Measurement of modified biologic including coagulation factors with extended half-life obtained, for example, by polysialylation pose an analytical challenge especially if both biological activity and presence of modification have to be determined. Analytical methods applied so far address only 1 of the 2 quality attributes of modified biologics. Here, we describe the development and bioanalytical validation of a polysialic acid-mediated factor VIII activity assay: Polysialic acid-specific capture of polysialylated recombinant factor VIII is combined with a chromogenic FVIII activity test using commercially available reagents. This assay principle enabled measurement of FVIII activity down to the pico mole-range without any interference by nonmodified factor VIII. To the best of our knowledge, this is the first method to selectively, accurately, and precisely measure simultaneously activity and modification integrity of a polysialylated biologic in complex matrices, as shown by the bioanalytical validation data. The convenience, robustness, and reliability of using this method has been demonstrated by its application for the nonclinical development of the polysialylated recombinant FVIII preparation. The method principle could be applied to protein modifications other than polysialylation and to activity tests other than the chromogenic FVIII assay.
Keywords: bioanalysis; combined integrity and functionality measurement; minimizing bias by endogenous protein; monitoring of pharmacokinetic studies; polysialylated recombinant factor VIII.
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