not be sterile at this stage.
• The product is passed through a (sterilizing) 0.22 μm filter.
• The sterile product is
• housed (temporarily) in a sterile-product holding tank, from where
it is aseptically filled into pre-sterile final product containers.
• The filling process normally employs highly automated liquid filling
systems.
• All items of equipment with which the sterilized product comes into
direct contact must obviously themselves be sterile.
• range of filter types
and their stainless
steel housing.
 Freeze-drying
• Freeze drying (lyophilization) refers to the removal of
solvent directly from a solution while in the frozen
state.
• Removal of water directly from (frozen)
biopharmaceutical products via lyophilization yields a
powdered product.
• Freeze-dried biopharmaceutical products usually
exhibit longer shelf lives than products sold in solution.
• Freeze-drying is also recognized by the regulatory
authorities as being a safe and acceptable method of
preserving many parenteral products.