This document discusses various downstream processing techniques used to purify proteins produced by biopharmaceutical manufacturing. It describes key steps like removal of insolubles, product isolation, purification, and polishing. Specific techniques covered include chromatography methods like ion exchange, size exclusion, hydrophobic interaction, and affinity chromatography. It also provides examples of using hydrophobic interaction chromatography and affinity chromatography to purify green fluorescent protein.

1. Biopharmaceutical
Manufacturing
Deanna Scott, B.S., RAC
MIP 480
Lab Basics for the Biotech Industry

2. Downstream
Processing
Deanna Scott, M.S., RAC
MIP480
Lab Basics for the Biotech
Industry

3. General Steps in Downstream
Protein Purification

4. Remember in Upstream
Processing…
Biological activity of a protein is based on
hydrophobicity and charge.
Post translational modifications play a key
role in activity.
Therefore, the expression system chosen has
a profound affect on protein localization, and
hence the down stream purification needs.

5. Use of Protein Production
Platforms

7. Upstream
Proteins are targeted to different
compartments of the cells and subcellular
localization of recombinant protein affects:
Post-translation modifications
Aggregation of protein
Refolding
Enzymatic activity
Secretion

8. Upstream, Fermentation,
Downstream Integration

9. E. Coli
Most important organism in bioprocessing.
Gram Negative – inner membrane – cell wall
– external membrane
Secretion mechanisms typically direct an
accumulation of the recombinant protein
between the two membranes.
This creates the need for a milder treatment
to release the product.

10. Stages in Downstream
Processing
Removal of Insolubles
Product Isolation
Product Purification
Product Polishing
A few product recovery methods may be considered
to combine two or more stages.
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.

11. Removal of Insolubles
Separation of cells, cell debris or other
particulate matter
Typical operations to achieve this:
Filtration
Centrifugation
Sedimentation
Flocculation a process where a solute
comes out of solution in the form of floc or
flakes.
Gravity settling

12. Product Isolation
Removal of those components whose
properties vary markedly from that of the
desired product.
Water is the chief impurity
Isolation steps are designed to remove it (i.e.
dialysis)
Reducing the volume
Concentrating the product.
Solvent extraction, adsorption, ultrafiltration, and
precipitation are some of the unit operations
involved.

13. Product Purification
Done to separate those contaminants that resemble
the product very closely in physical and chemical
properties.
Expensive to carry out
Require sensitive and sophisticated equipment
Significant fraction of the entire downstream
processing expenditure.
Examples of operations include affinity, size
exclusion, reversed phase chromatography,
crystallization and fractional precipitation.

14. Chromatography
Separation of mixtures
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.

15. Chromatography Terms:
Stationary Phase
The substance which is fixed in place for the
chromatography procedure.
Examples:
silica layer in thin layer chromatography

16. Chromatography Terms:
Mobile Phase
The phase which moves in a definite
direction.
Liquid (LC and CEC)
Gas (GC)
Supercritical fluid (supercritical-fluid
chromatography, SFC).
The mobile phase consists of the sample
being separated/analyzed and the solvent
that moves the sample through the column.

17. Chromatography Terms:
Analyte
The substance that is to be separated during
chromatography
Examples:
E. coli culture filtrate
Yeast cell extrations

18. Chromatography Terms:
Chromatograph
A chromatograph is equipment that enables a
sophisticated separation e.g. gas
chromatographic or liquid chromatographic
separation.

19. Chromatography Terms:
Chromatogram
The visual output of the chromatograph
In the case of an optimal separation, different
peaks or patterns on the chromatogram
correspond to different components of the
separated mixture.

