In 2018, Refacto AFR, a B-domain-deleted third-generation FVIII concentrate, became our preferential product. After the introduction, the development of inhibitors was prospectively monitored; retrospectively, we sought for risk factors in the patients who developed a de-novo inhibitor. Over a period of 15 months, 4/19 adult patients with non-severe haemophilia who were treated on demand for surgery, developed high titer antibodies to FVIII after administration of Refacto AFR; 5/52 mostly severe patients on prophylaxis, developed an inhibitor (3 ≥ 0.1 BU; 1 > 0.6 BU, 1 high titre) after they switched to Refacto AFR; all were children <14 years of age and with >100 exposure days, none related to surgery or intensive treatment; all received KovaltryR before. In conclusion: inhibitors were encountered in on demand patients and previously treated prophylaxis patients; this observation might be a coincidental finding, but also risk factors like genotype and surgery and/or that Refacto AFR is more immunogenic should be considered. For the patients on prophylaxis we hypothesize that loss of tolerance by preceding KovaltryR might have contributed to inhibitor development.

Symptomatic patients who test negative for common viruses are an important possible source of unrecognised or emerging pathogens, but metagenomic sequencing of all samples is inefficient because of the low likelihood of finding a pathogen in any given sample. We aimed to determine whether nasopharyngeal CXCL10 screening could be used as a strategy to enrich for samples containing undiagnosed viruses.

Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain-deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.

We retrospectively analysed the data files of 171 adults and 87 children/adolescents with severe haemophilia, except for 14 patients (moderate; minor) (1), to develop a global population pharmacokinetic (PK) model for eight factors VIII (FVIII) that could estimate individual PK parameters for targeting the desired level of FVIII activity (FVIII:C); and (2) to compare half-life (HL) in patients switching from a standard half-life (SHL) to an extended half-life (EHL) and evaluate the relevance of the switch. One-stage clotting assay for the measurement of FVIII activity (FVIII:C, IU/mL) was used for population PK modelling. The software, Monolix version 2019R1, was used for non-linear mixed-effects modelling. A linear two-compartment model best described FVIII:C. The estimated PK parameters (between-subject variability) were: 2640 mL (23.2%) for volume of central compartment (V1), 339 mL (46.8%) for volume of peripheral compartment (V2), 135 mL/h for Q (fixed random effect), and 204 mL/h (34.9%) for clearance (Cl). Weight, age, and categorical covariate EHL were found to influence Cl and only weight for V1. This model can be used for all of the FVIII cited in the study. Moreover, we demonstrated, in accordance with previous studies, that Elocta had longer half-life (EHL) than SHL (mean ratio: 1.48) as compared to Advate, Factane, Kogenate, Novoeight, and Refacto.

The current interest in recombinant factor VIII (rFVIII) products stems from the fact that they offer a technological solution to prolonging the half-life of and reducing the risk of formation of alloantibodies (inhibitors) against FVIII in treated patients with hemophilia A (HA). The Italian health care system has authorized the use of a wide range of rFVIII concentrates of the first, second, and third generation, as well as new innovative rFVIII preparates with an extended half-life (EHL) (Kogenate FS®-Bayer, belonging to the second generation and replaced since 2017 by a product consisting of the same modified molecule; because it is only available until the end of the current year, it will not be considered in this review). Some of these products have unique pharmacodynamic and pharmacokinetic (PK) profiles, including an EHL. The first-generation full-length rFVIII (FL-rFVIII), octocog alfa (Recombinate® Baxter/BIOVIIIx), although the oldest rFVIII product, has several desirable features. Third-generation products include two modified octocog alfa molecules (Advate®, Shire; Kovaltry®, Bayer) as well as the B domain-deleted rFVIII (BDD-rFVIII) moroctocog alfa (ReFacto®-Pfizer). The B domain-truncated (BDT-rFVIII) turoctocog alfa (NovoEight®, Novo Nordisk), the BDD-rFVIII simoctocog alfa (Nuwiq®, Kedrion), the single-chain BDT-rVIII lonoctocog alfa (Afstyla®, CSL Behring), and the BDD-rFVIIIFc efmoroctocog alfa (Elocta®, Sobi-Biogen) are new, innovative products. Simoctocog alfa, because its peculiarities, is considered a fourth-generation rFVIII concentrate. Turoctocog alfa, simoctocog alfa, and lonoctocog alfa have a high affinity for von Willebrand factor (vWF) that reduces renal clearance and prolongs the half-life of rFVIII. Efmoroctocog alfa, a first-in-class rFVIII-Fc fusion protein (rFVIIIFc), has a half-life 1.5-1.8 times longer than that of conventional plasma-derived FVIII (pd-rFVIII) and other rFVIII products. Clinical studies have evaluated the efficacy, safety, and inhibitor development of all these innovative concentrates in both previously treated (PTPs) and untreated patients (PUPs). This review considers the rFVIII products that are indicated for the treatment of patients with severe HA, focusing on those that are commercially available in Italy. Their PK characteristics, immunogenicity, and clinical benefits are discussed and compared.

