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Human blood coagulation factor VIII (hFVIII) is used in hemostatic and prophylactic treatment of patients with hemophilia A. Biotechnological innovations have enabled purification of the culture medium of rodent or human cells harboring the hFVIII expression cassette. However, cell lines express hFVIII protein derived from an exogenous expression vector at a lower level than most other proteins. Here, we describe hFVIII production using piggyBac transposon and the human-derived expi293F cell line. Use of a drug selection protocol, rather than transient expression protocol, allowed cells harboring hFVIII expression cassettes to efficiently produce hFVIII. In heterogeneous drug-selected cells, the production level was maintained even after multiple passages. The specific activity of the produced hFVIII was comparable to that of the commercial product and hFVIII derived from baby hamster kidney cells. We also applied codon optimization to the hFVIII open reading frame sequences in the transgene, which increased production of full-length hFVIII, but decreased production of B-domain-deleted human FVIII (BDD-hFVIII). Low transcriptional abundance of the hF8 transgene was observed in cells harboring codon-optimized BDD-hFVIII expression cassettes, which might partially contribute to decreased hFVIII production. The mechanism underlying these distinct outcomes may offer clues to highly efficient hFVIII protein production., (© 2022. Japanese Society of Hematology.)

Introduction: Moroctocog alfa albumin-free cell culture (AF-CC) increases plasma levels of factor VIII (FVIII) activity and, in China, is indicated for the control and prevention of bleeding episodes in patients with hemophilia A. This study aimed to evaluate the efficacy, safety, and recovery data of moroctocog alfa (AF-CC) in patients with hemophilia participating in two open-label studies, both conducted in China., Methods: The authorization study (clinicaltrials.gov identifier NCT00868530) enrolled patients aged ≥6 years, previously treated with ≥1 exposure day of FVIII replacement therapy. The real-world study (clinicaltrials.gov identifier NCT02492984) enrolled patients of any age who were previously untreated or requiring surgical prophylaxis. In both studies, on-demand treatment was administered over 6 months. Key assessments included response to treatment, FVIII inhibitor development, and recovery., Results: In the authorization study (N = 53; mean age, 23.2 years; severe hemophilia, 23%), response was excellent/good for 90% of infusions at 24 hours. Seven patients developed inhibitors. Mean (SD) FVIII recovery at the initial and final visits was 1.77 (0.50) and 1.67 (0.45) (IU/dL)/(IU/kg), respectively. In the real-world study (N = 85; mean age, 9.5 years; severe hemophilia, 58%), response was rated as excellent or good for most (87%) on-demand infusions and for all surgical prophylaxis patients (n = 14). Seven patients developed FVIII inhibitors. Mean (SD) FVIII recovery at the initial and final visits was 1.71 (0.50) and 1.68 (0.31) (IU/dL)/(IU/kg), respectively. No new safety signals were observed in either study., Conclusion: On-demand treatment and surgical prophylaxis with moroctocog alfa (AF-CC) is safe and effective for both previously treated and previously untreated Chinese patients with hemophilia A., Competing Interests: R. Yang, Y. Zhao, X. Wang, J. Sun, R. Wu, C. Jin, J. Jin, and D. Wu have no interests that might be perceived as posing a conflict or bias. J. Rupon, J. M. Korth-Bradley, and B. Luo, are employees of Pfizer Inc and may own stock/options in the company. F. Huard, P. Rendo, F. Sun, L. Xu, and Y. C. Liu were employees of Pfizer Inc at the time of this study. The authors report no other conflicts of interest in this work., (© 2020 Yang et al.)

Purpose: To identify international units (IUs) dispensed and consequent expenditures for standard half-life (SHL) versus extended half-life (EHL) recombinant factor VIII (rFVIII) replacement products in hemophilia A patients in a real-world setting., Design: Two U.S. claims databases were analyzed., Methodology: Number of IUs dispensed and quarterly expenditures for rFVIII products were collected from the Optum Clinformatics Data Mart and Truven Health MarketScan Databases. Truven claims were also analyzed for factor IUs dispensed and expenditures for patients with data for ≥3 months before and after switching to an EHL product., Results: The Optum and Truven databases, respectively, included 276 (SHL, n=243; EHL, n=33) and 500 (SHL, n=409; EHL, n=91) hemophilia A patients. Median quarterly factor IUs dispensed in Optum were 10% higher with EHL versus SHL products over nine quarters, and 45% higher with EHL versus SHL products in Truven over 10 quarters. Median quarterly expenditures in the EHL cohort were 51% (individual quarterly medians range, 1%-101%) higher than in the SHL cohort in Optum and 122% higher (individual quarterly medians range, 1%-189%) in Truven. Twenty-nine Truven patients switched to an EHL product; median factor IUs dispensed varied quarterly. The lowest SHL and highest EHL values occurred in the quarter immediately before switching and the first quarter post-switch, respectively. Overall median quarterly expenditures were higher post-switch; this was consistent over seven quarters., Conclusion: We found higher expenditures over two years for hemophilia A patients using EHL versus SHL products. Switching to an EHL rFVIII product was associated with variable factor IUs dispensed and consistently higher expenditures.

Hemophilia A is a bleeding disorder characterized by the absence or dysfunction of blood coagulation factor VIII (FVIII). Patients are treated with regular infusions of FVIII concentrate. In response to treatment, approximately 30% of patients with severe hemophilia A develop inhibitory antibodies targeting FVIII. Both patient and treatment related risk factors for inhibitor development have been described. Multiple studies comparing the immunogenicity of recombinant and plasma-derived FVIII have yielded conflicting results. The randomized controlled SIPPET (Survey of Inhibitors in Plasma-Product Exposed Toddlers) trial demonstrated an increased risk of inhibitor development of recombinant FVIII when compared to von Willebrand factor (VWF)-containing plasma-derived FVIII. Presently, it is unclear which mechanism underlies the reduced immunogenicity of plasma-derived FVIII. In this review we address the potential role of VWF on FVIII immunogenicity and we discuss how VWF affects the immune recognition, processing and presentation of FVIII. We also briefly discuss the potential impact of glycan-composition on FVIII immunogenicity. It is well established that VWF shields the uptake of FVIII by antigen presenting cells. We have recently shown that VWF binds to the surface of dendritic cells. Here, we present a novel model in which surface bound FVIII-VWF complexes regulate the internalization of FVIII. Binding of FVIII to VWF is critically dependent on sulfation of Tyr1699 (HVGS numbering) in the light chain of FVIII. Incomplete sulfation of Tyr1699 has been suggested to occur in several recombinant FVIII products resulting in a loss of VWF binding. We hypothesize that this results in alternative pathways of FVIII internalization by antigen presenting cells which are not regulated by VWF. This hypothetical mechanism may explain the reduced immunogenicity of VWF containing plasma-derived FVIII concentrates as found in the SIPPET study., (Copyright © 2017. Published by Elsevier Ltd.)