20. Chromatography Terms:
Preparative vs. Analytical
Preparative chromatography
Separate the components of a mixture for further
use (and is thus a form of purification).
Analytical chromatography
Operates with smaller amounts of material
Seeks to measure the relative proportions of
analytes in a mixture.
The two are not mutually exclusive

21. Techniques by chromatographic
bed shape
Column Chromatography
Planar Chromatography
Paper Chromatography
Thin layer Chromatography

22. Column Chromatography
Column chromatography is a separation technique
in which the stationary bed is within a tube.
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:
packed column
open tubular column
Differences in rates of movement through the
medium are calculated to different retention times of
the sample.

23. Column Chromatography-
Expanded Bed Adsorption
Fluidized bed vs. solid phase
This allows omission of initial clearing steps:
Centrifugation
Filtration, for culture broths
Slurries of broken cells
The principle of EBA is to allow the
chromatography beads to fluidize in the
feed stream which is pumped at low
pressure.
The expanded bed allows particulate
impurities in the feed stream to pass freely –
and at very high flow rates – through the
system without any clogging or pressure
building up throughout the process.

24. Planar Chromatography
The stationary phase is present as or on a
plane.
Paper, serving as such or impregnated by a
substance as the stationary bed
Paper chromatography
Layer of solid particles spread on a support such
as a glass plate
Thin layer chromatography

25. Paper Chromatography
Compounds in the sample mixture
travel different distances according
to how strongly they interact with
the paper.
Cellulose (aka “paper’):
Is a polar molecule
Compounds within the mixture
travel farther if they are non-
polar.
More polar substances bond with
the cellulose paper more quickly,
and therefore do not travel as far.

26. Thin Layer Chromatography
Similar to paper chromatography
Instead of using a stationary phase of paper, it involves a
stationary phase of a thin layer of adsorbent:
Silica gel, alumina, or cellulose on a flat, inert
substrate.
Compared to paper:
Runs faster
Better separations
Choice between different adsorbents.
Different compounds in the sample mixture travel
different distances according to how strongly they
interact with the adsorbent.

27. Techniques by physical state of
mobile phase
Gas chromatography
Liquid chromatography

28. Gas Chromatography
Mobile phase is a gas.
Carried out in a column
Based on a partition equilibrium of analyte between
a solid stationary phase (often a liquid silicone-
based material) and a mobile gas (most often
Helium).
The stationary phase is adhered to the inside of a
small-diameter glass tube (a capillary column) or a
solid matrix inside a larger metal tube (a packed
column).
This causes a difference in retention time

29. Gas Chromatography
It is widely used in
analytical chemistry
High temperatures
used in GC make it
unsuitable for high
molecular weight
biopolymers or
proteins (heat will
denature them)

30. Liquid Chromatography
Mobile phase is a liquid.
Carried out either in a column or a plane.
HPLC
In the HPLC technique, the sample is forced
through a column that is packed with
irregularly or spherically shaped particles or a
porous monolithic layer (stationary phase) by
a liquid (mobile phase) at high pressure.

31. HPLC Configuration

32. Techniques by separation
mechanism
Ion exchange chromatography
Size exclusion chromatography (SEC)
Reversed-phase chromatography (RP-HPLC)
Two-dimensional chromatography (2D)
Hydrophobic Interaction chromatography
(HIC)

33. Ion Exchange Chromatography
Used charged stationary
phase to separate charged
compounds
Resin that carries charged
functional groups which
interact with oppositely
charged groups of the
compound to be retained.
FPLC

34. Definition: Ion
Ion is an atom or molecule which has lost or gained
one or more valence electrons, giving it a positive or
negative electrical charge.
Anions are negatively charged ions, formed when
an atom gains electrons in a reaction. Anions are
negatively charged because there are more
electrons associated with them than there are
protons in their nuclei.
Cations are positively charged ions, formed when an
atom loses electrons in a reaction, forming an
'electron hole'.

35. Size Exclusion Chromatography
(SEC)
Gel permeation/filtration
chromatography (GPC)
Separates molecules
according to their size
Low resolution
"polishing"
Tertiary/Quaternary structure
(native)

36. Size Exclusion Chromatography
(SEC)

37. Reverse-Phase Chromatography
Reversed-phase
chromatography is an
elution procedure used
in liquid
chromatography in
which the mobile
phase is significantly
more polar than the
stationary phase.

