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 immobilized on the solid phase

the counterligand passing the chromatographic column

stages : adsorption, washing, elution and column regeneration

 Biological interactions
used in affinity chromatography
Ligand Counterligand
Antibody antigen, virus, cell
Enzyme substrate analogue, inhibitor, co-factor
Lectin polysaccharide, glycoprotein, cell surface receptor, cell
Nucleic acid nucleic acid-binding protein (enzyme or histone)
Hormone, vitamin receptor, carrier protein
Sugar lectin, enzyme or other sugar binding protein
 Special consideration
on Affinity chromatography
association strength, between ligand and counterligand
if it is too weak no adsorption
if it is too strong 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 Protein

Ritonavir Organic molecule
Plant-scale process of protein
Working cell bank vial removed from storage propagation of working bank cells,
generating starting cultures

cell harvesting and recovery of crude protein 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 filtration
diamine (white solid) compound X (white solid) epoxide (white solid)
distillation

chromatography
resin compound compound XXXIIIa Ritonavir
Downstream purification of Ritonavir

Hydrophobic interaction Affinity

chromatography chromatography

dryer

final product formulation

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 mate
rial
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. 2 nd ed. : Pergamon Press. 1980.
White HL. Introduction to industrial chemistry : John Wiley & Sons, Inc. 1986
Thank you