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. 

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 eukaryotic 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 

CH12 RECOMBINANT BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 

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 use of monoclonal antibodies to purify factor VIII (or any other serum/therapeutic protein) exhibits a number of associated advantages, the most important of which are  

However, a number of potential disadvantages are also associated with this approach, including  

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 

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Another by-product of Factor VIII processing having clinical value is von Wikebrand factor. It has been recovered from side fractions using ion-exchange and affinity chromatography (196). 

Persons suffering from haemophilia A exhibit markedly reduced levels (or the complete absence) of factor VIII complex in their blood. This is due to the lack of production of factor VIII C. 

Josic D, Buchacher A, Kannicht C, et al. Degradation products of factor VIII which can lead to increased immunogenicity. Vox Sang 1995 77 90. 

After a certain time period asparagine supplementation was stopped (day 0), as a consequence of which asparagine became exhausted from the culture medium and the production of factor VIII dropped (days 1-4). Asparagine (200 xmol) was later added to the medium (day 4), the result of which was an increase in the levels of factor VIII. 

Physical methods cryopredpitation is often used as the initial step for the production of Factor VIII and fibrinogen. It has no impact on the viral safety of such products. Subsequent purification techniques, such as precipitation by agents other than ethanol or chromatographic separation as well as procedures for viral inactivation are used to obtain the finished products. Cryoprecipitate-depleted plasma can be used for the separation of other coagulation factors or plasma protein solutions. 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Determination of the potency of Factor VIII is also difficult. This is normally measured by the abiUty of the sample to correct the clotting time of plasma deficient in Factor VIII. A number of methods and practices have evolved for this purpose (231), but these give very different results, particularly when activation of products may also occur (232). International standards have been used, but further standardization of the analytical method and harmonization of working standards is underway (233,234) under the auspices of the ISTH and the EC. 

The intrinsic pathway (Figure 51-1) involves factors XII, XI, IX, VIII, and X as well as prekallikrein, high-molecular-weight (HMW) kininogen, Ca, and platelet phospholipids. It results in the production of factor Xa (by convention, activated clotting factors are referred to by use of the suffix a). 

Because it is a small protein, the factor IX molecule passes into the intravascular and the extravascular spaces. Therefore, the volume of distribution of recombinant factor IX is twice that of factor VIII. Consequently, 1 unit of factor IX administered per kilogram of body weight yields a 1% rise in the plasma factor IX level (0.01 unit/mL, or 1 IU/dL). Thus 1750 units of factor IX provides an incremental increase of 50% of normal (0.5 unit/mL, or 50 IU/dL), that is, 1750 units/70 kg x 0.01 unit/mL (1 IU/dL).12 Additionally, the recovery rate with recombinant factor IX is 20% lower than that with the plasma-derived products. Therefore, initial and subsequent maintenance doses should be adjusted accordingly. 

Bleeding episodes Pis Onset Few weeks Symptoms TSpontaneous bleeding tendency (in joints, muscles, soft tissues, and hematuria) PI use in hemophiliac patients TPV and anticoagulants (risk of intracranial hemorrhage) Consider using a NNRTI-based regimen May require increased use of factor VIII products... 

Persons expressing 5 per cent or above of the normal complex levels experience less severe clinical symptoms. Treatment normally entails administration of factor VIII complex purified from donated blood. More recently, recombinant forms of the product have also become available. Therapeutic regimens can require product administration on a weekly basis, for life. About 1 in 10 000 males are born with a defect in the factor VIII complex and there are approximately 25 000 haemophiliacs currently resident in the USA. 

Different mutations in the disease-causing locus may cause more or less severe expression. For example, mis-sense mutations in the factor VIII gene tend to produce less severe hemophQia than do nonsense mutations, which result in a truncated protein product and little, if any, expression of factor VIII. The presence of different mutations, or alleles, in the same locus is termed allelic heterc eneity. 

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)... 

Cryoprecipitate should be used to treat bleeding in the setting of factor VIII deficiency and von Willebrand disease only in an emergency in which pathogen-inactivated products are not available. 

Cryoprecipitate may also be used for patients with factor VIII deficiency and von Willebrand disease if desmopressin is not indicated and a pathogen-inactivated, recombinant, or plasma-derived product is not available. The concentration of factor VIII and von Willebrand factor in cryoprecipitate is not as great as that found in the concentrated plasma fractions. Moreover, cryoprecipitate is not treated in any manner to decrease the risk of viral exposure. For infusion, the frozen cryoprecipitate unit is thawed and dissolved in a small volume of sterile citrate-saline solution and pooled with other units. Rh-negative women with potential for childbearing should receive only Rh-negative cryoprecipitate because of possible contamination of the product with Rh-positive blood cells. 

There has been some concern about the possible effects of factor VIII formulations on the immune system. In vitro experiments with coagulation factor concentrates have shown immunosuppressive effects (16,17), such as the impairment of Fc receptor-mediated phagocytosis and intracellular bacterial killing (18). Inhibition of IL-2 production, an impaired MLR, and impairment of PHA transformation have been demonstrated (19). A fall in the number of T4 lymphocytes has also been found. Whether these findings reflect functional impairment of the immune system is still unclear. 

In 58 previously treated patients with hemophiha treated with a recombinant factor VIII product for more than 5 years, there were neither allergic reactions to murine or hamster proteins nor any de novo formation of inhibitors of factor VIII (20). 

Yee TT, Lee CA. Is a change of factor VIII product a risk factor for the development of a factor VIII inhibitor Thromb Haemost 1999 81(5) 852. 

Another possibility is that some other clotting factor is increased or activated in such a way that the assay system responds fortuitously to it in a way indistinguishable from the usual response to factor VIII, e.g., factors XI and XII factor XII is known to rise on exercise (II). That this might occur over a limited range of the dose-response curve in the thromboplastin generation test system was shown by experiments in which the addition of activation product (Wl) simulated an increased factor VIII concentration (author s unpublished observations, 1960 FI), although statistical invalidity would probably be detectable over a series of experiments if this were the explanation. This also was looked for, but was not found (14). It is interesting that, in a patient with severe factor VIII deficiency and partial factor XI deficiency (SI), adrenaline infusion was followed by a marked rise in factor XI concentration and the appearance of a trace of factor VIII (K. Schulz, personal communication, 1964). Furthermore, the confusion that arose some years ago over factor IX assay now seems to have been due to activation of the contact factors (P4), hence... 

Protein purification, introduced in the 1990s, produced high-purity concentrates with increased amounts of factor VIII or factor IX relative to the product s total protein content. Recombinant factor VIII and then factor IX also became available. The first-generation recombinant factor VIII products utilize human and animal proteins in culture and add human albumin as a protein stablilizer. Second-generation recombinant factor VIII concentrates removed albumin as a protein stabilizer, and the third-generation products lack human and animal proteins in the culture media. Finally, gene therapy for the treatment of hemophilia is now in the early stages of clinical trials. 

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