Production of α2,6-sialylated IgG1 in CHO cells

  title={Production of $\alpha$2,6-sialylated IgG1 in CHO cells},
  author={C{\'e}line Raymond and Anna C Robotham and Maureen A. Spearman and Michael Butler and John F Kelly and Yves Durocher},
  pages={571 - 583}
The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type… 

Production of IgGs with a human-like sialylation in CHO cells

In this study, it is shown that the a2,6-sialylation of IgG1’s Fc domain can be efficiently achieved by the transient coexpression of the human b1,4-galactosyltransferase 1 (GT) and a3,3-sIALyltransferases 1 (ST6) in CHO cells, whereas the expression of one or the other glycosyl transferase alone does not significantly improve sialylations.

Impact of Fc N-glycan sialylation on IgG structure

Investigating the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange and limited proteolysis experiments found only alpha 2,3-linked sialic acid on the 6-arm destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction.

Enhanced sialylation of a human chimeric IgG1 variant produced in human and rodent cell lines.

Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors

Integrated Genome and Protein Editing Swaps α-2,6 Sialylation for α-2,3 Sialic Acid on Recombinant Antibodies from CHO.

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.

Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

General methods to produce recombinant proteins of any desired glycoform in eukaryotic cells are described and the biological effect of these glycosylation traits for IgG and other glycoproteins are systematically explored.

Metabolic engineering of CHO cells to prepare glycoproteins.

The interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Cell engineering for the production of hybrid-type N-glycans in HEK293 cells.

The results suggest that KO cell lines are suitable for the production of therapeutic proteins with hybrid-type N-glycans, and would be useful as models for researching the mechanism of antimetastatic effects in human tumors by swainsonine treatment.



Sialylation of human IgG-Fc carbohydrate by transfected rat alpha2,6-sialyltransferase.

A recombinant IgG3 antibody with Phe-243 replaced by Ala (FA243) having both alpha2,6 andalpha2,3-sialylation restored recognition to wild-type IgG 3 levels for human FcgammaRI, FcGammaRII, and target cell lysis by complement.

NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.

Surprisingly, ST6Gal1 sialylated the two termini of the complex-type binantennary glycan in a manner remarkably similar to that observed for the free N-glycan, suggesting the Fc polypeptide does not greatly influence ST6 Gal1 specificity.

Identification of a receptor required for the anti-inflammatory activity of IVIG

The studies thus identify an antibody receptor specific for sialylated Fc, and present the initial step that is triggered by IVIG to suppress inflammation.

Branch-specific sialylation of IgG-Fc glycans by ST6Gal-I.

MS and NMR methodology demonstrates glycan modification occurs in a branch-specific manner with the alpha1-3Man branch of the complex, biantennary Fc glycan preferentially sialylated, suggesting that the apparent occlusion of glycan termini in Fc crystal structures does not dominate specificity.

Lectin analysis of human immunoglobulin G N-glycan sialylation

SNA and Ricinus communisagglutinin were used to study the occurrence, linkage and distribution of human immunoglobulin G (IgG) sialylation and SNA displayed strong binding to the IgG Fab fragment in both its native and denatured state.

Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues.

In vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction.

Analysis and Functional Consequences of Increased Fab-Sialylation of Intravenous Immunoglobulin (IVIG) after Lectin Fractionation

Biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation and found that SNA fractionation of IVIG yields a minor fraction of highly sialylated IgG, wherein the sIALic acid is mainly found in the Fab region.

The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity*

The results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.

Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity

The activity of IgG-G0 antibodies in mice with a genetic deletion of MBL (MBL-null mice) is analyzed and it is demonstrated that IgG 0 antibodies are unimpaired in MBL- null mice.