Validation of a method for plasma quantification of emicizumab

Abstract

Introduction: Emicizumab (Hemlibra®, ROCHE) is a bispecific, recombinant and humanized IgG4 monoclonal antibody, that mimics the function of FVIIIa. Its subcutaneous administration is effective and safe in preventing bleeding in patients with severe hemophilia A with or without inhibitor. Although emicizumab has been approved with an administration regimen based on body weight and without monitoring of plasma levels, emicizumab plasma quantification may be useful in different clinical and research circumstances. Since emicizumab- specific commercial calibrators and controls are not yet available in Argentina, we have validated a modified one-stage FVIII assay (modOSA) for the quantification of emicizumab in plasma (Epq) using in-house prepared calibrators and controls. Materials and methods: For Epq a 1/80 sample predilution with Owren Koller buffer was used. In house emicizumab calibrator (150 μg/mL emicizumab) was prepared by adding emicizumab 150 mg/mL to FVIII deficient plasma. Two in house control levels and a 6-point calibration curve were prepared. The validation procedures were performed in a STA Compact Max2 analyser (Diagnostica Stago). Repeatability: controls were processed 20 times in the same analytical run. Intra-laboratory Imprecision:- controls were processed in triplicate for 5 days. Linearity: 11 levels were measured in triplicate. Limit of quantitation: 15 measurements of a commercial FVIII deficient plasma were assessed. Bias were estimated based on an external quality assessment program (UK NEQAS). The interference of FVIII and rFVIIa in Epq and of emicizumab in other hemostasis determinations were evaluated. Results: Epq showed acceptable imprecision and bias, a linear range of 5-150 μg/ml and a quantification limit of 5 μg/ml. Epq was interfered with FVIII but not with rFVIIa. Emicizumab shortened aPTT values below the lower limit of the local reference interval. FVIII measured by chromogenic method with components of bovine origin was not affected. The one-stage FVIII assay (OSA) was falsely increased but showed a strong linear correlation with Epq in this instrument reagent system. Discussion: The automated “in-house” modOSA allows the plasma quantification of emicizumab using a simple, reproducible, low-cost method that is easy to integrate into routine processes.