Characterization of PEGylated Factor VIII Molecules.

Patients with hemophilia A typically require frequent treatment with replacement Factor VIII (FVIII) products due to the relatively short circulating half-life of FVIII. Increasing the half-life of FVIII would profound impact the lives of patients with hemophilia A, especially children, by reducing the frequency of infusions. The addition of polyethylene glutamate (PEG) to therapeutic molecules or their carriers has been demonstrated to increase the survival of such molecules in the circulation. To create a longer circulating product, we have developed novel FVIII molecules using site specific PEGylation techniques. We present a characterization of these PEGylated FVIII molecules in terms of the site of PEGylation, the binding of von Willebrand Factor (vWF), and the effect of PEGylation on thrombin digestion, and we compare the one-stage and chromogenic potency test results. The site of PEGylation was determined using MALDI-mass spectrometry. A vWF binding assay was developed using surface plasmon resonance technology (Biacore). The effect of PEGylation on thrombin digestion of the novel constructs was evaluated using MALDI-mass spectrometry, thrombin activation, and potency decay curves following thrombin activation. All novel constructs were PEGylated on the expected target site based on the site of introduced mutation. PEGylation of FVIII appeared to have a slight reduction on vWF-binding capability. The site of PEGylation reduced the ability of thrombin to digest FVIII, as assayed by MALDI-mass spectrometry analysis and by thrombin digestion and decay curves. The size and position of the PEG molecule influenced the extent of FVIII thrombin sensitivity. The one-stage potency assay was more sensitive to the size and position of the attached PEG molecule than the chromogenic potency assay. We report that some PEGylated FVIII molecules are less sensitive to thrombin activation than unmodified FVIII, and that the size and position of the PEG molecule can significantly affect the potency of FVIII. The findings of this study suggest that site-directed PEGylation of FVIII may be a useful technique for the development of FVIII molecules with extended activity in circulation and that careful choice of size and position of the PEG molecule is critical.