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• high degree of purity obtained by a single chromatographic step;
• viral particles and other potential pathogens are rarely retained on an immuno-affinity column along with the product being purified; this provides added insurance with regard to removal of undetected pathogens from the product stream during downstream processing.
However, a number of potential disadvantages are also associated with this approach, including:
• possibility of contamination of the product stream with monoclonal antibody;
• conditions used to elute the protein off the immunoaffinity column can be harsh and, therefore, can cause limited protein denaturation.
Immobilized ligands (in this case, monoclonal antibody) may be attached to support beads by a variety of chemical means. Slow leakage of immobilized ligand characterize many affinity systems, although this has been minimized by newer immobilization coupling techniques. The potential clinical implications of leakage are unclear. Many would, however, feel that product contamination, with even very low levels of the monoclonal antibody, would be inappropriate, due to the regularity with which the product is normally administered. However, to date no significant clinical complications associated with ligand leakage have been noted in practice.
The binding between antibody and antigen is generally quite tight; thus, relatively harsh conditions are normally required to subsequently elute antigen off immuno-affinity columns. Several standard approaches may be adopted in this regard. Elution, using a buffer of low pH, is
BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 369
Anti-factor VIII ' antibody
Additional blood factors or other plasma proteins which fail to bind to the antibody
Figure 9.10. Purification of factor VIII complex using immunoaffinity chromatography. The immobilized anti-factor VIII antibody is of mouse origin. Antibodies raised against specific epitopes on both the VIII:C and vWF components have both been successfully used. Industrial-scale columns would often exhibit a bed volume of several litres. Note that the different elements in this diagram are not drawn to the correct scale relative to each other
often attempted, followed by rapid neutralization of the eluate to pH 7-8. Elution, using buffers of high pH, can also sometimes be successfully employed. In many instances, even harsher elution conditions may be required. These can include the use of chaotropic agents or detergents. Such conditions can obviously partially/completely denature the protein being eluted. Although many clotting factors are very stable when exposed to various denaturing influences, factor VIII is quite labile. Thus, the use of a monoclonal antibody with high, but not extreme, avidity for factor VIII would likely yield the most successful results.
Although fractionation can reduce very significantly the likelihood of pathogen transmission, it cannot entirely eliminate this possibility. Blood-derived factor VIII products, including those prepared by immunoaffinity chromatography, generally undergo further processing steps designed to remove/inactivate any virus present. The raw material is often heated for up to 10 h at 60°C or treated with solvent or dilute detergent prior to chromatography. Recombinant factor VIII is also often treated with dilute detergent in an effort to inactivate any viral particles potentially present.
Production of recombinant factor VIII (Table 9.6) has ended dependence on blood as the only source of this product and eliminated the possibility of transmitting blood-borne diseases specifically derived from infected blood. In the past, over 60% of haemophiliacs were likely to be accidently infected via contaminated products at some stage of their lives.
Several companies have expressed the cDNA coding for human factor VIII:C in a variety of eukaryotic production systems (human VIII:C contains 25 potential glycosylation sites).
Chinese hamster ovary (CHO) cells and baby hamster kidney (BHK) cell lines have been most commonly used, in addition to other cell lines, such as various mouse carcinoma cell lines. The recombinant factor VIII product generally contains only VIII:C (i.e. is devoid of vWF). However, both clinical and pre-clinical studies have shown that administration of this product to patients suffering from haemophilia A is equally as effective as administering blood-derived factor VIII complex. The recombinant VIII:C product appears to bind plasma vWF with equal affinity to native VIII:C, upon its injection into the patient’s circulatory system. Animal and human pharmacokinetic data reveal no significant difference between the properties of recombinant and native products.
Slight variation in the glycosylation patterns of native and recombinant product were to be expected, due to the differing production systems. While this has turned out to be the case, thus far it has not proved to be of any clinical significance. Significant differences could have made the recombinant product immunogenic. Bearing in mind that most recipients would receive several thousand injections of the product over their lifetime, even a mildly immunogenic product would promote a significant immunological response.