Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation

M. Thaysen-Andersen, E. Chertova, C. Bergamaschi, E. S. X. Moh, O. Chertov, J. Roser, R. Sowder, J. Bear, J. Lifson, N. H. Packer, B. K. Felber, G. N. Pavlakis

Research output: Contribution to journalArticleResearchpeer-review

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.

LanguageEnglish
Pages417-433
Number of pages17
JournalGlycoconjugate Journal
Volume33
Issue number3
DOIs
Publication statusPublished - Jun 2016

Fingerprint

Glycosylation
Interleukin-15
Polysaccharides
human IL15 protein
Interleukin-15 Receptor alpha Subunit
Pharmacokinetics
Lymphocytes
Glycopeptides
Interleukin-2 Receptors
Lymphocyte Activation
Purification

Keywords

  • N-glycosylation
  • O-glycosylation
  • LC-MS/MS
  • Glycoprotein
  • IL-15
  • IL-15Rα

Cite this

Thaysen-Andersen, M. ; Chertova, E. ; Bergamaschi, C. ; Moh, E. S. X. ; Chertov, O. ; Roser, J. ; Sowder, R. ; Bear, J. ; Lifson, J. ; Packer, N. H. ; Felber, B. K. ; Pavlakis, G. N. / Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation. In: Glycoconjugate Journal. 2016 ; Vol. 33, No. 3. pp. 417-433.
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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.",
keywords = "N-glycosylation, O-glycosylation, LC-MS/MS, Glycoprotein, IL-15, IL-15Rα",
author = "M. Thaysen-Andersen and E. Chertova and C. Bergamaschi and Moh, {E. S. X.} and O. Chertov and J. Roser and R. Sowder and J. Bear and J. Lifson and Packer, {N. H.} and Felber, {B. K.} and Pavlakis, {G. N.}",
year = "2016",
month = "6",
doi = "10.1007/s10719-015-9627-1",
language = "English",
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Thaysen-Andersen, M, Chertova, E, Bergamaschi, C, Moh, ESX, Chertov, O, Roser, J, Sowder, R, Bear, J, Lifson, J, Packer, NH, Felber, BK & Pavlakis, GN 2016, 'Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation', Glycoconjugate Journal, vol. 33, no. 3, pp. 417-433. https://doi.org/10.1007/s10719-015-9627-1

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation. / Thaysen-Andersen, M.; Chertova, E.; Bergamaschi, C.; Moh, E. S. X.; Chertov, O.; Roser, J.; Sowder, R.; Bear, J.; Lifson, J.; Packer, N. H.; Felber, B. K.; Pavlakis, G. N.

In: Glycoconjugate Journal, Vol. 33, No. 3, 06.2016, p. 417-433.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation

AU - Thaysen-Andersen, M.

AU - Chertova, E.

AU - Bergamaschi, C.

AU - Moh, E. S. X.

AU - Chertov, O.

AU - Roser, J.

AU - Sowder, R.

AU - Bear, J.

AU - Lifson, J.

AU - Packer, N. H.

AU - Felber, B. K.

AU - Pavlakis, G. N.

PY - 2016/6

Y1 - 2016/6

N2 - 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.

AB - 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.

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KW - Glycoprotein

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