Measurement of modified biol. including coagulation factors with extended half-life obtained, for example, by polysialylation pose an anal. challenge especially if both biol. activity and presence of modification have to be determined Anal. methods applied so far address only 1 of the 2 quality attributes of modified biologics. Here, we describe the development and bioanal. 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 com. 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 biol. in complex matrixes, as shown by the bioanal. validation data. The convenience, robustness, and reliability of using this method has been demonstrated by its application for the nonclin. 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.