Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation - PubMed (original) (raw)

doi: 10.1007/s10719-015-9627-1. Epub 2015 Nov 12.

E Chertova 2, C Bergamaschi 3 4, E S X Moh 5, O Chertov 6, J Roser 2, R Sowder 2, J Bear 4, J Lifson 2, N H Packer 5, B K Felber 4, G N Pavlakis 3

Affiliations

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

M Thaysen-Andersen et al. Glycoconj J. 2016 Jun.

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: Glycoprotein; IL-15; IL-15Rα; LC-MS/MS; N-glycosylation; O-glycosylation.

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Conflict of interest statement

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

Figures

Fig. 1

Fig. 1

Overview of the expression, cellular presentation, function and analysis of the soluble human hetIL-15 complex. a. Engineered human IL-15 and IL-15Rα were co-expressed and secreted by HEK293 cells as a soluble heterodimeric complex (hetIL-15) after proteolytic cleavage from the cell surface. The complex binds to the IL-2Rβ/γ receptor complex located on target cells, where it initiates a cellular response. b. The hetIL-15 complex was isolated and its purity monitored using reducing (upper) and native (lower) SDS-PAGE with Coomassie blue staining (left gels) and Western blotting using anti-IL-15 and anti-IL-15Rα antibodies (right gels). The two clinically relevant preparations of hetIL-15 (i.e. the EN and cGMP lots) are shown. c. IL-15 and sIL-15Rα were separated from their heterodimeric complex by non-reductive RP-HPLC and individually subjected to LC-MS/MS-based glycan (top), glycopeptide (bottom) and glycoprotein (right) profiling in order to characterize their _N_- and _O_-glycosylation in a detailed and site-specific manner

Fig. 2

Fig. 2

Glycome profiling demonstrates extensive and reproducible _N_-and _O_-glycosylation of IL-15 and sIL-15Rα in the large-scale preparations of hetIL-15 (cGMP and EN lots). _N_-glycans of IL-15 (a) and sIL-15Rα (b) were structurally characterized and quantitatively profiled using PGC-LC-ESI-negative ion-CID-MS/MS. c. Several isobaric _N_-glycan isomers were identified as exemplified by the extracted ion chromatogram (EIC) of the abundant Man3GlcNAc5Fuc1 composition (m/z 832.9 [M – 2 H]2−, upper panel) and the corresponding CID-MS/MS (bottom panel, see Fig. 3 for key to monosaccharide symbols) demonstrating three isobaric GlcNAc-terminating _N_-glycan isomers _i.e. N_-glycan structure 5a (shown), 5b and 5c (fragment spectra for the two latter _N_-glycans are presented in Supplementary Fig. S1). ‘*’ represents a non-glycan signal interference. d. The _O_-glycome profiling of sIL-15Rα showed less micro-heterogeneity. *Structure 2a/2b could not be consistently separated and were thus combined for quantitation purposes. No _O_-glycosylation was detected for IL-15 (data not shown). The _N_- and _O_-glycosylation profiles of IL-15 and sIL-15Rα of the EN (red bars) and cGMP (blue bars) lots of hetIL-15 were similar as evaluated by their high correlation coefficients (_R_2 = 0.815–0.982). The relative glycan quantities are averages of technical duplicates (see Supplementary Table S1 and S2 for exact values). The corresponding _N_- and _O_-glycan structures and their biosynthetic relationship are depicted in Fig. 3

Fig. 3

Fig. 3

Structures and biosynthetic relationship of the observed IL-15 and sIL-15Rα _N_- and _O_-glycans. The designated numbers of the individual _N_-linked (left) and _O_-linked (right) glycans correspond to the numbering used in Fig. 2, Fig. 5 and Supplementary Tables S1–S2. Their biosynthetic interconnectivity is presented with arrows symbolizing single glycosylation enzyme reactions. The most abundant _N_- and _O_-glycans are shaded in dark grey. Monosaccharide symbols are presented according to the Essentials of Glycobiology/Consortium for Functional Glycomics nomenclature. Key: fucose (red triangle), mannose (green circle), GlcNAc (blue square), sialic acid (NeuAc) (purple diamond), galactose (yellow circle) and HexNAc (unspecified GlcNAc or GalNAc) (open square)

