Site-specific N- and O-glycosylation analysis of atacicept

Figures & data

Figure 1. Stepping-energy CID spectra of pronase-treated N-glycopeptides (covering glycosylation site Asn163) of atacicept with the peptide sequence ₁₆₃NST₁₆₅. Compounds with the same mass and retention time of two different production batches were compared featuring the same fragmentation pattern and thus, could be identified as the same structure. (a + b) Spectra of the glycopeptide carrying a diantennary core-fucosylated structure. (c + d) Spectra of the glycopeptide carrying a diantennary and disialylated core-fucosylated structure.

Table 1. N-glycan compositions and proposed structures with retention times (RT) identified on N-glycosylation site Asn163 of atacicept. Samples were treated with pronase and glycopeptides were analyzed by C18-PGC-LC-ESI-MS/MS. Hex = white circle = hexose, HexNAc = N-acetylhexosamine, Fuc = red triangle = fucoses, NeuAc = purple diamond = N-acetylneuraminic acid, yellow circle = galactose, blue square = N-acetylglucosamine, green circle = mannose.

Monosaccharide composition	RT [min]	Proposed structure	Monosaccharide composition	RT [min]	Proposed structure
HexNAc2Hex5	40.1		HexNAc4Hex3	37.4
HexNAc2Hex6	38.6		HexNAc4Hex3Fuc1	39
HexNAc2Hex6	40.8		HexNAc4Hex4	38.3
HexNAc2Hex7	38.6		HexNAc4Hex4Fuc1	39.8
HexNAc2Hex7	40.4		HexNAc4Hex4Fuc1 NeuAc1	10.3/51.6
HexNAc2Hex8	39.8		HexNAc4Hex5	39.1
HexNAc2Hex10	43.6		HexNAc4Hex5Fuc1	40.5
HexNAc3Hex3	37.2		HexNAc4Hex5 NeuAc1	51.2
HexNAc3Hex3Fuc1	39		HexNAc4Hex5Fuc1 NeuAc1	10.3/51.6
HexNAc3Hex3Fuc1	39.9		HexNAc4Hex5Fuc1 NeuGc1	51.2
HexNAc3Hex3Fuc1	41.1		HexNAc4Hex5Fuc1 NeuAc2	11.6
HexNAc3Hex4	38.2		HexNAc4Hex5Fuc2 NeuAc2	11.4
HexNAc3Hex4Fuc1	39.7		HexNAc4Hex5Fuc1 NeuAc1NeuGc1	11.4
HexNAc3Hex4Fuc1	40.4		HexNAc5Hex3Fuc1	39.2
HexNAc3Hex4Fuc1	41.8		HexNAc5Hex3Fuc1	40.2
HexNAc3Hex4Fuc1 NeuAc1	51.6		HexNAc5Hex4Fuc1	39.9
HexNAc3Hex5Fuc1	38.6		HexNAc5Hex4Fuc1	40.8
HexNAc3Hex5Fuc1	39.2		HexNAc5Hex5Fuc1	40.4
HexNAc3Hex5Fuc1	40.4		HexNAc5Hex5Fuc1	41.2
HexNAc3Hex6Fuc1	39.4		HexNAc5Hex6Fuc1	41.6
HexNAc3Hex6Fuc1	40.4		HexNAc5Hex6Fuc1	42.5
HexNAc4Hex6Fuc1	41.4		HexNAc5Hex6Fuc1 NeuAc1	10.6
HexNAc3Hex5Fuc1 NeuAc1	51.4		HexNAc5Hex6Fuc1 NeuAc2	11.1
HexNAc3Hex6Fuc1 NeuAc1	51.7

Figure 2. Relative quantitation of N-glycoforms of atacicept. (a) Released N-glycans were analyzed by MALDI-TOF-MS after linkage-specific sialic acid derivatization. Peak intensities were normalized based on the total intensities of all N-glycans in a sample. Sialic acids (purple diamond) pointing to the right indicate α2,3-linkage. (b) Tryptic N-glycopeptides were analyzed by C18-RP-LC-MS, and peak intensities were normalized on the total peak intensity of all N-glycopeptides in a sample. Standard deviations are shown.

Figure 3. MS/MS spectra of pronase-treated O-glycopeptides of atacicept. (a) Stepping-energy CID spectra of the O-glycopeptide ₈₆SDKT₈₉ covering glycosylation site Ser86 with a monosialylated core 1-type glycan. (b) Stepping-energy CID spectra of the O-glycopeptide ₉₁TCPPCPAPE₉₉ covering the glycosylation site Thr91 with a monosialylated core 1-type glycan. (c + d) ETD spectra of the O-glycopeptide ₈₆SDKT₈₉ covering glycosylation site Ser86 carrying a monosialylated core 1-type glycan from two different production batches. Compounds with the same mass and retention time from two different production batches were compared featuring the same fragmentation pattern and thus, could be identified as the same glycopeptides.

Figure 4. O-glycosylation analysis of the de-N-glycosylated intact protein using CESI-MS. (a) Deconvoluted spectra of the monomeric reduced de-N-glycosylated protein species. (b) Deconvoluted spectra of the dimeric de-N-glycosylated protein species. (c) Relative quantitation of the unglycosylated and O-glycosylated monomeric and (d) dimeric protein based on triplicate analysis. Standard deviations are shown.

Figure 5. Hinge region O-glycosylation of IgG1 Fc-fusion proteins. A part of the hinge region and C_H2-domain sequences of IgG1 and the Fc-fusion proteins etanercept, atacicept and CTLA4-Ig are shown. Disulfide bonds and reported O-glycosylation sites for etanercept,^28,29 CTLA4-Ig³⁰ and atacicept are highlighted. Amino acid numbering by Edelman et al.³¹ OG, O-glycan.

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