Production of factor VIII

Native factor VIII is traditionally purified from blood donations first screened for evidence of the presence of viruses such as hepatitis B and HIV. A variety of fractionation procedures (initially mainly precipitation procedures) have been used to produce a factor VIII product. The final product is filter-sterilized and filled into its finished product containers. The product is then freeze-dried and the containers are subsequently sealed under vacuum, or are flushed with an inert gas (e.g. N2) before sealing. No preservative is added. The freeze-dried product is then stored below 8 °C until shortly before its use.

Although earlier factor VIII preparations were relatively crude (i.e. contained lower levels of various other plasma proteins), many of the modern preparations are chromatographically purified to a high degree. The use of immunoaffinity chromatography has become widespread in this regard since 1988 (Figure 12.7). The extreme bioselectivity of this method can yield a single-step purification factor of several thousand-fold.

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 12.2) 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 per cent of haemophiliacs were likely to be accidentally infected via contaminated products at some stage of their life.

Several companies have expressed the cDNA coding for human factor VIII:C in a variety of eu-karyotic production systems (human VIII:C contains 25 potential glycosylation sites). CHO cells and 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 preclinical 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

Additional blood factors or other plasma proteins which fail to bind to the antibody

Figure 12.7 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 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.

Some patients, particularly those suffering from severe haemophilia A (i.e. those naturally producing little or no VIII:C), will mount an immune response against injected factor VIII:C whatever its source.

The production of anti-factor VIII:C antibodies renders necessary administration of higher therapeutic doses of the product. In severe cases, the product may even become ineffective. Several approaches may be adopted in order to circumvent this problem. These include:

• Exchange transfusion of whole blood. This will transiently decrease circulating anti-factor VIII: C antibodies.

• Direct administration of factor Xa, thus bypassing the non-functional step in the coagulation cascade (Figures 12.2 and 12.3).

• Administration of high levels of a mixture of clotting factors II, VII, IX and X, which works effectively in 50 per cent of treated patients.

• Administration of factor VIIa, as discussed subsequently.

• Administration of porcine factor VIII, which may or may not cross-react with the antibodies raised against human factor VIIIa. (However, the immune system will soon begin to produce antibodies against the porcine clotting factor.)

• Administration of factor VIII, with concurrent administration of immunosuppressive agents.

Owing to the frequency of product administration, the purification procedure for recombinant factor VIII:C must be particularly stringent. Unlike the situation pertaining when the product is purified from human blood, any contaminant present in the final product will be non-human and, hence, immunogenic. Sources of such contaminants would include:

• animal cell culture medium;

• antibody leaked from the immunoaffinity column.

Emphasis is placed not only upon ensuring the absence of contaminant proteins, but also other potential contaminants, particularly DNA. (Host cell-line-derived DNA could harbour oncogenes; Chapter 7.)

Recombinant factor VIII is gaining an increasing market share of the factor VIII market. Researchers are also attempting to develop modified forms of VIII:C (by site-directed mutagen-esis) that display additional desirable characteristics. Particularly attractive in this regard would be the development of a product exhibiting an extended circulatory half-life. This could reduce the frequency of injections required by haemophilia A sufferers. However, any alteration of the primary sequence of the molecule carries with it the strong possibility of rendering the resultant mutant immunogenic.

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