High Performance Anion Exchange and Hydrophilic Interaction Liquid

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High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N-glycome of a Recombinant Human Erythropoietin Zoltan Szabo, James R. Thayer, Dietmar Reusch, Yury Agroskin, Rosa Viner, Jeff Rohrer, Sachin P. Patil, Michael Krawitzky, Andreas Huhmer, Nebojsa Avdalovic, Shaheer H. Khan, Yan Liu, and Christopher Pohl J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00862 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 17, 2018

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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Proteome Research

High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the Nglycome of a Recombinant Human Erythropoietin

Zoltan Szabo†,*, James R. Thayer†, Dietmar Reusch‡, Yury Agroskin†, Rosa Viner$, Jeff Rohrerǁ, Sachin P. Patilǁ, Michael Krawitzky$, Andreas Huhmer$, Nebojsa Avdalovic†, Shaheer H. Khan§, Yan Liu† and Christopher Pohl†

†

ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA 94088, USA

‡

Roche Diagnostics GmbH, 2 Nonnenwald, Penzberg 82377, Germany

$

ThermoFisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134, USA

ǁ

ThermoFisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA 94085, USA

§

ThermoFisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080, USA

*Corresponding author: Zoltan Szabo, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA 94088, USA, phone: +1(408)484-4625, email: [email protected]

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Keywords: Glycan Analyses, Mass Spectrometry, Native Glycans, Labeled Glycans, Ultra High Performance Liquid Chromatography, High Performance Anion Exchange Chromatography, Recombinant Erythropoietin, Glycoproteomics

ABSTRACT Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g. phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance towards phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, revealed


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multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS2 based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry.

INTRODUCTION The continuing growth of the pharmaceutical industry requires use of multiple complementary

techniques

to

fully

characterize

therapeutic

glycoproteins.1-3

Glycosylation, one of the most studied post translational modifications can be complex due to the presence of branch, linkage and positional isomers.4-5 Glycan modifications, such as phosphorylation, sulfation, acetylation, and methylation also occur on different specific glycan motifs6, impacting biological functions contributed by the glycan to the glycoprotein.

Due

to

the

effects

of

glycosylation

on

pharmacokinetic

and

pharmacodynamic attributes, regulatory agencies demand the characterization of 3 ACS Paragon Plus Environment


glycosylation to ensure both safety and efficacy of biologics. The need to characterize these features presents significant analytical challenges. Mass spectrometry (MS), coupled to chromatographic methods, is now routinely used to elucidate complex glycan structures.7-8 These chromatographic methods hyphenated to mass spectrometry are often combined with complementary approaches such as exoglycosidase digestion9-10 and/or capillary electrophoresis11-12 in order to better identify and quantify structural details of the glycome. As such, hydrophilic interaction liquid chromatography (HILIC) on 1.7 µm resin substantially reduced analyses time and increased efficiency for glycan analyses compared to previously popular HILIC phases.13 Hence, it has become a leading technology for glycan analyses. Glycan separation by HILIC is primarily governed by glycan hydrodynamic volume, which resolves many linkage and positional isomers, facilitating glycan annotation together with mass spectrometric analyses.14-15 HILIC separations are generally preceded by glycan derivatization via reductive amination or glycosylamine capturing with fluorophores (2-AB, 2-AA, Rapifluor, Procainamide) and the selection of fluorophore tag determines the ionization mode for MS.16-18 Derivatization is often followed by clean up protocols, introducing potential biases during sample preparation. UHPLC mobile phases are directly compatible with MS, facilitating coupling of these techniques. High performance anion exchange chromatography (HPAE) is also frequently used for glycan analysis based on its ability to resolve many native glycan isomers.19-20 Native glycan HPAE assays are not subject to glycan derivative biases.


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The HPAE platform performs best with sialylated (or otherwise charged) glycans. The HPAE separation mechanism depends primarily on the glycan charge, so a glycome comprised of structures terminated by different numbers of sialic acids is resolved into charge classes. This charge classification is useful to calculate glycan sialylation levels. A second prominent attribute of HPAE separation is that fucosylation (either core or outer arm) results in earlier elution compared to glycans of equivalent sialylation without fucose.21 Glycan separations usually occur at high pH with a sodium hydroxide and sodium acetate mobile phase that is not directly compatible with MS. Therefore, a high capacity desalter is used to replace the sodium ion with hydronium ion thus producing MS-friendly water and acetic acid.19 Mass spectrometric analyses of both 2-AB labeled and native glycans employ negative electrospray ionization

