The assay sensitivity was 0

The assay sensitivity was 0.1 IU/dL FVIII:Ag. Assay to Measure Total Albumin Concentration Albumin content of Kogenate was determined by an automated immunoassay (Hitachi 704/Roche Albumin Reagent). Results UV Spectra of Recombinant Products The main characteristics of the recombinant FVIII formulations used in this study are listed in Table 1. phenomenon was less evident for the Advate product. Molecular aggregation negatively affects the in vitro pharmacodynamics of the concentrates with higher aggregates’ content. Conclusions ?This study shows that the three pharmaceutical formulations of recombinant FVIII contain Germacrone variable amounts of molecular aggregates after their reconstitution at therapeutic concentrations. This phenomenon negatively affects the in vitro potency of the products with higher aggregates’ content and might be invoked as a contributing cause of their increased risk to induce the formation of FVIII inhibitors. strong class=”kwd-title” Keywords: recombinant FVIII, molecular aggregation, Hemophilia A, dynamic light scattering, FVIII inhibitors Introduction Patients with hemophilia A are currently treated with FVIII concentrates prepared with both recombinant technology and fractionation/purification from plasma of healthy donors. Recombinant FVIII products are produced by different cell lines, which synthesize FVIII molecules with the same primary sequence of the human factor (except the B-deleted and B-truncated molecules). However, these recombinant molecules inevitably go through different posttranslational modifications, such as glycosylation and tyrosine sulfation processes. 1 Furthermore, the process of expression and purification of recombinant FVIII products may potentially cause the accumulation of misfolded and aggregated proteins. These aspects may be responsible for perturbation of the efficiency and safety (regarding the inhibitor formation) of the recombinant products, as suggested in official files by regulatory agencies. 2 The purification process of recombinant FVIII products includes a solvent/detergent virus inactivation step in addition to the use of ion exchange chromatography, and monoclonal antibody immunoaffinity chromatography to remove contaminating substances. 3 4 Chemical stabilizers such as amino acids, sugars, and nonionic surfactants are added for the maintenance of the structural/functional integrity of recombinant FVIII products. 3 It has been questioned whether the production of FVIII in nonhuman cells and the manufacturing processes could induce structural changes in the FVIII molecules and whether this might be the cause of different properties of products in terms of immunogenicity. Previous findings from randomized clinical trials (RCTs) and national hemophilia registries provided evidence that recombinant FVIII products are associated with high risk of inhibitor formation and that the recombinant second-generation FVIII products were associated with an even higher risk of inhibitor formation than the third-generation recombinant products. 5 6 7 In this study, we investigated some biochemical properties and, using size exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) spectroscopy, the aggregation status of three recombinant concentrates belonging to the second (Kogenate) and third-generation products (Advate and Germacrone Refacto AF). We addressed the issue of whether the molecular aggregation status of these products in solution after their reconstitution could significantly differ among products, affect their activity, and be invoked as a potential cause of inhibitor formation in hemophilia A patients. Materials and Methods FVIII Products Three recombinant products (Advate [Baxalta/Shire], Refacto AF [Pfizer], and Kogenate [Bayer]) were studied. Three different lots of each product were used. In some experiments, Recombinate [Baxalta], the first-generation product of Advate and thus containing albumin, was also studied. FVIII preparations were reconstituted in distilled water for injection and passed through the particle filters (5 m) contained in the pharmaceutical kit. The samples were immediately used for the experiments described below. UV Spectra of Recombinant FVIII Preparations Ultraviolet Germacrone (UV) absorbance scans of reconstituted Advate, Kogenate, and Refacto and genuine polysorbate 80 (TWEEN 80, purchased from Merck), histidine (25?mM, purchased from Sigma-Aldrich), and PEG 3350 (U.S. Pharmacopeia [USP] Reference Standard, Sigma-Aldrich) were performed over a 220 to 340?nm wavelength range in a 1-cm-pathlength quartz cell lodged in a thermostated cell holder of a Cary 60 spectrophotometer (Agilent Technologies Italia S.p.A. Milano, Italy). All the products were reconstituted with sterile water for injection by the use of the kits supplied by the manufacturers for therapeutic delivery and used at a final concentration of nominal 3,000 IU/mL (? 