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 counterligandpassing 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 RitonavirOrganic 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 material

References  Berthold, W. and Walter, J. Protein purification: aspects of processes for pharmaceutical products. Biologicals 1994;22:  Janson JC. And Ryden L. Protein purification; principles, high- resolution methods, and application: VCH publishers, Inc  Hesse F. and Wagner R. Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. Trends in Biotechnology ;18(4):  WHO study group. Acceptability of cell substrates for production of biologicals. WHO technical report series; 1987: 747,  Walter, J and Allgaier, H. Validation of downstream processes. In: Mammalian Cell Biotechnology in Protein Product.1997;  Perrin DD., Armarego WLF. and Perrin DR. Purification of laboratory Chemicals. 2 nd ed. : Pergamon Press  White HL. Introduction to industrial chemistry : John Wiley & Sons, Inc. 1986

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