22

. Compared with positive ionization, glycan fragmentation in

negative ESI using high energy collisional dissociation (HCD) generates the typical Band Y-type glycosidic fragment ions, as well as highly informative A-type cross ring fragments and D- and E-ions, which facilitate assignment of the antennae.23-25 Detection sensitivity of neutral glycans in negative ESI is lower than that using positive ESI26, but is enhanced by adduct formation.23,

27

Sialylated (negatively charged) glycans exhibit

better detection sensitivity than neutral glycans in negative ESI.19 In this study, recombinant human erythropoietin (rhEPO), a heavily glycosylated protein with three N-glycosylation sites expressed by a Chinese Hamster Ovary (CHO) cell line was chosen to compare N-linked glycome characterization by the two chromatographic platforms. The rhEPO contains sialylated glycans, the majority being tetraantennary.28 Sialic acids (mostly N-acetylneuraminic acid, Neu5Ac) are attached to galactoses via α5 ACS Paragon Plus Environment


2,3 linkages.29-30 O-Acetylated silalic acid and a small amount of N-glycolylneuraminic acid (Neu5Gc) on rhEPO further increase glycan microheterogeneity.31-33 Neu5Ac acetylation impacts specific molecular recognition (binding and degradation)34 and Neu5Gc is known to be immunogenic in humans.35,36 The presence of Neu5Gc was already known37 but no attention was paid to it. However, recent research showed the humans have variable, sometimes high level of circulating anti-Neu5Gc antibodies.38 Cetuximab, a chimeric antibody produced in mouse myeloma cells known to have Neu5Gc-containing epitopes. This antibody has been frequently studied and found to form immune complexes with anti-Neu5Gc antibodies. Small amount of such epitope is present in rhEPO and can stimulate antibody production in patient. The antibody may remove the drug containing non-human epitope from the blood stream but the immune complex can accumulate in renal glomeruli, leading to developing symptoms of serumsickness.39 Phosphorylated, mostly high mannose glycans are also present on rhEPO at low, but not negligible levels32 and contain mannose 6-phosphate. One of their key roles is in protein transport from the Golgi to the lysosomes.40 Many literatures account for the important role of mannose 6-phospahte receptors (MPRs) targeting targeting soluble acid hydrolases that contains phosphorylated high mannose glycans.41 Fibroblast, such as cytokine producing cells in kidney have MPRs on its surface and upon recognition and internalization of a substrates bearing phosphorylated high mannose glycans, they are transported to lysosomes.22 Phosphorylated glycans on rhEPO may have similar role but scientific literature lacks studies that would specifically investigate the role of phosphorylated glycans linked to this therapeutic glycoprotein.


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Sulfation is an important modification in biological recognitions. Sulfated glycans have been reported to impart important effects during lymphocyte homing since endothelial ligands for L-selectins are sulfated by two specific modifications, Gal-6-SO4 and GlcNAc-6-SO4.42 Sulfation is also implicated in removal of pituitary hormons from circulation.43 These findings suggest the potential role of sulfated N-glycans attached on rhEPO but no literature found that would explicitly support these statements. Given the complexity of the N-glycan pool of rhEPO, the present study was designed to evaluate the two chromatographic platforms for their ability to characterize the N-linked glycome of this biologic. A prior report indicated significant desialylation during reductive amination, especially when glycans harbor more than two sialic acids.44 This may shift the resulting glycan profiles to lower apparent sialylation levels when compared to native glycan analyses. Conversely, O-acetylation is lost at high pH during HPAE but glycans terminated by O-acetylated sialic acids remained intact in the 2-AB labeled glycome. Phosphorylated high mannose glycans and phosphorylated hybrid structures are well resolved by HPAE, as are sulfated glycans. Analysis of the rhEPO glycome highlights the strengths and weaknesses of both HILIC-MS and HPAE-MS platforms. They appear to be complementary methods, so both are recommended when the glycoprotein of interest carries glycans with challenging modifications.