Fig. 4

Fig. 4

Site-specific _O_-glycoprofiling of sIL-15Rα of clinical-grade hetIL-15 (cGMP lot) using RP (C18)-LC-ESI-positive ion-CID/ETD-MS/MS. a. The MS1 level profile (right) indicated multiple Thr81- and Thr86 - glycoforms on the tryptic O - glycopeptide R - ] 74 PA P PAPPSTV

T

TAGV

T

PQPESLSPSGK97[−E. ETD and CID fragmentation (left spectra) confirmed the _O_-glycosylation sites and the structure of the two conjugated core 1-type _O_-glycansans (HexHexNAcNeuAc, corresponding to structure 2b, Fig. 3), m/z 905.4 (4+). Additional examples of ETD-MS/MS fragment spectra of two other sIL-15Rα tryptic _O_-glycopeptides i. e. b. The N-terminal-1I

T

CPPPMSVEHADIWVK17-[S peptide conjugated with a single HexNAc (corresponding to structure i, Fig. 3) m/z 728.9 (3+) and c. the C-terminal peptide K-]152NWEL

T

A

S

A

S

HQPPGVYPQG170[−conjugated with two core 1-type _O_-sialoglycans (HexHexNAcNeuAc, corresponding to structure 2a or 2b, latter shown) and one core 1-type _O_-asialoglycan (HexHexNAc, structure ii, Fig. 3) m/z 930.8 (4+). Key fragment ions for exact site localization are presented in red. See Fig. 3 for monosaccharide key

Fig. 5

Fig. 5

Site-specific _N_-glycoprofiling of IL-15 of clinical-grade hetIL-15 (cGMP lot) using RP (C18)-LC-ESI-positive ion-CID/ETD-MS/MS. a. The MS1 level profile (right, numbering corresponds to structures in Fig. 3) indicated multiple glycoforms of the _N_-glycopeptide 65NLIILANNSLSSNG

N

VTE82. ETD- (left large spectrum, key site-localizing c/z-ions shown in red) and CID- (inserted spectrum) based fragmentation confirmed the N-glycosylation site on the Asn79 sequon as opposed to the Asn71 sequon and confirmed the structure of the conjugated complex-type GlcNAc-terminating _N_-glycan (Hex3HexNAc5Fuc, corresponding to structure 5a (shown), 5b or 5c, Fig. 3), m/z 881.2 (4+). b. Accurate mass analysis of intact IL-15 using RP (C4)-LC-ESI-positive ion-QTOF-MS confirmed the _N_-glycosylation of IL-15 (glycoforms observed in the glycome profile shown in red, numbering corresponds to structures in Fig. 3) and indicated some under-glycosylation of IL-15. Zoom of deconvoluted MS spectra (right) of the isotopic distribution of i) non-glycosylated and ii) glycosylated (_N_-glycan #5) IL-15. Overlaid, a theoretical isotope distribution (red bars). The ‘best fit’ showed partial Asn77-deamidation of IL-15, which appeared to be dependent on the glycosylation status of Asn79, see also Supplementary Fig. S4. See Fig. 3 for key to monosaccharide symbols

Fig. 6

Fig. 6

Spatial map of the _N_- and _O_-glycosylation sites of hetIL-15 in its quaternary complex with IL-2Rβ and IL-2Rγ. The three putative _N_-glycosylation sites of IL-15 (green) are shown in blue. Only Asn79 was found to be occupied; Asn71 and Asn112 found on the interface to IL-2Rβ (cyan) and IL-2Rγ (magenta), respectively, were not utilized as _N_-glycosylation sites when expressed in HEK293. The available crystal structure (PDB: 4GS7) covered only the lightly _O_-glycosylated N-terminal region of the sIL-15Rα polypeptide chain (yellow) [34]. The occupied _O_-glycosylation site at Thr2 covered by this region is shown in orange. See also Fig. 1a. for schematic illustration of the quaternary complex

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