title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.
abstract: Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and β-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia. morten.andersen@mq.edu.au.
authors.forename: M
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD, 21702, USA.
authors.forename: E
authors.initials: E
authors.lastname: Chertova
authors.affiliation: Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, 21702, USA.|Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, 21702, USA.
authors.forename: C
authors.initials: C
authors.lastname: Bergamaschi
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: E S X
authors.initials: ES
authors.lastname: Moh
authors.affiliation: Cancer Research Technology Program, Leidos Biomedical, Inc., Frederick National Laboratory, Frederick, MD, 21702, USA.
authors.forename: O
authors.initials: O
authors.lastname: Chertov
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD, 21702, USA.
authors.forename: J
authors.initials: J
authors.lastname: Roser
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD, 21702, USA.
authors.forename: R
authors.initials: R
authors.lastname: Sowder
authors.affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, 21702, USA.
authors.forename: J
authors.initials: J
authors.lastname: Bear
authors.affiliation: AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD, 21702, USA.
authors.forename: J
authors.initials: J
authors.lastname: Lifson
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: N H
authors.initials: NH
authors.lastname: Packer
authors.affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, 21702, USA.
authors.forename: B K
authors.initials: BK
authors.lastname: Felber
authors.affiliation: Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, 21702, USA.
authors.forename: G N
authors.initials: GN
authors.lastname: Pavlakis
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D019409:Interleukin-15 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D053647:Receptors, Interleukin-15 / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins
chemical_list: C569274:IL15 protein, human; C508024:IL15RA protein, human; D019409:Interleukin-15; D053647:Receptors, Interleukin-15; D011994:Recombinant Proteins; D000117:Acetylglucosamine
keywords: Glycoprotein; IL-15; IL-15Rα; LC-MS/MS; N-glycosylation; O-glycosylation