Transcript Intoduction - Chulabhorn Research Institute

Purification strategies
By : Miss Thida Chanyachukul
AIDs Production group
Outline
General concepts in Purification strategies
Principle of purification operations
Special considerations in our biopharmaceutical products
Ritonavir (Protease inhibitor)
Vaccine
IL-2
Conclusions
References
General concepts
Purification strategies
important prerequisite in pharmaceutical manufacture
more predictable and controllable
not all problems in purification system are solved by the acquisition of
sophisticated laboratory equipment and column packing
difficult to find optimum conditions of choosing suitable methods
General concepts
base the purification of biopharmaceutical product on knowledge on
structure / function / particular structural details
Conversely, results from the application of a particular purification
method can often be interpreted in terms of molecular properties
General concepts
Production can be divided into
upstream processing
the initial fermentation process ,
which results in the initial generation of product.
downstream processing
the actual purification of the product
and generation of finished product format
followed by sealing of the final product containers
Categories of unwanted
Components present due to process conditions :
 Host-cell-derived components
 Process-derived components
Components present due to contamination :
 Adventitious agent
In Plant-scale working
volumes of between 2-5 liters
the most useful methods of purification
recrystallization for solids
distillation or steam distillation for liquids
chromatography should be avoided
but if it is necessary, then medium pressure liquid chromatography
(mplc) is the method of choice
Principle of purification operations
Filtration
Chromatography
Distillation
Crystallization or Recrystallization
Basic concept of Filtration
Technique : pass the solution, cold or hot,
through a fluted filter paper in a conical funnel
removes particulate impurities from liquids
or collect insoluble or crystalline solids from solution
the solid particles are too fine
centrifugation should be used
Basic concept of Gel filtration
different amount of time different solute stay within the liquid phase
that is entrapped by the matrix
pore dimension
gel structure
solute size
uncomplicated / straightforward
technique
Industrial filtration devices
Basic concept of chromatography
= a group of separation techniques, which are characterized by a
distribution of the molecules to be separated between two phases,
one stationary and the other mobile phase
molecules with a high tendency to stay in the stationary phase will
move through the system at a lower velocity than will those which
favor the mobile phase.
the shape, rigidity and particle size distribution profile of the gel
matrix are important parameters
Basis chromatography
Ion exchange
differences in protein surface charge at a given pH
Gel filtration
differences in size/shape of different protein
Hydrophobic interaction chromatography
differences in the size and
extent of hydrophobic patches on the surface of proteins
Affinity chromatography
ability of a protein to bind in a
bio- specific manner to a Chosen immobilized ligand
mplc
simplified
cheaper
easy to predict which fractions
will contain each component
silica / alumina / ion exchange
resin,
the appropriate size of column
and the correct solvent
linked to a fraction collector / An
UV / refractive index detector /
TLC
Basic concept of
Ion exchange chromatography (IEC)
the more highly charged a protein
the more strongly it will bind to a given,
oppositely charged ion exchanger
the more highly charged ion
exchangers
bind proteins more effectively than weakly
charged
Advantages of
Ion exchange chromatography (IEC)
versatility
high resolving power
high loading capacity
multiple inlets
column with large diameter
straight forward basic principle.
Basic concept of affinity chromatography
= the exploitation of various biological affinities for adsorption to a
solid phase
the ligand
the counterligand
immobilized on the solid phase
passing the chromatographic column
stages : adsorption, washing, elution and column regeneration
Biological interactions
used in affinity chromatography
Ligand
Antibody
Enzyme
Lectin
Nucleic acid
Hormone, vitamin
Sugar
Counterligand
antigen, virus, cell
substrate analogue, inhibitor, co-factor
polysaccharide, glycoprotein, cell surface receptor, cell
nucleic acid-binding protein (enzyme or histone)
receptor, carrier protein
lectin, enzyme or other sugar binding protein
Special consideration
on Affinity chromatography
association strength, between ligand and counterligand
if it is too weak
if it is too strong
no adsorption
difficult to elute the protein adsorbed
pH
salt concentration
dissociation substances
appropriate specificity
affecting the protein to be isolated
Basic concept of distillation
The distillation process involves
boiling a liquid
condensing the vapors
the resulting liquid
suitable for all organic liquids and
most of the low-melting organic
solids
the efficiency depends on
the difference in boiling points of the
pure material and its impurities
Steps of
crystallization or recrystallization
The impure material
dissolved at or near the boiling point to form a near-saturated solution.
the hot solution is filtered to remove any insoluble particles
allowed to cool
fast cooling
generate many nuclei of small crystals
the dissolved substance crystallizes out
centrifuge or filter crystals from mother liquor
washed free of mother liquor with a little fresh cold solvent
dried
Basic concept of
crystallization or recrystallization
only as a last step
yield of about 90% at best
10% yield loss is quite high
purified with additional
processing which are much more
quantitative such as extraction or
chromatography
Special considerations in our
Biopharmaceutical products
Vaccine / IL-2
Ritonavir
Protein
Organic molecule
Plant-scale process of protein
Working cell bank vial removed from storage
cell harvesting and recovery of crude protein
propagation of working bank cells,
generating starting cultures
production-scale cell culture
concentration (if necessary) and initial purification
main purification (chromatography)
product filling, freezing and sealing
final product formulation
labeling and
packaging
Upstream purification of Ritonavir
+
chromatography
2-aminoaldehyde
diols (white solid)
bromoacetate (white solid)
pricipitation
filtration
chromatography
diamine (white solid)
compound X (white solid)
filtration
epoxide (white solid)
distillation
chromatography
resin compound
compound XXXIIIa
Ritonavir
Downstream purification of Ritonavir
Hydrophobic interaction
chromatography
dryer
final product formulation
Affinity
chromatography
Conclusion
purity is a matter of degree
sufficient pure for some intended purpose
absolute purity is an ideal which can never be shown to be attained
the starting material should be of the grade commercially available
In general, at least two different methods, such as recrystallization and
distillation, should be used in order to ensure maximum purification
the decision to market the product in liquid or powder form must be determined
experimentally, as there is no way to predict the outcome for any particular
material
References
 Berthold, W. and Walter, J. Protein purification: aspects of processes for pharmaceutical products. Biologicals
1994;22:135-150.
 Janson JC. And Ryden L. Protein purification; principles, high-resolution methods, and application: VCH
publishers, Inc. 1989.
 Hesse F. and Wagner R. Developments and improvements in the manufacturing of human therapeutics with
mammalian cell cultures. Trends in Biotechnology. 2000 ;18(4):173-180
 WHO study group. Acceptability of cell substrates for production of biologicals. WHO technical report series;
1987: 747, 1-29.
 Walter, J and Allgaier, H. Validation of downstream processes. In: Mammalian Cell Biotechnology in Protein
Product.1997; 453-48.
 Perrin DD., Armarego WLF. and Perrin DR. Purification of laboratory Chemicals. 2nd ed. : Pergamon Press. 1980.
 White HL. Introduction to industrial chemistry : John Wiley & Sons, Inc. 1986
Thank you