1 CPPC Group, NIBRT - National Institute for Bioprocessing Research & Training, Ireland. 2 Physical and Theoretical Chemistry Laboratory, University of Oxford. 3 Waters Corporation, Milford Abstract Materials & Methods In-Depth Glycan Analysis Of The Biotherapeutic Enbrel (Etanercept) Using HILIC UPLC/FLR And Mass Spectrometry Mark Hilliard 1 , Weston Struwe 2 , Pauline M. Rudd 1 , Ying Qing Yu 3 • Enbrel N-Glycans were enzymatically liberated with N-linked glycans where released with peptide-N-glycosidase F in the presence of 0.1% RapiGest SF. O-linked glycans where released via reductive amination and all glycan's were labelled with 2-aminobenzamide (2AB). All labelled glycans were analysed on a Waters ACQUITY UPLC with a BEH (Bridged Ethyl Hybrid particles) glycan chromatography column HILIC (Hydrophilic interaction chromatography) (2.1 mm X 150 mm 1.7 µm) and fluorescent detection was achieved using a Waters FLR Fluorescence Detector all controlled by Empower 3. • For glycopeptide analysis Enbrel was subjected to tryptic digestion in the presence of 0.1% RapiGest SF. All glycopeptide sample where analysis by UPLC-RP-XEVO G2 (MS E ) analysis with BEH C18 columns (15cm) and data analysis was preformed by BiopharmaLynx,™ 1.3. Manual interpretation of data was performed with MassLynx software. Enbrel (Etanercept) is a fusion protein comprised of tumour necrosis factor α (TNF-α) and the Fc of immunoglobulin G1 (IgG1). It is primarily used for treatment of inflammatory conditions that affect the joints and skin, including rheumatoid arthritis and juvenile idiopathic arthritis. Enbrel contains three N-linked glycosylation sites, two of which are in the TNF-α component and one in the Fc region of IgG1. Furthermore Enbrel contains numerous O-glycan sites in the linker region, which are probably involved in protecting the biotherapeutic from proteolytic digestion. In collaboration with Waters Corporation, using UPLC BEH Glycan Column Chemistry combined with exoglycosidase array digestions, ion exchange chromatography and our experimental glycan reference database, Glycobase 3.1+ (http://glycobase.nibrt.ie), we have analysed in depth the N- and O- linked glycans present on Enbrel. The majority of the N-linked glycans were bi-antennary structures with varying amounts of core fucosylation and terminal sialylation. Furthermore, we also observed tri- and tetra-antennary N-glycans that are derived from the TNF-α component. Consistent with the production of Embrel in CHO cells, the O-glycans observed were all of the core 1 type and most contained one sialic acid, although a proportion were disialylated (10%). These structures were confirmed by UPLC HILIC-FLR-MS analysis of exoglycosidase array digestions, providing a comprehensive view of the total glycosylation present on this biotherapeutic. Furthermore, using UPLC-RP-MS E analysis of the tryptic glycopeptides, we present O-glycopeptide occupancy data. UPLC-HILIC Based Method For N-linked Glycan Analysis RELEASED LABELLED GLYCANS 2 7 Nomenclature • Specific symbols and abbreviations are used for each identified residue. • Linkage type is characterized by either a solid line (β-linkage) or a dashed line (α- linkage), where linkages have not been determined, a waved-line is used. • Linkage positions are indicated by the angle linking two sugar residues. Glycan Symbols Glycan Linkages Linkage Positions Glycan Release and labelling 1 HILIC UPLC Pooled Glycoprofiling 2 Glycobase 3.1 searching 5 Total N Linked Exoglycosidase Digestions 6 Structure Assignments 7 3 Searching Glycobase 3.1 N-linked Glycan Release Method S2 S1 S4 S3 S2 S1 + N-glycan Control Biopharmaceutical Neutrals Neutrals 6 3 Charged Based Separation and Fractionation Of Sialic Acid 4 4 Exoglycosidase Digestions Of Charged N-Glycan Fraction PNGase F 2-AB Label Formic Acid 1 Dextran Standard Biotherapeutic with RapiGest SF 5.0 6.0 7.0 8.0 9.0 10.0 ABS BKF BTG ABS ABS BKF Undigested ` Conclusions •ACQUITY UPLC with BEH Glycan column enables highly resolved 2AB-labeled N- and O- linked glycan separation in HILIC mode. •Glycobase 3.1 database improves the accuracy of the UPLC-Glycan data interpretation. Figure 1: Charged Based Separation Analysis Of Sialylated N-Linked Glycans From Enbrel Figure 2: Total Analysis Of N, O-glycans From Enbrel By UPLC-FLR/UPLC-FLR-MS Figure 3:UPLC-RP-MS (MS E ) Analysis Of O-Glycoppetides From Enbrel Figure 1A: Weak anion exchange (WAX) fractionation and analysis of sialylated N-linked glycan's from Enbrel. (i) Fetuin N linked glycan's used as a positive control to identify sialylated speciation. (ii) Total released N linked glycan's from Enbrel. (iii) ABS exoglycosidase digest, suggest that all changed glycan's present on Enbrel are sialylated (mono and di sialylated). Percentage areas of each of the glycan species is presented in the embedded table 5.0 6.0 7.0 8.0 9.0 10.0 WAX fractions overlaid Figure 1B Figure 2A:UPLC analysis of total N linked released glycans and exoglycosidase array by HILIC-fluorescence from