Factor VIII concentrates, recombinant

In past years, treatment for patients with hemophilia A has consisted of administration of cryoprecipitates (enriched in factor VIII) prepared from individual donors or lyophilized factor VIII concentrates prepared from plasma pools of up to 5000 donors. It is now possible to prepare factor Vlll by recombinant DNA technology. Such preparations are free of contaminating viruses (eg, hepatitis A, B, G, or HlV-1) found in human plasma but are at present expensive their use may increase if cost of production decreases. 

Clinical trials have demonstrated excellent efficacy with recombinant human factor VIII concentrates available as Recombinate and Kogenate. These recombinant factor VIII products are purified from the cell culture of plasmids, not viral DNA-transfected hamster cells and therefore do not express viral sequences. The addition of human serum albumin for stabilization, constitutes the sole possible source for human viral contamination. More recently recombinant factor IX has been genetically engineered by insertion of the human factor IX gene into a Chinese hamster ovary cell line. It has been proved to be safe and effective in the treatment of patients with hemophilia B. 

Scharrer I, Bray GL, Neutzling O. Incidence of inhibitors in haemophilia A patients—a review of recent studies of recombinant and plasma-derived factor VIII concentrates. Haemophilia 1999 5 (3) 145—54. 

For patients with high titre factor VIII inhibitors, standard factor IX complex products are first-line therapy but activated factor IX complex concentrates may be necessary for continued, more frequent or more severe bleeding. Porcine factor VIII or recombinant human factor Vila may be necessary for major bleeds or elective surgery. 

Recombinant and high-purity coagulation factor products appear to have a greater tendency to induce inhibitors than human-derived concentrates of intermediate or low purity (7). These intermediate-purity or low-purity human-derived concentrates are probably more suitable for inducing immune tolerance in patients with hemophilia with inhibitors. It has been suggested that for immune tolerance a high content of Von Willebrand factor in factor VIII concentrates is required, although direct comparisons of different products have not been made (8). 

In two patients with hemophilia with antibodies to both human and porcine factor VIII, continuous recombinant factor Vila resulted in hematuria (21). In neither case was a cause of the hematuria found. The author suggested that mucosal bleeds, such as hematuria, are characterized by high fibrinoljdic activity locally and may require higher peak concentrations of factor VII to generate sufficient thrombin to achieve and sustain hemostasis. The need for a full thrombin burst could relate to the role of thrombin in the activation of thrombin-activatable fibrinolysis inhibitor. 

Inhibitor bypassing agents include prothrombin complex concentrates, activated prothrombin complex concentrates (such as FEIBA), porcine factor VIII, and recombinant factor Vila. During 10 years usage of FEIBA (3.95 x 10 FEIBA units infused) a total of 16 thrombotic events were reported, corresponding to an incidence of 4.05 per 10 ... 

Plasma-derived factor VIII concentrates have been implicated in the transmission of the non-enveloped hepatitis A and parvovirus B19 (28). The virus-inactivating procedures now in use (chemical inactivation, wet-heat treatment, and nanofiltration) should provide coagulation factors without risk of transmitting HIV and with a very high safety for hepatitis virus. Nevertheless, recombinant factor VIII is considered a safer alternative. 

Figure 3.4.2 Culture of DON cells showing asparagine and recombinant factor VIII concentrations.

Azzi A, De Santis R, Morfini M, Zakrzewska K, Musso R, Santagostino E, et al. TT virus contaminates first-generation recombinant factor VIII concentrates. Blood 2001 98 2571-73. 

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. 

The treatment of choice for patients with types 2B, 2M, and 3 von Willebrand disease and for patients with type 1 or 2A von Willebrand disease who are unresponsive to desmopressin is replacement therapy with plasma-derived von Willebrand factor-containing products. Several virus-inactivated, intermediate- or high-purity factor VIII concentrates contain sufficient amounts of functional von Willebrand factor. Ultrahigh-purity (monoclonal antibody-derived) plasma-derived products and recombinant factor VIII products... 

Lusher JM. First and second generation recombinant factor VIII concentrates in previously untreated patients recovery, safety, efficacy, and inhibitor development. Semin Thromb Hemostas 2002 28 273-276. 

The major concern associated with the use of concentrated clotting factors is the risk of viral transmission (primarily HIV and hepatitis B). This fear has somewhat attenuated the use of concentrated plasma fractions, even In diseases such as hemophilia. Ultrapure factor VIII concentrates produced using recombinant DMA technology have been approved for use. Frequently, however, the expense of these recombinant agents is the reason why the more traditional plasma Isolates are used—despite the possibility of viral transmission. 

Comparative studies A recombinant VWF/factor VIII concentrate was compared with a plasma-derived form (Humate-P) in 32 patients. The recombinant form caused the following in four patients tremor, pruritus, psychomotor hyperactivity, hypertension, nausea and dizziness [1363. 