EXPERIMENTAL PROCEDURES Chemicals Sodium perfluorooctanoate, formic acid, ammonium formate (Optima, LC/MS grade), and iodoacetamide (Acros Organic) were purchased from Thermo Fisher Scientific (Ward Hill, MA). Anhydrous sodium acetate (electrochemical grade), acetic acid, sodium hydroxide solution (50 w/w %), Bond-Breaker® TCEP-Solution, trifluoroacetic acid, and acetonitrile (LC-MS grade) were from Thermo Scientific (Waltham, MA). HEPES, ammonium acetate, 10 x PBS buffer and 1,4-Dithio-DL-Threitol (DTT) were obtained from Sigma Aldrich (St Louis, MO). Lys-C enzyme (mass spec grade) was from Promega (Madison, WI). PNGase F enzyme (N-Glycanase® ULTRA, ≥ 10 U/mL), Sialidase A, Sialidase S a 2-AB labeling kit (GlykoPrep Rapid-Reductive-Amination 2AB Labeling Module) were purchased from Prozyme (Hayward, CA) and were used according to instructions provided by the vendor. Formulated rhEPO was obtained from Roche Diagnostics GmbH (Penzberg, Germany).

Release and Purification of N-linked Glycans The rhEPO N-linked glycans were released using the rapid protocol19 developed by the authors of the current study. Briefly, 160 µg of rhEPO was dispensed in a 96 well plate (Fisherbrand® PCR Plate, 96 well, polypropylene, 0.2 mL, skirted) followed by 8 µL deglycosylation buffer (0.5 M HEPES buffer, pH 7.9), 8 µL of reductant solution (200 mM TCEP), 8µL detergent solution (12 w/w % sodium perfluorooctanoate) and 56 µL of water. After heat denaturation at 95 ⁰C for 5 min, the solution was cooled to ambient 8 ACS Paragon Plus Environment

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temperature and 8 µL of undiluted PNGase F was added. Deglycosylation proceeded at 55 ⁰C for 15 min. After deglycosylation, the digested solution was acidified with 200 µL 0.2% TFA. The digest was transferred into wells of a Hypercarb™ Hypersep™ Filter Plate (40 µL) previously equilibrated (in order) with 200 µL acetonitrile, 200 µL of 40 % acetonitrile, 0.1% TFA (aq) and three washes with 200 µL 0.1% TFA (aq). The loaded samples were washed thrice with 200 µL 0.1% TFA (aq) and the glycans eluted using three 200µL washes with 40 % acetonitrile, 0.1% TFA (aq). These three fractions were pooled and evaporated to dryness in a speed vac. For HPAE analyses, dried glycan samples were dissolved in 100 µL of DI H2O and injected without further processing. Before labeling with 2-AB, glycan samples were dissolved in 100 µL of water, transferred into PCR tubes and dried in the Speed-Vac.

Labeling of N-linked Glycans The labeling mixture (10 µL of a 1 to 1 ratio of the kit dye and reductant solutions) was added to the dried rhEPO glycans in PCR tubes. The tubes were vortexed, centrifuged and incubated for 1 hour on a dry heating block at 60⁰C (per kit instructions). The 2-AB labeled glycan samples were purified on a Hypersep™ Hypercarb™ (40 µL) Filter Plate using the same protocol used for purification of native N-linked glycans after deglycosylation. Purified 2AB-labeled glycans were evaporated to dryness in a SpeedVac and dissolved in 100 µL of a mixture of 50 % acetonitrile and 50 mM ammonium formate buffer (pH 4.4) for UHPLC-MS analyses.



Determination of the Sialic Acid Content of Native and 2-AB Labeled N-linked Glycans

A 10 µl aliquot of native, or 2-AB-labeled rhEPO glycan prepared without glycan purification, was mixed with 90 µl DI water and 100 µl 4 M acetic acid to prepare a 2 M acetic acid reaction concentration. The glycans were hydrolyzed for 2 hours at 80⁰C in a heat block. After hydrolysis, the digests were dried in the speed-vac and resuspended in 400 µl DI water. Ten microliter of hydrolyzed samples were injected into a Dionex CarboPac PA20 (3 x 150 mm, 6.5 µm particle size). The chromatographic system was equipped with an electrochemical detector (ED) with pH-Ag/AgCl reference electrode and a 0.002” gasket to define the detection channel. Sialic acids were eluted at 0.5 mL/min using an isocratic segment of 7.5 min followed by a 6.5 min gradient of 30- to 270-mM sodium acetate (in 100 mM sodium hydroxide) and a further 2.5 min isocratic segment. The eluent returned to initial conditions in 30 sec and the column reequilibrated for 7 minutes prior to subsequent injections. Sialic acid content was quantified using calibration curves employing Neu5Ac and Neu5Gc standards.

