Bay 94-9027, a Site-Specifically Pegylated Recombinant Factor VIII: Preliminary Results from an International Comparative Laboratory Field Study
Authors: Church Nikki , Leong Lilley , Katterle Yvonne , Rao Danda Yadagiri , Ulbrich Hannes-Friedrich , Noerenberg Iris , Bruns Stefan , Michaels Lisa A. 
Published: October 2019

Abstract
Introduction: Accurate measurement of factor VIII (FVIII) in patients with hemophilia A after infusion of FVIII is important for patient monitoring and treatment decisions. Discrepancies in results using different assays or reagents to measure prolonged-half-life factor products have been recognized and highlight that effective monitoring of patient response to these products may require adjustments in clinical laboratory practices. Because of this recognized issue, a global field study was conducted to assess the ability of clinical laboratories to measure BAY 94-9027 activity in spiked hemophilic plasma samples using their in-house or specific assays. BAY 94-9027 is a prolonged-half-life FVIII that has been modified through site-specific addition of a 60-kDa polyethylene glycol (2×30 kDa branched).
Methods: Participating laboratories received sample sets of 26 blinded samples each in randomized order for analysis; 3-4 sample sets were provided to each laboratory to complete the study. Each set consisted of triplicate BAY 94-9027 or a comparator (antihemophilic factor [recombinant] plasma/albumin-free method [rAHF-PFM (Advate®); Shire, Westlake Village, CA, USA]) spiked at low (<10 IU/dL), medium (10-50 IU/dL), and high (50-100 IU/dL) concentrations in pooled hemophilic plasma. BAY 94-9027 and rAHF-PFM both use the chromogenic assay for potency labeling. Normal control plasma and unspiked pooled hemophilic plasma in triplicate served as positive and negative controls, respectively. Two additional blinded samples matching 2 of the other 24 samples in the set were included in each set to decrease the predictability of the sample sets and prevent observer bias. In the first stage of the study, laboratories analyzed test samples using their in-house assays, reagents, and standards. Laboratories that had the capability to use the chromogenic assay also measured an additional sample set using the chromogenic assay. In the second stage of the study, all participating laboratories tested 2 additional sample sets using 2 Bayer-provided activated partial thromboplastin time (aPTT) kits (Pathromtin® [Siemens, Marburg, Germany] and HemosIL® SynthASil [Instrumentation Laboratory, Bedford, MA, USA]), as these kits have previously been shown to accurately measure BAY 94-9027 and full-length FVIII. FVIII recovery and FVIII levels were primary and secondary endpoints, respectively. Results were analyzed for intra- and interlaboratory variation.
Results: 52 laboratories in North America, Europe, and Israel participated in the field study. Of these, 49 laboratories tested samples using their in-house one-stage assay, 16 tested samples using the chromogenic assay, and 13 used both assays. The reagents routinely used for measuring FVIII activity varied among participating laboratories. Mean FVIII recovery ranged from 75.1%‒103.2% for BAY 94-9027 and 94.6%‒114.7% for rAHF-PFM across all concentrations and reagents using the one-stage assay. As expected, the PTT-A (Stago, Asnières sur Seine, France) and HemosIL® APTT-SP kits (Instrumentation Laboratory), used by 6 and 2 laboratories, respectively, underestimated BAY 94-9027 at all concentrations. More accurate one-stage results were generated using the Pathromtin® and HemosIL® SynthASil kits, used by 6 and 15 laboratories, respectively, as shown in the second stage of the study. For the chromogenic assay, mean FVIII recovery ranged from 104.4%‒117.1% for BAY 94-9027 and 87.7%‒107.8% for rAHF-PFM across all concentrations. Interlaboratory variability was low for measurement of BAY 94-9027 with chromogenic assays (particularly Chromogenix Coamatic® and HemosIL® ELECTRACHROME™ [Instrumentation Laboratory]).
Conclusions: The results of this global field study indicate that chromogenic assays are an accurate method for measurement of BAY 94-9027. BAY 94-9027 can also be accurately monitored using many commonly used one-stage assay kits, as gauged by their representation in the field study. Understanding the limitations and advantages of specific assay kits is important for choosing the correct assay systems to measure individual FVIII products in clinical practice.

Disclosures
Church: Bayer: Employment. Leong:Bayer: Employment. Katterle:Bayer Pharma AG: Employment. Rao Danda:Bayer: Employment. Ulbrich:Bayer Pharma AG: Employment. Noerenberg:Bayer Pharma AG: Employment. Bruns:Bayer: Consultancy. Michaels:Bayer: Employment.