The molar hydrodynamic volume changes of factor VIIa due to GlycoPEGylation

The effects of GlycoPEGylation on the molar hydrodynamic volume of recombinant human rFVIIa were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10 kDa PEG and a branched 40 kDa PEG, respectively. Molar hydrodynamic volumes were determined by capillary viscometry and mass spectrometry. The intrinsic viscosities of rFVIIa, its two GlycoPEGylated compounds, and of linear 8 kDa, 10 kDa, 20 kDa and branched 40 kDa PEG polymers were determined. The measured intrinsic viscosity of rFVIIa is 6.0 mL/g, while the intrinsic viscosities of 10 kDa PEG-rFVIIa and 40 kDa PEG-rFVIIa are 29.5 mL/g and 79.0 mL/g, respectively. The intrinsic viscosities of the linear PEG polymers are 20, 22.6 and 41.4 mL/g for 8, 10, and 20 kDa, respectively, and 61.1 mL/g for the branched 40 kDa PEG. From the results of the intrinsic viscosity and MALDI–TOF measurements it is evident, that the molar hydrodynamic volume of the conjugated protein is not just an addition of the molar hydrodynamic volume of the PEG and the protein. The molar hydrodynamic volume of the GlycoPEGylated protein is larger than the volume of its composites. These results suggest that both the linear and the branched PEG are not wrapped around the surface of rFVIIa but are chains that are significantly stretched out when attached to the protein.