title: Comparison of the glycosylation of in vitro generated polyclonal human IgG and therapeutic immunoglobulins.
abstract: We have recently developed an in vitro culture model enabling the large-scale expansion of switched-memory B lymphocytes, producing a polyclonal human IgG repertoire. Given the importance of glycosylation for the functions of immunoglobulins, we analyzed the N-glycosylation profiles of the immunoglobulin G (IgG) in this model. Switched-memory B cells were cultured for 38 days and, using liquid chromatography-mass spectrometry, we analyzed IgGs' glycosylation profiles which were then compared to the glycosylation patterns of commercial intravenous immunoglobulin (IVIG). We observed a reproducible proliferation rate, high viability through the cultures as well as a good maintenance of the switched-memory B cells repertoire. The glycosylation pattern analyses revealed a variety of the typical biantennary N-glycan structures with diverse terminal monosaccharides. While many similarities were detected in comparison to the glycosylation profile of IVIG, in vitro-produced polyclonal IgGs were bearing higher levels of bisecting GlcNAc known to affect the effector functions of therapeutic antibodies. This data highlights the need for monitoring of the glycoform distribution in antibodies produced in vitro.
authors.affiliation: Advanced Therapies and Product Development, Finnish Red Cross Blood Service, Kivihaantie 7, FI-00310 Helsinki, Finland. Electronic address: ilja.ritamo@thermofisher.com.
authors.forename: Ilja
authors.initials: I
authors.lastname: Ritamo
authors.forename: Marc
authors.initials: M
authors.lastname: Cloutier
authors.forename: Leena
authors.initials: L
authors.lastname: Valmu
authors.forename: Sonia
authors.initials: S
authors.lastname: Néron
authors.forename: Jarkko
authors.initials: J
authors.lastname: Räbinä
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D007156:Immunologic Memory / Q000276:immunology; D013058:Mass Spectrometry; D006023:Glycoproteins
chemical_list: D007074:Immunoglobulin G; C046179:glycosylated IgG; D006023:Glycoproteins
keywords: ADCC; Antibody; F; FBS; Fc receptor; Gal; GlcNAc; Glycosylation; H; IVIG; IgG; LC; LTQ; MS; MS/MS; Mass spectrometry; N; N-acetylglucosamine; N-acetylhexosamine, HexNAc; N-acetylneuraminic acid, NeuAc (sialic acid); N-glycolylneuraminic acid; NeuGc; S; antibody-dependent cellular cytotoxicity; deoxyhexose (fucose); fetal bovine serum; galactose; hexose, Hex; intravenous immunoglobulin; linear trap quadrupole; liquid chromatography; mass spectrometry; tandem mass spectrometry