Enbrel. (i) Whole N-glycan pool released by PNGase F, (ii) NAN1 (Recombinant Sialidase) releases α 2-3, (iIi) ABS (Athrobacter ureafaciens Sialidase) releases α2-3/6/8 sialic acids. (iv) BFK (Fucosidase from bovine kidney) releases α1- 2,1-6 fucose. (v) BTG (Bovine testes ß-galactosidase) releases galactose β1-3,1-4 linkages. (vi) SPG (Streptococcus pneumonia Galactosidase) releases β1-4 linked galactose residues (vii) GUH (hexosaminidase) release β GlcNAc but not GlcNAc linked to β 1-4 Man and (viii) JBM (Jack Bean Mannosidase) releases α1-2/1-6 and α1-3 linked mannose residues. Figure 1B: Comparison of WAX N linked fractions overlaid from Enbrel (i) to total released profile (ii). Figure 1C: UPLC analysis of each of the WAX fractions, neutral, mono and di sialylated glycans. *Diagnostic ions for O-linked glycans * * * * * Core 1 O-glycan Core 1 O-glycan+SA Figure 1D:UPLC analysis and exoglycosidase array by HILIC-fluorescence of WAX fraction S1 (mono sialylated) from WAX analysis to determine N glycan's present in each anionic fraction, (i) Whole N-glycan pool released by PNGase F, (ii) NAN1 (Recombinant Sialidase) releases α 2-3 sialic acids. (iii) ABS (Athrobacter ureafaciens Sialidase) releases α2-3 /6/8 sialic acids. (iv) BFK (Fucosidase from bovine kidney) releases α1-2,1-6 fucose. (v) BTG (Bovine testes ß-galactosidase) releases galactose β1-3,1-4 linkages and (vi) GUH (hexosaminidase) release β GlcNAc but not GlcNAc linked to β 1-4 Man. Figure 3B: Biopharmalynx analysis of tryptic O glycopeptide data from Enbrel . * indicated a selected the peptide SMAPGAVHLPQPVSTR (186-201 aa) with two core one O glycan’s with siclic acid and the same peptide without any O glycan modification. Rates of O glycan occupancy can be determined by comparison of the intensity values. (ii) (i) Figure 3C Figure 2B: Base peak ion (BPI) and fluorescent chromatogram (FLD) of 2-AB labeled Enbrel N-glycans analyzed by HILIC UPLC-FLR-MS. Figure 2B (ii,iii): C1 ions *at m/z 179 and 220 show both Hex and HexNAc terminating the antennae and the 1,3 A 3 ions *at m/z 262 and 424 show antennae consisting of HexNAc (GlcNAc) and Hex-HexNAc (Gal- GlcNAc). The set of D and [D-18]- ions *at m/z 688/670 in Figure 2B (ii) shows that the 6-antennae exists with a galactose (F(6)A2[6]G(4)1) which is represented by peak 4 * in Figure 2B (i). ULPC-HILCI-FLR-MS E confirms major glycan structures that we observed in the UPLC-FLR exoglycosidase array analysis. Figure 2B (ii) (i) SMAPGAVHLPQPVSTR (M+3H) +2 core 1 O- glycans w ith Sialic acid Figure 3C: Site occupancy analysis of peptide SMAPGAVHLPQPVSTR (186-201aa) containing two core one O glycan’s with siclic acid. This peptide contains three possible sites of modification S186, S199 and T200. (i) Summed chromatogram for peptide SMAPGAVHLPQPVSTR (M+3H) 3 for parent mass at retention time 31.9 minutes. (ii) Summed chromatogram MS E high energy fragmentation data for peptide SMAPGAVHLPQPVSTR, diagnostic ion confirm the present of a glycan moiety*and ions such as 366 m/z and 657m/z suggestion the addition of O glycan. As CID fragmentation provides little glycan-peptide linkage specific information its recommend to use alternative fragmentation such as electron-transfer dissociation (ETD) to locate the exact sites. (iii) XIC of peptide SMAPGAVHLPQPVSTR, 987 m/z suggest that this peptide is consistently modified at two residues by core 1 O glycans with sialic acid. Dextran Erythropoietin * Dextran Figure 1A Figure 2B (i) Figure 2C: UPLC analysis of total O glycan's from Enbrel. (i) Undigested released O linked glycans (ii) ABS exoglycosidase digest to determine sialylated O glycan's. Peak’s 3 and 4 where identified as mono and di sialylated core one O glycan's (iii) ABS and BTG digest (iv) O glycan release method control, green highlighted area is a reagent containment . Table 1 of percentage area and GU value associated with each O glycan identified Dextran Figure 2C Figure 3B Figure 3A: UPLC-RP-MS (MS E ) analysis of tryptic digest of Enbrel O gylcopeptides. (i) BPI parent ion chromatogram. (ii) Extracted ion chromatogram (XIC) for glycan diagnostic ion 204 m/z which corresponded to the HexNAc structure. Suggested area where N and O glycopeptide elute are represented. (iii) XIC for glycan diagnostic ion 366 m/z which can represent both N and O glycopeptide. (iv) XIC for glycan diagnostic ion 657 m/z can represent a core one O glycan with one sialic acid. Figure 1E (i) UPLC-UV BEH C4 (5cm) fractionation of Enbrel digestion product after FabRICATOR enzyme. Two components of Enbrel where identified, the TNF-α and FC region. (ii) UPLC-FLR analysis of released N linked glycan's from the TNF-α region. We identified that tri and tetra N glycan's occur on this region and not the FC (iii) UPLC-FLR analysis of released N linked glycan's from the FC region. (iv) Overlay of FC and TNF-α N-linked region glycan's. Figure 1E Mono sialylated glycans S1 Di sialylated glycans S2 Neutral glycans Figure 1C * Figure 2B (iii) * * * (ii) Undigested profile (iii)