HPAE-MS Analyses of Native Glycans Chromatography A Thermo Scientific Dionex ICS-5000+ HPIC system with a WPS-3000 TB autosampler was used to analyze released, purified glycans. The system was equipped with an electrochemical detector (ED for Pulsed Amperometry, with pH-Ag/AgCl reference electrode and a disposable Au working electrode). The analytical column was a 10 ACS Paragon Plus Environment

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CarboPac PA200 (3 x 250 mm, 5.5 µm particle size) developed for oligosaccharide separation. To minimize peak dispersion, the column outlet was connected to a 1/16 inch Micro-Tee (Idex, Oak Harbor WA) with a 4 cm length of 0.01” ID PEEK tubing. One outlet of the “T” was connected to the ED and the other delivered ~50 % of the flow to an electrolytically regenerated desalter (Thermo Scientific, Dionex ERD 500, 2 mm) that exchanges eluent Na+ for H+ before introduction to the mass spectrometer. A 90 cm long (0.005” ID) tube was placed at the ED cell outlet, to control flow from the “T” between the ED and the mass spectrometer. The system was plumbed to minimize dead volume using tubing described earlier.19 The current applied to the desalter was 400 mA and the regenerant was DI water flowing at 3.5 mL/min. The detector cell used a 0.002“ gasket to define the detection chamber. Data was acquired at 2 Hz using the standard four potential carbohydrate waveform. All separations were performed at 30 ⁰C and 0.5 mL/min. Separation of rhEPO glycans employed a linear gradient of sodium acetate in 100 mM sodium hydroxide. Mobile phase “A” was 100 mM NaOH and “B” was 100 mM sodium hydroxide and 250 mM sodium acetate. The gradient started at 2.4 % B, reaching 76% in 70 min. Mobile phase “B” increased to 80% by 70.1 min and held at 80 % for 5 min before returning to 2.4% at 85.1 min. The column was equilibrated for 15 min prior to subsequent injections. In contrast to earlier work, an IC PEEK Viper Fitted tubing (24” x 0.007”) was used between the desalter and mass spectrometer. Chromatographic

system

control,

data

acquisition

Chromeleon 7 CDS software.


and

processing

employed


Mass spectrometry A Q Exactive™ Classic Orbitrap™ mass spectrometer (Thermo Scientific, San Jose, CA) was coupled to the ion chromatography system to analyze native glycans in negative ion mode. The spray voltage was 3.2 kV and the capillary temperature was set to 300 °C. The probe heater temperature was 200 °C. The sheath and auxiliary gas flow were 40 and 20 respectively (arbitrary units). MS1 spectra were acquired at m/z 600-2000 over 85 min at 70,000 resolution. The MS1 AGC target was set to 1x106 and the maximum injection time was 60 msec. Data dependent MS2 experiments for top 3 ions were performed using 28 kV normalized collision energy. For MS2 experiments the AGC target value was 2x105, the maximum injection time was 350 msec and resolution was 17,500. The number of microscans in MS1 was 1 and in MS2 experiments was 3 and the quad isolation window was 2 T. The minimum AGC target and intensity threshold values were set at 2x104 and 5.7x104. For data acquisition and processing Xcalibur™ 3.0 software was used. MS/MS experiments were evaluated using SimGlycan™ 5.61 software (PREMIER Biosoft, Palo Alto, CA).

UHPLC-MS Analyses of 2-AB Labeled Glycans A Thermo Scientific UltiMate™ 3000 BioRS UHPLC system was equipped with a WPS3000 TB autosampler, a TCC-3000 column oven and an FLD 3400RS DualPMT fluorescence detector. Separation of 2-AB labeled rhEPO glycans employed a Waters ACQUITY UPLC Glycan BEH Amide column (130 Å, 1.7 µm, 2.1 x 150 mm). The column temperature was 60°C, fluorescence excitation and emission wavelengths were 330 and 420 nm, respectively and emission sensitivity was 7. FLD data acquisition was 12 ACS Paragon Plus Environment

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at 10 Hz and the flow cell temperature was 45 ⁰C. For 2-AB glycan separations, mobile phase “A” was acetonitrile and mobile phase “B” was 50 mM ammonium formate (pH 4.4). The gradient began at 0.5mL/min and 30 % “B” and increased to 55 % in 54 min. The flow rate was reduced to 0.25mL/min at 55 min and mobile phase held Isocratically for 2 min. At 57 min the flow rate was restored to 0.5mL/min and mobile phase “B” returned to 30% in 0.1 min. The column was equilibrated for 10 min at 0.5mL/min prior to subsequent injections. Mass spectrometry The same tune setting and instrument methods were used for 2-AB and native glycans using the conditions described in the HPAE-MS Analyses of Native Glycans section.

