title: Integrated Genome and Protein Editing Swaps α-2,6 Sialylation for α-2,3 Sialic Acid on Recombinant Antibodies from CHO.
abstract: Immunoglobin G with α-2,6 sialylation has been reported to have an impact on antibody-dependent cellular cytotoxicity and anti-inflammatory efficacy. However, production of antibodies with α-2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N-glycan site and the presence of exclusively α-2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α-2,6 sialyltransferase produced IgG with significant levels of both α-2,6 and α-2,3 sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α-2,3 sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α-2,6 sialylation level relative to α-2,3 sialylation for the α-2,3 sialyltransferase knockouts when combined with α-2,6 sialyltransferase overexpression. Indeed, α-2,3 linked sialic acids were not detected on IgG produced from the α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI-TOF and LC-ESI-MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N-glycans with different structural variations for examining the role of glycosylation on protein performance.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Chung
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Shuang
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Bojiao
authors.initials: B
authors.lastname: Yin
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Betenbaugh
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D057005:Bioengineering / Q000379:methods*; D016466:CHO Cells; D003412:Cricetulus; D019158:N-Acetylneuraminic Acid / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D019158:N-Acetylneuraminic Acid
keywords: CHO cells; Glycoengineering; Monoclonal Antibody; α -2,6 Sialylation; α-2,3 Sialylation