The effect of GlycoPEGylation on the physical stability of human rFVIIa with increasing calcium chloride concentration

Abstract

The effects of calcium chloride on the structural, kinetic and thermal stability of recombinant human factor VIIa (rFVIIa) were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10 kDa PEG and a branched 40 kDa PEG, respectively. Three different CaCl 2 concentrations were used: 10 mM, 35 mM and 100 mM. The secondary structure and tertiary structure of rFVIIa at 25 °C, measured by circular dichroism (CD), were maintained upon GlycoPEGylation as well as CaCl 2 content. In contrast, the thermal stability of the three rFVIIa compounds, measured by differential scanning calorimetry (DSC) and circular dichroism (CD), and aggregation behaviour, measured by light scattering (LS), were affected by the increasing calcium concentration. Increasing the CaCl 2 concentration from 10 mM to 35 mM resulted in a decrease in the apparent unfolding temperature, T m, of rFVIIa, whereas the concentration of CaCl 2 has to be raised to 100 mM in order to see the same effect on the GlycoPEGylated rFVIIa compounds. The temperature of aggregation of rFVIIa, T agg, increased as the CaCl 2 concentration increased from 35 mM to 100 mM, while T agg for the GlycoPEGylated rFVIIa compounds was practically independent of the CaCl 2 concentration. From the obtained results, it is concluded that GlycoPEGylation postpones the calcium induced thermal destabilisation of rFVIIa, and a much higher calcium concentration also postpones the thermally induced aggregation of rFVIIa. The thermally induced aggregation of the GlycoPEGylated rFVIIa compounds is unaffected by an increasing calcium chloride concentration.