Lys-C Digestion of rhEPO The Lys-C digestion protocol was modified in order to obtain larger size peptides (middle down analyses) for improved fragmentation in ETD mode. The enzyme was dissolved in 20 mM ammonium acetate (pH 6), the concentration of Lys-C solution was 0.5 µg/µL. The low MW components in formulated rhEPO containing 200 µg of glycoprotein were buffer exchanged using Amicon® Ultra 0.5 mL 10 kDa MW cutoff centrifugal filter (Billerica, MA). For buffer exchange, rhEPO was applied to the MWCO filter, centrifuged at 10,000g rinsed with 100 µL of ammonium acetate buffer and the process repeated three more times. The protein was recovered in 100 µL of ammonium acetate buffer, reduced with 3 µL 150 mM DTT and alkylated with 5µL 200 mM IAA. The reduced and alkylated protein was buffer exchanged as before. Reduced and alkylated 13 ACS Paragon Plus Environment


protein in 100 µL ammonium acetate buffer was transferred into PCR tubes and 2 µL Lys-C solution added to prepare a glycoprotein-to-enzyme ratio of 200:1. Digestion was performed at 37 ⁰C for 3 h. After incubation the samples were evaporated to dryness in a speed vac.

LC/MS Analyses of EPO Digest The peptides generated by rhEPO Lys-C digestion were separated on a 50cm Thermo ScientificTM EASY-Spray Column using a Thermo ScientificTM EASY-nLCTM1200 UPLC system. The gradient was 5% to 25 % acetonitrile in 60 min and 25 % to 90% in 15 min at a flow rate of 300 nL/min. One ug of rhEPO digest was injected and eluting peptides were analyzed with a Thermo Scientific™ Orbitrap Fusion Lumos™ mass spectrometer. Fragmentation employed FT-HCD, EThcD in DDDT or HCDpdETD/CID methods. FT MS1 was acquired at resolution settings of 60–120K at m/z 200 and FTMS2 at resolution of 30–60K at m/z 200.

Data Analyses Glycan MS2 spectra were entered into SimGlycan™ 5.61 software (Premier Biosoft, Palo Alto CA.) but many glycans with modifications (e.g. those shown as figures) were not recognized by the software. These were annotated manually from the MS2 spectra. Raw data files were searched in SimGlycan using the high throughput option. For these searches the ion mode was “Negative”, adduct formation was “H” (since highly


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sialylated EPO glycans do not adduct with acetate) and chemical derivatization was “underivatized”. Glycan end modification was “free” for native glycans from HPAE-MS data files and “2-AB” for the date generated by UHPLC-MS. Both precursor and fragment ion error tolerances were 5 ppm. The fragmentation pattern search employed “all types” of fragments (glycosidic, cross ring, glycosidic/glycosidic and cross ring/glycosidic). The Filter options applied were “Chinese hamster”, “CHO cells”, “EPO”, “erythropoietin”, “human” and “recombinant”. Data search was performed at both MS1 and MS2 levels. Proteome Discoverer 2.1 (Thermo Fisher Scientific) with the ByonicTM search node (Protein Metrics, San Carlos, CA) was used for glycopeptide data analysis. Data were searched against a rhEPO + contaminants database (246 proteins) with a 1% 1D peptide FDR criteria and custom glycan database created from glycomics experiments. The cleavage site was “K” at “C-terminal” and digestion specificity was “Semi-specific” with “2” missed cleavages for digestion conditions. Instrument parameters included 10 and 20 ppm mass tolerances for precursor and fragment ions, respectively. Fixed and variable modifications were “Carbamidomethylation” (fixed) and “methionine oxidation” and “deamidation” (variable).



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Results and Discussion Overview of Chromatography As shown in Figure 1A, the 2-aminobenzamide labeled glycan pool of rhEPO resolves into 46 peaks in 30 minutes. The base peak chromatogram reveals primarily tetraantennary glycans with or without N-acetyllactoseamine repeats (Table 1). Of the 46 peaks, eight are apparently dehydration artifacts generated by a loss of water during the labeling reaction. The dehydration with concomitant loss of sialic acid is attributed to the reductive amination process. Figure S1B in Supporting Information shows a distinctive ion in the HCD MS2 spectrum which is formed from B3 (Neu5Ac-Gal-GlcNAc) ion by a loss of water. This finding reveals that dehydration caused by the labeling reaction displays on the non-reducing end of the glycan.

