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Evolution of Recombinant Factor VIII: Progress in Patient Safety

This report is based on medical evidence presented at sanctioned medical congress, from peer reviewed literature or opinion provided by a qualified healthcare practitioner. The consumption of the information contained within this report is intended for qualified Canadian healthcare practitioners only.

XXVIII International Congress of the World Federation of Hemophilia

Istanbul, Turkey / June 1-5, 2008

Here at the 2008 World Federation of Hemophilia meeting, experts presented an update on antihemophilia factor VIII (FVIII) products for hemophilia A.

History of Recombinant Factor VIII

The use of freeze-dried FVIII concentrates made from pooled plasma beginning in the late 1960s and early 1970s was a significant advantage over previous attempts to stem bleeding in patients with hemophilia A using whole blood or fresh plasma, but by the 1980s, this led to a large proportion of patients becoming infected with blood-borne pathogens such as HIV and hepatitis C. Researchers then developed virus-free FVIII concentrates using improved donor selection and screening and special processes to kill any viruses that might remain in the plasma pools. However, some hypothetical risks still remained, such as prions. The development of recombinant protein expression biotechnology allowed for the production of recombinant FVIII (rFVIII) with good safety profiles for use in patients with hemophilia. A wide range of recombinant factor concentrates are now available, and treatment with some form of rFVIII is generally preferred today for patient safety.

Improving Safety

The use of first-generation recombinant antihemophilia products still carries some theoretical risk of exposure to blood-borne pathogens because the manufacturing processes employ a variety of human and mammalian proteins. Hypothetical risks of contamination include, for example, prions in fetal calf serum added to cell media used for the initial cell culture step; murine viruses contained in the monoclonal antibodies (MAbs) employed in the purification techniques; and human and mammalian viruses carried in serum albumin-based nutrients or stabilizers used in the production processes and in final product.

Research scientist Stacey B. Weston, PhD, Andover, Massachusetts, explained that the progress in the development of rFVIII second-generation products came about when albumin was removed from both the production processes and the final product. However, albumin is still needed in the initial cell culture phase, which still leaves some risk of FVIII contamination. The initial second-generation product was a full-length rFVIII (FLrFVIII). In addition, researchers have determined that the B domain of FVIII is not required for hemostatic function and can be deleted, producing a B-domain-deleted rFVIII (BDDrFVIII) of 170 kDa, compared to full-length wild-type rFVIII of approximately 280 kDa. While the initial impetus for developing a smaller, hemostatically functional rFVIII was the quest for a form to use with a viral vector for gene therapy, BDDrFVIII was made available as a second-generation process therapeutic agent.

Third-generation rFVIII products are now available. These have final formulations free of human and bovine serum albumin with no albumin required in the cell culture phase. These products have the lowest theoretical risk of contamination, a significant advance in patient safety. Recent potential safety enhancements to third-generation rFVIII production include the replacement of the one remaining protein-linked process step (the murine MAb) and the addition of a viral filtration step. Currently, only B-domain-deleted rFVIII is produced using purification enhancements, making it arguably a fourth-generation product.

Non-neutralizing Anti-FVIII Antibodies

Dr. Georges-Étienne Rivard, Centre hospitalier universitaire Sainte-Justine, Department of Hematology and Oncology, and Clinical Professor of Pediatrics, Université de Montréal, Quebec, investigated another potential difference among various rFVIII preparations. He and colleagues theorized that non-neutralizing anti-FVIII antibodies might be directed against nonfunctional domains of FVIII such as the B domain and could be responsible for low in vivo recuperation of FVIII observed in the absence of neutralizing antibodies. They tested plasma from 58 participants with congenital hemophilia A exclusively treated with FLrFVIII and seven participants with acquired hemophilia. The plasma assays included the Nijmegen-Bethesda assay (to detect neutralizing antibodies) and three enzyme-linked immunosorbent assays (ELISA, to detect non neutralizing antibodies) using two different FLrFVIII preparations or a BDDrFVIII preparation as antigens to capture antibodies present in the patients' plasma.

Of the seven patients with acquired hemophilia, all had Bethesda-positive antibodies and ELISA-positive antibodies directed against each of the three rFVIII preparations. All patients with congenital hemophilia with Bethesda-positive antibodies also had ELISA-positive antibodies to all three rFVIII preparations. However, of those patients whose plasma samples did not have Bethesda-positive antibodies, six and four, respectively, had ELISA-positive antibodies directed against the two different FLrFVIII preparations while none had ELISA-positive antibodies directed against the BDDrFVIII preparation.

Dr. Rivard explained, “The clinical significance of non-neutralizing anti-rFVIII antibodies is presently unknown but could imply shortened in vivo FVIII half-life. Investigation of a large number of subjects would be necessary to assess their clinical relevance.” He also emphasized that pharmacokinetic studies should be performed to confirm in vivo clinical relevance of these results. It is possible that the follow-up of patients with hemophilia A could include not only frequent testing with the Bethesda assay but also systematic testing with ELISA assays to allow more rational decisions about which rFVIII concentrate to select, but Dr. Rivard insisted that “more research is necessary” before physicians consider switching rFVIII concentrate based on in vitro tests.

Third-generation rFVIII Agents

Dr. Laszlo Nemes, Director, National Haemophilia Center, National Medical Center, Budapest, Hungary, presented pharmacokinetic, safety and efficacy data for a third-generation BDDrFVIII (albumin-free moroctocog alfa [AF-CC]).

Its pharmacokinetic profile was compared with that of a third-generation FLrFVIII. The profiles were assessed in a randomized double-blind crossover study in previously untreated patients. A standard bioequivalence approach (a one-stage clotting assay) was used. The prophylactic use of the products was initiated with a dose of 30 (+5) IU/kg three times per week and the protocol included dose-escalation procedures for patients experiencing breakthrough bleeding. The results for K-value, AUCt, and AUC were within the bioequivalence window of 80% to 125% for the two products. A follow-up assessment at six months to assess the stability of moroctocog alfa (AF-CC) in a subgroup of these patients did not reveal any changes in pharmacokinetic profile.

Safety and efficacy were also assessed in a second phase of this research, in an open-label, multicentre trial of moroctocog alfa (AF-CC) in routine prophylaxis and on-demand therapy in previously treated patients with severe or moderately severe hemophilia A. Participants received the BDDrFVIII for a minimum of 50 exposure days over six months; the drug was to be used exclusively for prophylaxis and for the treatment of any spontaneous or traumatic bleeds. Median routine prophylaxis dose was approximately 30 IU/kg. The overall adverse-event profile was consistent with that of second-generation BDDrFVIII and other rFVIII products. The development of transient, low-titre, clinically silent inhibitors was a rare occurrence (two of the initial 94 participants). In these two cases, the presence of the inhibitors was detected by routine surveillance on a single occasion; each patient was negative on follow-up testing. Corresponding ELISA tests for rFVIII antibodies were negative.

Dr. Nemes concluded that the third-generation BDDrFVIII is pharmacokinetically equivalent to third-generation FLrFVIII; its pharmacokinetic profile is stable over six months of use; and it is effective in the prevention and treatment of bleeding episodes in patients with hemophilia A. Inhibitor safety results did not show any evidence of neoantigenicity, and the adverse-event profile demonstrated safety in previously treated patients with hemophilia A.

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