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.

Graphical abstract

Figure 1.

Differences in glycosylation and sialylation between CHO-rVWF and pdVWF. (A) Diagrams illustrate common N- and O-linked glycan structures expressed on human pdVWF and lectin affinities. (B) Lectin plate–binding assays were performed to compare glycans expressed on CHO-rVWF to pdVWF. Lectins used included SNA, MAA-II, WGA, RCA-I, and ECA. All enzyme-linked immunosorbent assays (ELISAs) were performed in triplicate and results expressed as a percentage of binding normalized to pdVWF. Data were analyzed for normality using the Shapiro-Wilk test and compared using the Student t test. Data are presented as mean ± standard error of the mean (SEM). (C) A, B, and H blood group carbohydrate determinants on pdVWF and CHO-rVWF were assessed using plate-binding assays. (D) LC-MS was used to analyze the N-glycans on CHO-rVWF compared to pdVWF; chromatograms of pdVWF (top) and CHO-rVWF (bottom). Peaks are annotated with the most abundant N-glycan indicated per peak. In pdVWF, H5N4F1S2, highlighted in pink, was present in α2,3 α2,3; α2,3 α2,6; and α2,6 α2,6 forms. In contrast, CHO-rVWF only displays a single linkage form (α2,3 α2,3). (E) To investigate the clearance of CHO-rVWF in mice, VWF^–/– mice were infused with either pdVWF (blue) or CHO-rVWF (red), and blood was collected at 3, 30 minutes, and 1, 2, 3, 4, and 6 hours after infusion. At each time point, residual circulating VWF concentration was determined by VWF:Ag ELISA, and mean residence time was calculated. P value is the outcome of extra-sum-of-squares F test. (F) To study the importance of terminal sialylation in modulating the prolonged half-life of CHO-rVWF, in vivo studies were repeated in VWF^–/– mice after the digestion of CHO-rVWF and pdVWF with α2-3,6,8,9 neuraminidase (asialo-CHO-rVWF and asialo-pdVWF respectively). Three to 5 mice were used per point time, and data are presented as mean ± SEM. MAA-II, M amurensis lectin II; SNA, S nigra agglutinin; WGA, wheat germ agglutinin.

Figure 2.

Interaction of CHO-rVWF with macrophage and hepatocyte clearance receptors. (A) Macrophage and hepatocyte receptors implicated in regulating VWF clearance in vivo. (B) To determine whether macrophages play a role in regulating the clearance of CHO-rVWF, in vivo clearance studies were repeated in VWF^–/– mice 24 hours after clodronate-induced macrophage depletion. Data are graphed as percentage residual CHO-rVWF relative to the amount injected. P value is the outcome of extra-sum-of-squares F test. (C) Binding of CHO-rVWF (red) and pdVWF (blue) to murine BMDMs was assessed by flow cytometry. Representative histograms of binding relative to no VWF (gray) are shown. The y-axis represents the binding capacity normalized to the number of cells, and the x-axis represents the fluorescence intensity absorbance at the wavelength of 488 nm, in which higher values indicate more binding. (D) MGL binding was assessed for CHO-rVWF compared to pdVWF using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (E) BMDMs were isolated from WT and MGL1^–/– mice. Binding of pdVWF (blue bars) and CHO-rVWF (red bars) to BMDMs with or without MGL was then assessed by flow cytometry. Data are presented as MFI normalized to controls without VWF. (F) In vivo clearance of pdVWF (blue lines) and CHO-rVWF (red lines) was studied in WT (solid lines) and MGL1^–/– mice (dashed lines). Mice were sampled after injection (time = 0) and subsequently sampled at indicated time points. Data are graphed as percentage residual VWF relative to the VWF:Ag at time of 0. (G) Binding of CHO-rVWF (red) and pdVWF (blue) to human hepatocyte HepG2 cells was assessed by flow cytometry. Representative histograms of binding relative to no VWF (gray) are shown. Data were assessed for normality using the Shapiro-Wilk test and compared by the Student t test. The y-axis represents the binding capacity normalized to the number of cells, and the x-axis represents the fluorescence intensity absorbance at the wavelength of 488 nm, in which higher values indicate more binding. (H) ASGPR1 binding was assessed for CHO-rVWF compared to pdVWF using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (I) In vivo clearance of pdVWF (blue lines) and CHO-rVWF (red lines) was studied in WT (solid lines) and Asgr1^–/– mice (dashed lines). Mice were sampled after injection (time = 0) and subsequently sampled at indicated time points. Data are graphed as percentage residual VWF relative to the VWF:Ag at time of 0. (J) LRP1 cluster IV binding was assessed for CHO-rVWF (red) compared to pdVWF (blue) using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (K) Binding of CHO-rVWF (red) and pdVWF (blue) VWF to HEK293 cells stably transfected with LRP1 (HEK-LRP1) was assessed by flow cytometry. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. Abs, absorbance; MFI, mean fluorescence intensities; WT, wild-type.