Because the expression

system (CHO cell line) terminates complex and hybrid glycans with α-2,3-linked sialic acids, the glycans are rendered more susceptible to cleavage, as depicted in Figure 1B. An alternative to reductive amination labeling and HILIC separations is HPAE that requires no derivatization. Hence, neither dehydration nor loss of sialic acid residues were observed using HPAE. Glycans were separated into charge clusters by HPAE (Figure 2A) based on the content of sialic acids or other charged modifications. Compared to HILIC, HPAE separations took longer due to later eluting sulfated glycans (Figure 2B). Acetate concentration gradients elute glycans in order of increasing charge, as influenced by the high pH (12.5-13) contributed by the presence of NaOH. Residual sodium was removed using a membrane desalter prior to MS introduction. Because the pH is high, epimer formation occurs on the surface of the stationary phase and increases for glycans with longer retention. Differences between the migration times of 16 ACS Paragon Plus Environment

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different glycans and their epimers are consistent, which is useful in annotating epimer peaks of different oligosaccharide structures. The high pH mobile phase also releases O-acetyl groups from sialic acid, while this glycan modification persists upon reductive amination and is detected under HILIC chromatographic conditions (pH 4.4). Glycan phosphorylation and sulfation are also biologically important modifications6. HPAE-MS revealed that ~1.8 % of the total rhEPO glycan pool is phosphorylated (represented by one hybrid and two high-mannose glycans). Phosphorylated glycans were barely observed and sulfated glycans were not observed using HILIC chromatography, apparently due to poor solubility in the HILIC mobile phase. A small fraction of rhEPO glycans was revealed by HPAE/MS bearing α-2,3 Neu5Ac attached to GlcNAc. Although the two most abundant representatives of this linkage were also observed after 2-AB-labeling in HILIC, their relative amounts were considerably lower compared to the amounts observed for native glycans by HPAE. The low level of their detection by HILIC-MS is likely attributable sialic acid loss during reductive amination. Glycan structures observed in both chromatographic platforms are depicted in Scheme 1, structural annotations of the glycans are corroborated by MS2 spectra and diagnostic ions are listed in Table 3. Desialylation The sialic acid content of glycans released from 120 µg of rhEPO was analyzed with and without 2-AB labeling. Of the 3 replicates of each approach, 2AB-labeled glycans contained 100 ± 2.4 pmol sialic acid/µg glycoprotein (without clean up) and native 17 ACS Paragon Plus Environment


glycans contained 163 ± 3.7 pmol/µg glycoprotein showing a relatively consistent 39% loss of sialic acid content upon 2-AB labeling. A follow-on analysis comparing peak area by HPAE with pulsed amperometric detection for both native and 2-AB-labeled glycans (Supporting Information Figure S2) revealed that the native glycans produced ~ 2x the total peak area of the 2AB-labeled glycan pool. Some of this may have resulted from differences in the peak response between native and labeled glycan by PAD. However, the chromatograms revealed that there is also a change in the relative proportions of each charge class (mono-, di-, tri-, tetra-, and tetra- with Neu5Gc sialic acids. Most of the glycans in the native glycan pool were tetrasialylated (~47%), with decreasing amounts in trisialylated (~33%), disialylated (~12%), monosialylated (~4%) and tetrasialylated with Neu5Gc (~5%). Tri- to tetrasialylated (with Neu5Gc) amount to ~84% of the total, and ~16% are represented in the mono- and di-sialylated glycans. Conversely, most of the 2-AB-labeled glycans appeared in the trisialylated group (~33%) followed by the disialylated (~32%) tetrasialylated (~19%), monosialylated (~14%) and tetrasialylated with Neu5Gc (3%). In the 2AB-labeled pool, ~54% of the total appear in the tri- to tetrasialylated (including the Neu5Gc glycans) and ~ 46% were in the mono- and di-sialylated fractions. Assuming consistent peak response within each of the (native and labeled) pools, this difference would represent ~30% loss of sialic acid on the tri- to tetra-sialylated glycan fractions, a value similar to that of sialic acid loss observed by direct assay of sialic acids released from native and 2AB-labeled glycans (~39%). A decreasing trend in the relative distribution of tetrasialylated tetraantennary structures was observed by UHPLC in the 2-AB-labeled rhEPO glycans compared with the HPAE 18 ACS Paragon Plus Environment

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results for the native glycan pool (Figure 1B, Table S1 in Supporting Information). Peak area distributions were calculated from extracted ion traces using both HILIC-MS and HPAE-MS.

