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.

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.

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