Ciara Byrne, Soracha Ward, Jamie O'Sullivan, Alain Chion, Patricia Lopes, Bogdan Baci, Caoimhe Dowd, Darragh Jordan, Ross Baker, Roger J S Preston, Marco Monopoli, Peter L Turecek, Maximilianos Kotsias, Jack Cheeseman, Alan B Moran, Richard A Gardner, Daniel I R Spencer, Ferdows Atiq, James S O'Donnell
{"title":"α - 2-3链唾液酰化的增强决定了CHO-rVWF半衰期的延长。","authors":"Ciara Byrne, Soracha Ward, Jamie O'Sullivan, Alain Chion, Patricia Lopes, Bogdan Baci, Caoimhe Dowd, Darragh Jordan, Ross Baker, Roger J S Preston, Marco Monopoli, Peter L Turecek, Maximilianos Kotsias, Jack Cheeseman, Alan B Moran, Richard A Gardner, Daniel I R Spencer, Ferdows Atiq, James S O'Donnell","doi":"10.1182/blood.2024027038","DOIUrl":null,"url":null,"abstract":"<p><strong>Abstract: </strong>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.</p>","PeriodicalId":9102,"journal":{"name":"Blood","volume":" ","pages":"2768-2773"},"PeriodicalIF":21.0000,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Enhanced α2-3-linked sialylation determines the extended half-life of CHO-rVWF.\",\"authors\":\"Ciara Byrne, Soracha Ward, Jamie O'Sullivan, Alain Chion, Patricia Lopes, Bogdan Baci, Caoimhe Dowd, Darragh Jordan, Ross Baker, Roger J S Preston, Marco Monopoli, Peter L Turecek, Maximilianos Kotsias, Jack Cheeseman, Alan B Moran, Richard A Gardner, Daniel I R Spencer, Ferdows Atiq, James S O'Donnell\",\"doi\":\"10.1182/blood.2024027038\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Abstract: </strong>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. 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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.
期刊介绍：
Blood, the official journal of the American Society of Hematology, published online and in print, provides an international forum for the publication of original articles describing basic laboratory, translational, and clinical investigations in hematology. Primary research articles will be published under the following scientific categories: Clinical Trials and Observations; Gene Therapy; Hematopoiesis and Stem Cells; Immunobiology and Immunotherapy scope; Myeloid Neoplasia; Lymphoid Neoplasia; Phagocytes, Granulocytes and Myelopoiesis; Platelets and Thrombopoiesis; Red Cells, Iron and Erythropoiesis; Thrombosis and Hemostasis; Transfusion Medicine; Transplantation; and Vascular Biology. Papers can be listed under more than one category as appropriate.
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