Following

2-AB-derivatization,

the

most

abundant

glycan

was

F(6)A4G(4)4S(3)3, present as two isomers during HILIC chromatography (peaks 29 and 30, Figure 1A). Direct comparison with the native rhEPO glycan pool suggests part of the overabundance of these isomers derived from loss of sialic acid from α-1,3 antenna position of F(6)A4G(4)4S(3)4 which is the prevailing glycan in the native pool by HPAE/MS. There is also a significant decrease in the amount of F(6)A4G(4)4Lac1S(3)4 in the labeled glycan pool compared to native rhEPO glycan pool, the difference amounting to 3% of the total glycan. The amount of disialylated glycans represented by F(6)A2G(4)2S(3)2, F(6)A3G(4)3S(3)2 and F(6)A4G(4)4S(3)2 increased from 19.9 % (native-HPAE) to 25.4 % (2-AB-HILIC). This increase is attributed to the conversion of tri- and tetrasialylated to di- and trisialylated structures by desialylation during derivatization.44

Furthermore,

there

was

a

drop

in

the

relative

amount

of

F(6)A2G(4)2S(3)2 in the labeled pool, its relative amount decreased from 12.9 % to 6.36 %. 2,3-linked sialic acids attached to GlcNAc One of the best known examples of this linkage is the N-glycome of fetuin, the tetrasialylated, triantennary structures bear α-2,3 linked sialic acid attached to GlcNAc. 45

The most abundant glycan containing this linkage was F(6)A2G(4)1S(3)2 and Figure

3 revealed separation of two positional isomers with annotation shown for one of the isomers. This linkage seems susceptible to labeling-induced degradation as the relative amount of both isomers was 0.61 ± 2 % when native glycans were analysed by HPAE19 ACS Paragon Plus Environment


MS, and 0.2 ± 6 % for labeled glycans by UHPLC-MS. Extraction of the 1102.4 m/z ion (doubly charged precursor) from the spectra acquired by HPAE-MS revealed two primary peaks (Figure 3A) at 28.04 and 29.76 min. Two minor peaks were also noticeable indicating epimer formation at high pH. The HCD fragmentation of the precursor at 28.04 min confirmed the presence of α-2,3 linked sialic acid on GlcNAc (Figure 3B). Fragment ions were comprised mostly of cross ring and glycosidic/cross ring cleavages. Cross ring fragment

1,3

A5 (or

2,4

A5 at m/z 424.1461) highlights the

composition of α-1,3 antenna. This type of cross ring fragmentation-occuring on the central mannose- can be very useful to predict antenna composition since the cleavage isolates the two antennae from each other. Additionally, single glycosidic C4α and B4α ions at m/z 382.1368 and 364.1278 further corroborate that α-1,3 antenna is terminated by a GlcNAc residue. Further investigation of the MS2 fragments revealed a double glycosidic ion (m/z 891.3159, C6/Z4 or B6/Y4)-generated by Z- or Y-type glycosidic cleavages of α-1,2 linkage (between mannose and GlcNAc) on α-1,6 arm and C- or Btype cleavages of β-1,4 linkage of the chitobiose core (between two GlcNAcs). These ions render confirmation of antenna composition, however does not support the annotation of antenna position. Furthermore, ions comprised of Y5- or Z5-type glycosidic fragments such as

3,5

A6/Y5,

2,4

A7/Y5 and Z1γ/Z5 at m/z 1256.4373, 1445.5090 and

1570.8311 provide evidence of the presence of α-2,3 linked sialic acid attached to GlcNAc. Both of these glycosidic cleavages occur at β-1,4 linkage between galactose and GlcNAc, and the resulting ions bear the α-2,3 linked sialic acid on the GlcNAc Sialidase S digestion performed on released, native glycan pool removes of all sialic acids, evidencing all sialic acids are

α-2,3

linked, either to galactose or GlcNAc.


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Although fragment ions provided evidence for the composition of two antennae, the D ion (or E-ion) that would definitely assign certain composition of antennae to either α-1,3 or α-1,6 position was not observed. Cross ring fragment

1,3

A5 is affirmative but further

evidence is rendered from chromatographic data since the peak of interest at 28.04 min is further followed by the elution of an isobaric peak at 29.76 min, corroborating that the structure eluting earlier bears the two sialic acids (galactose and GlcNAc linked) on the α-1,6 arm.21 Other glycans with the GlcNAc linked α-2,3 sialic acid motif were identified in HPAE-MS base peak traces (Table 2, peaks 31 and 36, Figure 2B, sulfated triantennary glycan eluting at 78.52 min, HCD fragmentation is elaborated in Figure 6)

but were not

observed in the 2-AB labeled UHPLC-MS traces. Phosphorylation of N-linked glycans Glycans bearing phosphorylation play crucial roles in protein transport and human disease. Some of the commonly recognized diseases include deficiencies in acid hydrolase function (lysosomal storage disorders) which can be treated by functional enzyme

replacement

therapy

(fERT).

