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

Abstract

The half-life of recombinant human von Willebrand factor (rVWF) expressed in CHO cells (CHO-rVWF; Vonicog alfa; and Vonvendi/Veyvondi) is significantly longer than that of plasma-derived VWF (pdVWF). This finding is intriguing because 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-galactose (β-Gal) compared to pdVWF. Consistent with human data, CHO-rVWF clearance was significantly (P < .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 = .012) and HepG2 cells (P = .001) was significantly decreased compared to pdVWF. Furthermore, CHO-rVWF binding to purified macrophage-galactose-type lectin (MGL) receptor and asialoglycoprotein receptor (ASGPR) was also significantly reduced. In contrast to pdVWF, in vivo studies in MGL1-/- mice and Asgr1-/- mice demonstrated that neither MGL nor ASGPR plays 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 half-life.