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 SpectrometryMark 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

(MSE) 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-MSE 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 labelling1

HILIC UPLC Pooled

Glycoprofiling2

Glycobase 3.1 searching

5Total N Linked

Exoglycosidase

Digestions

6

Structure Assignments7

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 F2-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 (MSE) 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 Nlinked 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 fromEnbrel. (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 (Streptococcuspneumonia 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 mannoseresidues.

Figure 1B: Comparison of WAX N linked fractions overlaid from Enbrel (i) to total released profile (ii). Figure 1C: UPLCanalysis 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 WAXanalysis 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) releasesgalactose 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 aselected the peptide SMAPGAVHLPQPVSTR

(186-201 aa) with two core one O glycan’s withsiclic acid and the same peptide without any O

glycan modification. Rates of O glycan occupancycan 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 byHILIC UPLC-FLR-MS. Figure 2B (ii,iii): C1 ions *at m/z 179 and 220 show both Hex and HexNAc terminating theantennae and the 1,3A3 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 agalactose (F(6)A2[6]G(4)1) which is represented by peak 4 * in Figure 2B (i). ULPC-HILCI-FLR-MSE confirms major

glycan structures that we observed in the UPLC-FLR exoglycosidase array analysis.

Figure 2B (ii)

(i)

SMAPGAVHLPQPVSTR (M+3H)3 +2 core 1 O-

glycans with Sialic acid

Figure 3C: Site occupancy analysis of peptideSMAPGAVHLPQPVSTR (186-201aa) containing two coreone 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 atretention time 31.9 minutes. (ii) Summed chromatogram

MSE high energy fragmentation data for peptideSMAPGAVHLPQPVSTR, diagnostic ion confirm the

present of a glycan moiety*and ions such as 366 m/z and657m/z suggestion the addition of O glycan. As CID

fragmentation provides little glycan-peptide linkagespecific information its recommend to use alternative

fragmentation such as electron-transferdissociation (ETD) to locate the exact sites. (iii) XIC of

peptide SMAPGAVHLPQPVSTR, 987 m/z suggest thatthis peptide is consistently modified at two residues bycore 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) Undigestedreleased O linked glycans (ii) ABS

exoglycosidase digest to determinesialylated O glycan's. Peak’s 3 and 4

where identified as mono and di sialylatedcore one O glycan's (iii) ABS and BTG

digest (iv) O glycan release methodcontrol, green highlighted area is a

reagent containment . Table 1 ofpercentage area and GU value associated

with each O glycan identified

Dextran

Figure 2C

Figure 3B

Figure 3A: UPLC-RP-MS (MSE) analysis of trypticdigest of Enbrel O gylcopeptides. (i) BPI parent ionchromatogram. (ii) Extracted ion chromatogram

(XIC) for glycan diagnostic ion 204 m/z whichcorresponded to the HexNAc structure. Suggested

area where N and O glycopeptide elute arerepresented. (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 onesialic acid.

Figure 1E (i) UPLC-UV BEH C4 (5cm) fractionation of Enbreldigestion product after FabRICATOR enzyme. Two components ofEnbrel where identified, the TNF-α and FC region. (ii) UPLC-FLR

analysis of released N linked glycan's from the TNF-α region. Weidentified 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 FCregion. (iv) Overlay of FC and TNF-α N-linked region glycan's.

Figure 1E

Mono sialylated glycans S1

Di sialylated glycans S2

Neutral glycansFigure 1C

*

Figure 2B (iii)

**

*

(ii)

Undigested profile

(iii)