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

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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.

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Abbreviations

ACN:

Acetonitrile

cGMP:

Current good manufacturing practices

CID:

Collision induced dissociation

EIC:

Extracted ion chromatogram

ESI:

Electrospray ionization

ETD:

Electron transfer dissociation

GlcNAc:

N-acetylglucosamine

HexNAc:

N-acetylhexosamine

HILIC:

Hydrophilic interaction liquid chromatography

HEK293:

Human embryonic kidney 293

hetIL-15:

Heterodimeric IL-15:sIL-15Rα complex

IL-2R:

Interleukin 2 receptor

IL-15:

Interleukin 15

IL-15Rα:

Interleukin 15 receptor alpha

sIL-15Rα:

Soluble interleukin 15 receptor alpha

LacdiNAc:

N,N-diacetyllactosamine

LC:

Liquid chromatography

MALDI:

Matrix assisted laser desorption ionization

MS/MS:

Tandem mass spectrometry

NeuAc:

N-acetylneuraminic acid

PGC:

Porous graphitized carbon

QTOF:

Quadrupole time-of-flight

RP-HPLC:

Reversed-phase high performance liquid chromatography

SPE:

Solid phase extraction

TFA:

Trifluoroacetic acid

TOF:

Time-of-flight

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Acknowledgments

This research was facilitated through access to the Australian Proteomics Analysis Facility (APAF). ESXM was supported by an international Macquarie University Research Scholarship (iMQRES). M.T.-A. was supported by an Early Career Fellowship from the Cancer Institute, NSW, Australia. This project was funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E and by the Intramural Research Program of the National Cancer Institute, National Institutes of Health (NCI/NIH). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. We thank Admune Therapeutics for supplying hetIL-15 preparations.

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CB, BKF and GNP are inventors on US Government-owned patents and patent applications related to hetIL-15 and gene expression optimization.

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Thaysen-Andersen, M., Chertova, E., Bergamaschi, C. et al. Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation. Glycoconj J 33, 417–433 (2016). https://doi.org/10.1007/s10719-015-9627-1

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