Enhanced α2-3 linked sialylation determines the extended half-life of CHO-rVWF

• α2-3 linked sialylation is significantly increased on CHO-rVWF compared to human plasma-derived VWF.

• Enhanced α2-3 linked sialylation attenuates in vivo clearance through the macrophage galactose lectin and asialoglycoprotein receptor.

The half-life of recombinant VWF (rVWF) expressed in CHO cells (CHO-rVWF; Vonicog alfa, VonvendiÒ / VeyvondiÒ) is significantly longer than plasma-derived (pd) VWF concentrates. This finding is intriguing since CHO cells do not generate α2-6 sialylation, which constitutes the majority of human pdVWF sialylation. We hypothesized that glycan differences might regulate the longer half-life of CHO-rVWF. In lectin plate binding assays and liquid chromatography mass spectrometry analysis, we confirmed that CHO-rVWF lacked α2-6 linked sialylation. Conversely, however, α2-3 linked sialylation was significantly increased on CHO-rVWF, which also had reduced exposed β-Gal compared to pdVWF. Consistent with human data, CHO-rVWF clearance was significantly (p<0.001) reduced in VWF-/- mice compared to pdVWF. However, clearance of asialo-pdVWF and asialo-CHO-rVWF were identical. In keeping with the in vivo half-life prolongation, CHO-rVWF binding to murine macrophages (p=0.012) and HepG2 cells (p=0.001) was significantly decreased compared to pdVWF. Furthermore, CHO-rVWF binding to purified MGL and ASGPR receptors was also significantly reduced. In contrast to pdVWF, in vivo studies in MGL1-/- mice and Asgr1-/- mice demonstrated that neither MGL nor ASGPR play significant roles in regulating CHO-rVWF clearance. Together, our findings demonstrate that enhanced α2-3 linked sialylation on CHO-rVWF is responsible for its extended in vivo half-life.