Characterization of F VIII concentrates produced by two methods incorporating double virus inactivation

Abstract

The trend toward the production of high purity factor VIII concentrates for clinical use is still in progress. Although all plasma derivatives must undergo viral inactivation procedures, the possibility of transmission of viral diseases is not completely eliminated. In order to reduce such risk, we have included double virus inactivation in the procedure of factor VIII concentrate production. In a scale-up procedure for isolation of factor VIII cryoprecipitate, two methods were used. The first is based on the chromatographic purification of factor VIII after pasteurization of cryoprecipitate solution and solvent/detergent (S/D) inactivation of viruses. The second is based on multistep precipitation of factor VIII by sodium chloride and glycine. Viral inactivation was performed by combination of S/D treatment and heating of final freeze-dried product 30 min at 100 degrees C. The typical yield of factor VIII activity in the freeze-dried product was about 20% for the first method, and 25-30% for the second. Electrophoretic analyses of both factor VIII preparations by SDS-PAGE and IEF show very low content of contaminant proteins, in accordance with observed 400-650-fold increase of their specific activity over plasma. Both factor VIII products were stable in the liquid state for more than 24 h at room temperature. The final products, after double viral inactivation, are considered to be suitable for clinical evaluations.

Plasma‐derived clotting factor VIII: Heterogeneity evaluation in the quest for potential inhibitory‐antibody stimulating factors

“…Western blot analyses targeting the FL FVIII molecule yielded comparable results for all the tested products (Fig. 2, upper right panel), with two main bands appearing at MW higher than 205 kDa, as already reported in literature 19. In literature, these two bands have been attributed either to differential glycosylation or alterations of within‐molecule disulfide bonds 19.…”

“…SDS‐PAGE was performed according to the method of Laemmli 24. The rFVIII preparations (30 mg), before and after thrombin digestion, were loaded onto a 0.75‐mm‐thick 7.5% w/v acrylamide gradient gel, which was the optimal concentration for pdFVIII, as previously reported 13, 19. The apparent molecular weight of bands was determined using a wide molecular weight range calibration kit for SDS‐PAGE (Sigma‐Aldrich).…”

“…FVIII is finally purified via chromatographic techniques using multiple precipitation, affinity chromatography or ion‐exchange chromatography 14–16. Freeze‐dried and lyophilized FVIII then undergoes virucidal steps, such as heat treatment and/or solvent/detergent treatment 18, 19, to inactivate both enveloped and non‐enveloped viruses. In some commercial pdFVIII concentrates, albumin is added to stabilize and protect FVIII molecules, although all pdFVIII, albeit not rFVIII, already contain significant concentrations of vWF, which is complexed with the FVIII protein.…”

Protein modification during anti‐viral heat‐treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose

“…Although there are now new generations of recombinant and plasma-derived protein sources with improved purity and viral safety, these products still bear the theoretical potential risk of inhibitor development and carrying/transmitting viruses (Radosevich, 2000). The bioprocessing industry presently utilizes viral inactivation methods including heating of proteins in solution and lyophilized states, nano®ltration, and solvent/detergent treatment (Biesert et al, 1996;Branovic et al, 1998;Chandra et al, 1999;Hilfenhaus and Weidmann, 1986). However, heat treatment is the``gold standard'' against which other methods are judged and many therapeutic proteins are treated in this way (e.g., factor VIII, factor IX, albumin).…”

Evaluation of protein modification during anti‐viral heat bioprocessing by electrospray ionization mass spectrometry

“…Considerable interest exists in methods of viral inactivation in therapeutic biopharmaceutical protein products derived from both plasma and recombinant sources, primarily due to the outbreak of HIV infection in hemophiliac patients during the early 1980s. Currently favored viral inactivation methods include heating of protein solutions and lyophilizates, nanofiltration, solvent/detergent treatments, and UV irradiation 1–3. However, heat‐treatments are the current ‘gold standard’ against which other methods are judged and many therapeutic protein products are treated in this way (e.g.…”