Background Recombinant (rec-) coagulation factor VIII concentrates available for hemophilia A (HA) treatment differ in cell line production and structure, which could affect their pharmacodynamics and immunogenicity. Clinical trials showed that previously untreated patients with severe HA present higher rates of inhibitor development if treated with rec-FVIII products and that differences do exist as to inhibitor's formation among different rec-FVIII products. This finding could arise from several causes, such as absence of von Willebrand factor, different glycosylation profiles, or processes of molecular aggregation of the recombinant FVIII molecules. Objectives/Methods In this study, using size exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS) spectroscopy, and functional biochemical assays, we investigated the purity grade, FX activating ability, and aggregation status of three recombinant marketed products (Advate [Baxalta], Refacto AF [Pfizer], and Kogenate [Bayer]). Results The overall analysis of the results obtained with SE-HPLC and DLS spectroscopy showed that the three recombinant FVIII concentrates contain low but significant amounts of 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 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.

The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)-associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor's MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although >12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)-recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain-deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII.

Replacement therapy with plasma-derived or recombinant FVIII (pdFVIII or rFVIII) concentrates is the standard of treatment in patients with hemophilia A. The reference method used for measuring factor VIII (FVIII:C) levels in patients treated by FVIII concentrates is the chromogenic substrate assay (CSA). However, the one-stage clotting assay (OSA) is predominantly used in current clinical practice, but this method depends on the activated partial thromboplastin time (APTT) reagent and the coagulation analyzer used, and wide variations in the measurements of FVIII recovery have been reported with some factor concentrates. The French study group on the biology of hemorrhagic diseases (a collaborative group of the GFHT and MHEMO network) presents a review of the literature and proposals for the monitoring of FVIII:C levels in treated hemophilia A patients. The use of CSA calibrated with a plasma reference tested against the current FVIII WHO (World Health Organization) International Standard is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. OSA are adequate for the monitoring of patients treated with pdFVIII or with most of rFVIII concentrates. However, preliminary comparison with CSA is mandatory before measuring FVIII:C by OSA in patients treated by Refacto AF®. For rFVIII-EHL, OSA are only acceptable for Elocta®. Great caution is therefore required when measuring FVIII:C levels by OSA in patients substituted by other EHL-rFVIII. Indeed, most of recent studies reported data obtained with spiked plasmas, which deserve to be confirmed on plasma samples collected in treated patients.

We report a 10-year-old patient with haemophilia A developing anaphylaxis to recombinant factor VIII (octocog alfa). Allergic reactions, and especially anaphylactic events, are rare in patients with haemophilia A. The nature of these reactions is not fully understood. Here, we demonstrate a type I hypersensitivity reaction using sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting assay. This test revealed itself as an essential diagnostic tool, as it allowed us to choose an alternative treatment (moroctocog alfa). Its safety was later confirmed by an uneventful challenge test.