Development, validation and application of a polysialylation-dependent FVIII activity assay to measure SHP656 (BAX 0826), a next generation extended half-life recombinant factor VIII product

A. Weber, S. Haindl, G. Mohr, A. Engelmaier (Vienna, Austria)

Laboratory tests
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: Hemophilia A is a rare genetic bleeding disorder caused by missing or defective factor VIII (FVIII), a crucial factor in blood coagulation. SHP 826 (BAX 826) is currently developed as the next generation extended half-life (EHL) recombinant (r)FVIII replacement for treatment of hemophilia A. SHP 826 is the first EHL rFVIII in which polysialic acid (PSA) is conjugated to ADVATE (rFVIII) to extend the circulatory half-life of FVIII. Here we describe the development, validation and application of a chromogenic FVIII activity assay to exclusively measure polysialylated rFVIII. During nonclinical characterization this selectivity not only enabled unambiguous measurement of SHP 826 in the presence of endogenous animal FVIII, but also provided information on its integrity.

Methods: An anti-PSA antibody was coated to a polystyrene plate and used to selectively capture SHP 826 from the sample. After a washing step, a conventional chromogenic assay was carried out, for which calibration was achieved using a SHP 826 preparation with a defined FVIII activity. Development focused on defining adequate antibody coating conditions, in particular pH and antibody concentration, and demonstrated the assay’s selectivity in plasma samples from various animal species and human plasma. Assay validation was performed in the matrices of citrated rat, cynomolgus monkey and FVIII-deficient mouse plasma in accordance with the EMA guideline for bioanalytical assay validation. Finally, the assay was used in nonclinical studies of SHP 826 in FVIII-deficient mice, rats and cynomolgus monkeys.

Results: Sensitive and reproducible calibration curves of 1.1 to 34 mU/mL were generated. The results of the method validation proved the assay suitable for its intended use. Mean accuracies given as the recovery of SHP 826 activity spiked at five relevant concentrations (0.05 - 15 U/mL) were 95% to 101%. Intra- and inter-run precision, expressed as relative standard deviations, were lower than 10%. These values were also obtained for samples spiked with concentrations at the lower limit of quantification.

Conclusion: Successful application of the assay during the nonclinical SHP 826 studies with no interference by endogenous FVIII confirmed the validation data,