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Table 3.18. Various chromatographic techniques that may be used to separate proteins from each other. The basis upon which separation is achieved is also listed
Basis of separation
Hydrophobic interaction chromatography (HIC)
Metal chelate chromatography Hydroxyapatite chromatography
Differences in protein surface charge at a given pH Differences in size/shape of different proteins Differences in the size and extent of hydrophobic patches on the surface of proteins Ability of a protein to bind in a bio-specific (selective) manner to a chosen immobilized ligand Ability of certain proteins to complex with zinc and copper Mechanism not fully understood. Involves ability of some proteins to bind to calcium and phosphate ions on the surface of hydroxyapatite crystals
Table 3.19. Some forms of affinity chromatography that may be employed to purify a selected protein. Virtually all proteins carry out their biological effects by interacting in some way with other molecules, which can be given the general title Ďligandsí. This interaction is often quite biospecific. Immobilization of such ligands (or molecules which mimic ligands) onto chromatographic beads thus facilitates selective purification of the molecule of interest
Protein A chromatography Lectin affinity chromatography Immunoaffinity chromatography
Dye affinity chromatography
Protein A, produced by Staphylococcus aureus, binds IgG. It is used extensively in antibody purification protocols Lectins are a group of proteins capable of binding carbohydrates.
Lectin affinity chromatography may be used to purify glycoproteins Immobilized antibodies may be used as affinity absorbants for the antigen that stimulated their production (e.g. purification of factor VIII using immobilized anti-factor VIII antibodies)
Purification of proteins that display ability to interact tightly with selected dyes
ē filtration of the final product through a 0.22 mm absolute filter in order to generate sterile product, followed by its aseptic filling into final product containers;
ē freeze-drying (lyophilization) if the product is to be marketed in a powdered format.
The decision to market the product in liquid or powder form is often dictated by how stable the protein is in solution. This in turn must be determined experimentally, as there is no way to predict the outcome for any particular protein. Some proteins may remain stable for months or even years in solution, particularly if stabilizing excipients are added and the solution is refrigerated. Other proteins, particularly when purified, may retain biological activity for only a matter of hours or days when in aqueous solution.
Some influences that can alter the biological activity of proteins
A number of different influences can denature or otherwise modify proteins, rendering them less active/inactive. As all protein products are marketed on an activity basis, every precaution must
THE DRUG MANUFACTURING PROCESS 143
Table 3.20. The various molecular alterations that usually result in loss of a proteinís biological activity
Partial/complete protein denaturation Covalent alterations Hydrolysis Deamidation Imine formation Racemization Oxidation
be taken to minimize loss of biological activity during downstream processing and subsequent storage. Disruptive influences can be chemical (e.g. oxidizing agents, detergents, etc.), physical (e.g. extremes of pH, elevated temperature, vigorous agitation) or biological (e.g. proteolytic degradation). Minimization of inactivation can be achieved by minimizing the exposure of the product stream to such influences, and undertaking downstream processing in as short a time as possible. In addition, it is possible to protect the protein from many of these influences by the addition of suitable stabilizing agents. The addition of such agents to the final product is often essential in order to confer upon the product an acceptably long shelf-life. During initial development, considerable empirical study is undertaken by the formulators to determine what excipients are most effective in enhancing a productís stability. Detailed treatment of the topic of protein stability and stabilizers is in itself worthy of a complete book. Only a summary overview is provided below and the interested reader is referred to the Further Reading section at the end of this chapter.
A number of different molecular mechanisms can underpin the loss of biological activity of any protein. These include both covalent and non-covalent modification of the protein molecule, as summarized in Table 3.20. Protein denaturation, for example, entails a partial or complete alteration of the proteinís 3-D shape. This is underlined by the disruption of the intramolecular forces that stabilize a proteinís native conformation, viz hydrogen bonding, ionic attractions and hydrophobic interactions. Covalent modifications of protein structure that can adversely effect its biological activity are summarized below.