Individuals who display a deficiency of factor IX develop haemophilia B, also known as Christmas disease. Although its clinical consequences are very similar to that of a deficiency of factor VIII, its general incidence in the population is far lower. Persons suffering from haemophilia B are treated by i.v. administration of a concentrate of factor IX. This was traditionally obtained by fractionation of human blood. Recombinant factor IX is now also produced in genetically engineered CHO cells (Table 12.2 and Box 12.1). 

VIII Hemophilia A 30-50% 100% for major bleeding or trauma 12 hours Recombinant factor VIII products Plasma-derived high purity concentrates Cryoprecipitate1 Some patients with mild deficiency will respond to DDAVP... 

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. 

The visualization method also worked with a 500-L perfusion reactor system for production of recombinant human coagulation factor VIII (hFVIII) in Chinese hamster ovary (CHO) cells [36,37]. Despite the diluted concentration of CHO cells and low titer of hFVIII in the medium, the nose could differentiate between the batch phase, medium replacement phase, and the high and low productivity phases during the five-week long cultivations (Fig. 10). The low concentration of hFVIII makes it credible to believe that there are other components associated with the product formation that the electronic nose responds to. 

Homologous recombinant proteins have had to undergo a more extensive evaluation preclinically. For example, a recombinant factor VIII product was evaluated in acute and subacute studies in mice, rats, rabbits, dogs, and nonhuman primates over a range of concentrations and dose frequency [12],... 

In the past, hemophiliacs were treated with transfusions of a concentrated plasma fraction containing factor VIII. This therapy carried the risk of infection. Indeed, many hemophiliacs contracted hepatitis and AIDS. A safer preparation of factor VIII was urgently needed. With the use of biochemical purification and recombinant DNA techniques, the gene for factor VIII was isolated and expressed in cells grown in culture. Recombinant factor VIII purified from these cells has largely replaced plasma concentrates in treating hemophilia. 

Recombinant factor VIII or IX concentrates are recommended for all previously untreated patients, those who have been previously treated but remain hepatitis C virus or HIV seronegative, and for mild to moderate severity disease when desmopressin is not sufficient. 

Treatment options for patients with inhibitors are high dosages of clotting factor or recombinant factor Vila for both hemophilia A and B or, in the case of hemophiha A, porcine factor VlllrC or activated prothrombin complex (37). Regular administration of intermediate or low-dose factor Vin concentrates leads to the rapid disappearance of factor VIII inhibitors in some high responders (27) this is thought to be due to the development of immune tolerance. 

If the inhibitor activity is under 10 Bethesda units/ml, patients can be treated with increased doses of factor VIII or IX concentrates (43). In addition, patients with hemophilia A with low or intermediate antibody titers can also be treated with porcine factor VIII (43). However, hemorrhagic episodes in patients with antibody activity over 10 Bethesda units/ml may result in life-threatening hemorrhage that cannot be treated by conventional therapy (26,43). Prevention or treatment of clinically significant bleeding episodes in these patients can be achieved by using so-called bypassing therapies, such as recombinant factor Vila and activated prothrombin complex (23/26,43). Recombinant factor Vila is both effective and safe in the treatment of inhibitors directed to either factor VIII or IX (44,45). 

To emphasize the importance of controlling the amino acid concentration, an example illustrating the importance of asparagine for the production of recombinant factor VIII in DON cells will be discussed (Figure 3.4.2). 

Several different plasma-derived factor VEI products are avaUable (see Table 100 ). These products are derived from the plasma of thousands of donors, and therefore potentially can transmit infection. Donor screening, testing plasma pools for evidence of infection, viral reduction through purification steps, and viral inactivation procedures (e.g., dry heat, pasteurization, and solvent detergent treatment) have all resulted in a safer product. No cases of HIV transmission from factor concentrates have been reported since 1986. However, there have been isolated reports of hepatitis C infection with the use of plasma-derived products. Additionally, there have been outbreaks of hepatitis A viral infections associated with plasma-derived products, likely because solvent detergent treatment does not inactivate this nonenveloped virus. Parvovirus has also been reported to be present in both plasma-derived and recombinant factor VIII products. " Finally, there remains concern about the possibility for infection with as yet unidentified viruses that currently used methods would not inactivate. 

Cryoprecipitate is a blood product that is manufactured from fresh frozen plasma. It contains a subset of coagulation factors fibrinogen (minimum of 150mg/unit), factor VIII (minimum 80IU/unit), von Willebrand factor (VWF) and factor XIII. With the production of recombinant and purified factor concentrates, the classical indications of haemophilia A and von Willebrand disease are no longer appropriate. Currently, cryoprecipitate is used to treat hypofibrinogemia. 

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