38. Definitions: Polarity
The dipole-dipole intermolecular forces
between the slightly positively-charged end of
one molecule to the negative end of another
or the same molecule.
Molecular polarity is dependent on the
difference in electronegativity between atoms
in a compound and the asymmetry of the
compound's structure.

39. Two-dimensional
chromatography
Insufficient separation of some analytes.
It is possible to direct a series of unresolved
peaks onto a second column with different
physico-chemical
Since the mechanism of retention on this new
solid support is different from the first
dimensional separation, it can be possible to
separate compounds that are
indistinguishable by one-dimensional
chromatography.

40. Hydrophobic Interaction
Chromatography (HIC)
When the analyte, is passed over a HIC
column in a highly salty buffer (Binding
Buffer), the hydrophobic regions
Stick to the HIC beads.
Other proteins which are less hydrophobic (or
more hydrophilic) pass right through the
column.
This procedure allows the purification of one
protein from a complex mixture of bacterial
proteins.

41. Green Fluorescent Protein
Purification Using HIC
Equilibration buffer
A medium salt buffer (2 M (NH4)2SO4) which
is used to "equilibrate" or "prime" the column

42. Green Fluorescent Protein
Purification Using HIC
Binding buffer
An equal volume of high salt
Binding Buffer (4 M (NH4)2SO4) is
added to the bacterial lysate.
Supernatant containing GFP has
the same salt concentration as the
equilibrated column.
When in a high salt solution, the
hydrophobic regions of proteins
are more exposed and are able to
interact with and bind the
hydrophobic regions of the
column.

43. Green Fluorescent Protein
Purification Using HIC
Wash buffer
A medium salt Wash Buffer
(1.3 M (NH4)2SO4) is used
to wash weakly associated
proteins from the column
Proteins which are strongly
hydrophobic (GFP) remain
bound to the column.

44. Green Fluorescent Protein
Purification Using HIC
Elution buffer
A low salt buffer (TE Solution; 10 mM
Tris/EDTA) is used to wash GFP from
the column.
In low salt buffers (which have a
higher concentration of water
molecules), the conformation of GFP
changes so that the hydrophilic
residues of GFP are more exposed to
the surface, causing the GFP to have
a higher affinity for the buffer than for
the column, thereby allowing the GFP
to wash off the column.

45. Affinity Chromatography
Immunoadsorbent
Antibody binding
Elution
Dialysis

46. Affinity Chromatography
Immunosorbant
Column Chromatography
Solid matrix:
Agarose
Sephadex
Derivatives of cellulose, or other
polymers

47. Affinity Chromatography
Antibody Absorption
The serum is passed over
the immunoadsorbent.
Antibodies bind
noncovalently to matrix
Antibodies of other
specificities (green) and
other serum proteins
(yellow) will pass through
unimpeded.

48. Affinity Chromatography
Elution
Disruption of non covalent interaction
Buffers
High [NaCl] and/or
Low pH
Denaturing agents: 8 M urea
Soluble form of the antigen.
These compete with the immunoadsorbent for the
antigen-binding sites of the antibodies and
release the antibodies to the fluid phase.

49. Affinity Chromatography
Dialysis
The eluate is then dialyzed against, for
example, buffered saline in order to remove
the reagent used for elution.

50. Product Polishing
End with packaging of the product in a form that is
stable, easily transportable and convenient.
Crystallization
Desiccation
Lyophilization
Spray drying
May include:
Sterilization of the product
Remove or deactivate trace contaminants which might
compromise product safety
viruses or depyrogenation

51. Green Fluorescent Purification

52. Green Fluorescent Purification

53. Green Fluorescent Purification

54. Green Fluorescent Purification