0.6 mg/mL, considering an activity of ? 5,000 IU/mg for the FVIII reference). The approximate concentrations of the above products were measured based on the extinction coefficient at 280?nm using em /em (01%) ?=?1.3 for the full-length FVIII products Advate and Kogenate, 8 and em /em (01%) ?=?1.55 for the B-deleted FVIII Refacto AF. 9 SE-HPLC of the Recombinant FVIII Concentrates FVIII.at 25C just before the measurements start. molecular aggregates. This phenomenon was less evident for the Advate product. Molecular aggregation negatively affects the in vitro pharmacodynamics of the concentrates with higher aggregates’ content. Conclusions ?This study shows that the three pharmaceutical formulations of recombinant FVIII contain Mef2c variable amounts of molecular aggregates after their reconstitution at therapeutic concentrations. This phenomenon negatively affects the in vitro potency of the products with higher aggregates’ content and might be invoked as a contributing cause of their increased risk to induce the formation of FVIII inhibitors. strong class=”kwd-title” Keywords: recombinant FVIII, molecular aggregation, Hemophilia A, dynamic light scattering, FVIII inhibitors Introduction Patients with hemophilia A are currently treated with FVIII concentrates prepared with both recombinant technology and fractionation/purification from plasma of healthy donors. Recombinant FVIII products are produced by different cell lines, which synthesize FVIII molecules with the same primary sequence of the human factor (except the B-deleted and B-truncated molecules). However, these recombinant molecules inevitably go through different posttranslational modifications, such as glycosylation and tyrosine sulfation processes. 1 Furthermore, the process of expression and purification of recombinant FVIII products may potentially cause the accumulation of misfolded and aggregated proteins. These aspects may be responsible for perturbation of the efficiency and safety (regarding the inhibitor formation) of the recombinant products, as suggested in official documents by regulatory agencies. 2 The purification process of recombinant FVIII products includes a solvent/detergent virus inactivation step in addition to the use of ion exchange chromatography, and monoclonal antibody immunoaffinity chromatography to remove contaminating substances. 3 4 Chemical stabilizers such as amino acids, sugars, and nonionic surfactants are added for the maintenance of the structural/functional integrity of recombinant FVIII products. 3 It has been questioned whether the production of FVIII in nonhuman cells and the manufacturing processes could induce structural changes in the FVIII molecules and whether this might be the cause of different properties of products in terms of immunogenicity. Previous findings from randomized clinical trials (RCTs) and national hemophilia registries provided evidence that recombinant FVIII products are associated with high risk of inhibitor formation and that the recombinant second-generation FVIII products were associated with an even higher risk of inhibitor formation than the third-generation recombinant products. 5 6 7 In this study, we investigated some biochemical properties and, using size exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) spectroscopy, the aggregation status of three recombinant concentrates belonging to the second (Kogenate) and third-generation products (Advate and Refacto AF). We addressed the issue of whether the molecular aggregation status of these products in solution after their reconstitution could significantly differ among products, affect their activity, and be invoked as a potential cause of inhibitor formation in hemophilia A patients. Materials and Methods FVIII Products Three recombinant products (Advate [Baxalta/Shire], Refacto AF [Pfizer], and Kogenate [Bayer]) were studied. Three different lots of each product were used. In some experiments, Recombinate [Baxalta], the first-generation product of Advate and thus containing albumin, was also studied. FVIII preparations were Germacrone reconstituted in distilled water for injection and passed through the particle filters (5 m) contained in the pharmaceutical kit. The samples were immediately used for the experiments described below. UV Spectra of Recombinant FVIII Preparations Ultraviolet (UV) absorbance scans of reconstituted Advate, Kogenate, and Refacto and genuine polysorbate 80 (TWEEN 80, purchased from Merck), histidine (25?mM, purchased from Sigma-Aldrich), and PEG 3350 (U.S. Pharmacopeia [USP] Reference Standard, Sigma-Aldrich) were performed over a 220 to 340?nm wavelength range in a 1-cm-pathlength quartz cell lodged in a thermostated cell holder of a Cary 60 spectrophotometer (Agilent Technologies Italia S.p.A. Milano, Italy). All the products were reconstituted with sterile water for injection by the use of the kits supplied by the manufacturers for therapeutic delivery and used at a.