n strategie s

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

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

General concepts
base the purification biopharmaceutical product knowledge on structure / function / particular structural details Conversely, application results of a from the particular of on

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

Categories of unwanted
Components present due to process conditions :
 Host-cell-derived components  Process-derived components

Components present due to

working
volumes of between 2-5 liters the most useful purification methods of

recrystallization for solids distillation or steam distillation for liquids

chromatography avoided

should

be

purification operations
Filtration Chromatography Distillation Crystallization or Recrystallization

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

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

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

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

mplc
simplified cheaper easy to predict which fractions will contain each component silica / alumina / ion exchange resin, the appropriate size of column

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

chromatography (IEC)
versatility high resolving power high loading capacity
multiple inlets column with large diameter

straight forward basic principle.

affinity chromatography
= the exploitation of various biological affinities for adsorption to a solid phase immobilized on the solid phase the counterligand passing the chromatographic column the ligand

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

on Affinity chromatography
association strength, between ligand and counterligand
if it is too weak adsorption if it is too strong difficult to elute the protein adsorbed no

pH salt concentration

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

crystallization or recrystallization
v The impure material v dissolved at or near the boiling point to form a near-saturated solution. v the hot solution is filtered to remove any insoluble particles v allowed to cool
v fast cooling nuclei of small crystals generate many

v the dissolved substance crystallizes out

crystallization or recrystallization
only as a last step yield of about 90% at best 10% yield loss is quite high purified with additional processing

our Biopharmaceutic al products

Vaccine / IL-2 Protein Ritonavir Organic molecule

n
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)

purification of Rito navir

+

chromatography
solid) 2-aminoaldehyde bromoacetate (white solid) diols (white

pricipitation

filtration chromatography

filtration
diamine (white solid) compound X (white solid) epoxide (white solid)

distillation

purification of Rito navir

Hydrophobic interaction Affinity chromatography chromatography dryer

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

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

Than k yo