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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)