Refacto af

The presence of FVIII activity was evaluated in plasma of treated mice starting from 2 weeks after injection, as described previously.⁴² (link) The percentage of FVIII activity was quantified by one-stage aPTT using HemosIL Synthasil aPTT reagents (Instrumentation Laboratory) in a Coatron M4 coagulometer (TECO Medical Instruments). Standards for FVIII activity quantification were generated by serially diluting recombinant human BDD-FVIII (RefactoAF, Pfizer) in HA mouse plasma.
The presence of anti-FVIII IgG antibodies in plasma of LV-injected mice was evaluated by indirect ELISA on a 96-well plate coated with 0.2 μg/mL FVIII-BDD (RefactoAF) as described previously.¹⁹ (link) Anti-FVIII IgG antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgG Fc secondary antibody (Thermo Fisher Scientific). Plasma from HA mice previously immunized with LV.PGK-FVIII and reacting against FVIII was used as positive control, while pooled plasma from untreated HA mice served as negative control.

In this study, data from severe and moderate paediatric (age < 18 years) haemophilia A patients undergoing a minor or major elective surgery were gathered retrospectively at the Great Ormond Street Hospital in London, UK. Surgeries were conducted to remove, replace and or insert a CVAD to facilitate FVIII concentrate administration.⁹In the perioperative period, patients received replacement therapy with one of the following products: recombinant FVIII concentrates (Advate and Recombinate: Baxter Bioscience, Thousand Oaks, CA, USA; Kogenate FS: Bayer, Berkeley, CA, USA; Refacto AF: Pfizer, New York, NY, USA; Helixate FS: CSL Behring, Marburg, Germany; Octanate and Nuwiq: Octapharma AB, Stockholm, Sweden; Innovate: Biomed Lublin, Lublin, Poland) or plasma‐derived FVIII concentrates (Monoclate‐P: CSL Behring, Kankakee, IL, USA). Other patient characteristics are described in Table 1.
The study was not subject to the Medical Research Involving Human Subjects Act and was approved by all Medical Ethics Committees in the Netherlands. In the United Kingdom, the study was approved by the Research Ethics Committee (NRES committee South Central‐Berkshire, REC reference 15/SC/0367); an opt‐out consent procedure was used to collect anonymized clinical data.

The test product was moroctocog alfa (AF‐CC), manufactured using the albumin‐free process (ReFacto AF, Pfizer, Collegeville, PA). The reference product was moroctocog alfa (ReFacto, Pfizer) manufactured using the process containing albumin.2 Each subject received single doses of 50 IU/kg as 2‐min infusions of each of the study medications in a randomized fashion. The dose was calculated on the basis of the subject's actual body weight as measured at the screening visit and on the labeled potency of the study drug. After completion of the first study period and a minimum washout of 5 days, subjects received the alternate treatment.