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Deanna Scott, B.S., RAC
MIP 480
Lab Basics for the Biotech Industry
Downstream
Processing
Deanna Scott, M.S., RAC
MIP480
Lab Basics for the Biotech
Industry
General Steps in Downstream
Protein Purification
Remember in Upstream
Processing…
z Biological activity of a protein is based on
hydrophobicity and charge.
z Post translational modifications play a key
role in activity.
z Therefore, the expression system chosen has
a profound affect on protein localization, and
hence the down stream purification needs.
Use of Protein Production
Platforms
Upstream
z Proteins are targeted to different
compartments of the cells and subcellular
localization of recombinant protein affects:
z Post-translation modifications
z Aggregation of protein
z Refolding
z Enzymatic activity
z Secretion
Upstream, Fermentation,
Downstream Integration
E. Coli
z Most important organism in bioprocessing.
z Gram Negative – inner membrane – cell wall
– external membrane
z Secretion mechanisms typically direct an
accumulation of the recombinant protein
between the two membranes.
z This creates the need for a milder treatment
to release the product.
Stages in Downstream
Processing
z Removal of Insolubles
z Product Isolation
z Product Purification
z Product Polishing
z A few product recovery methods may be considered
to combine two or more stages.
z For example, expanded bed adsorption accomplishes
removal of insolubles and product isolation in a single step.
Affinity chromatography often isolates and purifies in a
single step.
Removal of Insolubles
z Separation of cells, cell debris or other
particulate matter
z Typical operations to achieve this:
z Filtration
z Centrifugation
z Sedimentation
z Flocculation a process where a solute
comes out of solution in the form of floc or
flakes.
z Gravity settling
Product Isolation
z Removal of those components whose
properties vary markedly from that of the
desired product.
z Water is the chief impurity
z Isolation steps are designed to remove it (i.e.
dialysis)
z Reducing the volume
z Concentrating the product.
z Solvent extraction, adsorption, ultrafiltration, and
precipitation are some of the unit operations
involved.
Product Purification
z Done to separate those contaminants that resemble
the product very closely in physical and chemical
properties.
z Expensive to carry out
z Require sensitive and sophisticated equipment
z Significant fraction of the entire downstream
processing expenditure.
z Examples of operations include affinity, size
exclusion, reversed phase chromatography,
crystallization and fractional precipitation.
Chromatography
z Separation of mixtures
z Passing a mixture dissolved in a "mobile
phase" through a stationary phase, which
separates the analyte to be measured from
other molecules in the mixture and allows it to
be isolated.
Chromatography Terms:
Stationary Phase
z The substance which is fixed in place for the
chromatography procedure.
z Examples:
z silica layer in thin layer chromatography
Chromatography Terms:
Mobile Phase
z The phase which moves in a definite
direction.
z Liquid (LC and CEC)
z Gas (GC)
z Supercritical fluid (supercritical-fluid
chromatography, SFC).
z The mobile phase consists of the sample
being separated/analyzed and the solvent
that moves the sample through the column.
Chromatography Terms:
Analyte
z The substance that is to be separated during
chromatography
z Examples:
z E. coli culture filtrate
z Yeast cell extrations
Chromatography Terms:
Chromatograph
z A chromatograph is equipment that enables a
sophisticated separation e.g. gas
chromatographic or liquid chromatographic
separation.
Chromatography Terms:
Chromatogram
z The visual output of the chromatograph
z In the case of an optimal separation, different
peaks or patterns on the chromatogram
correspond to different components of the
separated mixture.
Chromatography Terms:
Preparative vs. Analytical
z Preparative chromatography
z Separate the components of a mixture for further
use (and is thus a form of purification).
z Analytical chromatography
z Operates with smaller amounts of material
z Seeks to measure the relative proportions of
analytes in a mixture.
z The two are not mutually exclusive
Techniques by chromatographic
bed shape
z Column Chromatography
z Planar Chromatography
z Paper Chromatography
z Thin layer Chromatography
Column Chromatography
z Column chromatography is a separation technique
in which the stationary bed is within a tube.
z The particles of the solid stationary phase or the
support coated with a liquid stationary phase may fill
the whole inside volume of the tube:
z packed column
z Better separations