Glycosylation

of

these

enzymes

with

phosphorylated high-mannose glycans support fERT as they target lysosomes by binding to Man6 receptors.40 Phosphorylated high-mannose glycans appeared to show decreased solubility in mobile phase containing organic eluent (acetonitrile). Phosphorylated high-mannose glycans taken up in 50 % organic precipitated at the column inlet and slowly re-dissolved,



resulting in broad and diminishing peaks (Supporting Information Figure S3B for 2-AB labeled phosphorylated Man5 and Man6 in Figure S3C). HPAE-MS revealed three separate phosphorylated glycans characterized as harboring mannose- 6 phosphate (Figure 4). Phosphorylated Man5 and Man6, each bearing two negative charges, eluted at the end of the disialylated glycan cluster at 33.2 and 33.4 min (Figures 4A, B and C). A hybrid glycan bearing the phosphorylated high-mannose on the α-1,6 branch elutes much later (45.3 min) at the end of the cluster of triple charged glycans (Figures 4A & D). Because phosphate confers additional charge, phosphorylation increases glycan retention in HPAE. The native glycan pool was treated with Sialidase A and the resulting desialylated glycans were run in an additional HPAE-MS experiment (Figure 4E). The larger peak eluting at 33.47 min harbored both Man5 and Man6 phosphorylated glycans (Figure 4F and G). After sialic acid removal, the phosphorylated hybrid glycan (now doubly charged) eluted considerably earlier (32.95 min Figure 4H), and slightly before the co migrating phosphorylated high-mannose structures, consistent with the effects of sialic acid loss and presence of fucose on glycan retention by HPAE.21 Structural characterization of phosphorylated glycans employed MS2 spectral interpretation. Figure 5 details annotation of the phosphorylated hybrid glycan. Formation of the D-ion (m/z 889.2249, C5/Z3)- revealed the composition of the α-1,6 antenna and assigned the phosphate to the α-1,6 antenna. A single glycosidic, doubly charged Y4α ion (m/z 976.3028) located the phosphate on the α-1,3 linked mannose of the α-1,6 antenna. It is important to mention that although phosphorylation is expected


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to be on α-1,2 mannose, the formation of Y4α (precursor reduced by two hexoses) can only be possible if a single glycosidic cleavage removes the two mannoses. If the sialic acid on α-1,6 arm was attached to GlcNAc, removal of two hexoses by the formation of a double glycosidic ion (galactose on α-1,6 arm and mannose on α-1,3 arm) would yield the same mass as Y4α This would result in possibly annotating . phosphorylation on α1,2 mannose as well. This latter structure is however omitted by the presence of of B2 ion at m/z 452.1425 in the spectrum, clearly indicating the terminal sialic acid. Based on the HCD MS2 fragmentation of phosphorylated Man5, phosphorlylation was assigned on the α-1,6 antenna by diagnostic

0,4

A3 at m/z 625.1400 on the central

mannose of trimannosil core. If the phosphorylation occurred on α-1,3 arm, this ion would not appear to be observable in the spectrum. The appearance of double glycosidic C3/Z3β (C3/Z3) ion called D-ion at m/z 727.1753 reveals the phosphorylation is localized on one of the terminal mannoses on the α-1,6 antenna. Figure S4A in Supporting Information reports on the HCD fragmentation of phosphorylated Man5 assuming the phosphate is arbitrarily assigned on the α-1,6 linked mannose of the α-1,6 arm, Figure S4B in Supporting Information accounts for annotation of fragment ions assuming the α-1,3 linked mannose of the α-1,6 arm is phosphorylated. The comparison of these two annotations are summarized in Figure S4C in Supporting Information. The appearance of 0,4A3 and C3/Z3β explicitly assigned the phosphorylation on the α-1,6 antenna. However, in the absence of other diagnostic fragment ions, it is not possible to further assign the phosphate to either of two terminal mannoses. Cross ring fragment ions, such as 1,3A2 (assuming phosphorylation is located on α-1,3 terminal mannose) and

0,4

A2 (assuming phosphorylation is assigned on α-1,6 terminal mannose) 23 ACS Paragon Plus Environment


Page 24 of 52

are present but bear no structural information concerning position, given the fact they are isobaric. The MS2 spectrum of phosphorylated Man6 (Supporting Information Figure S5) yielded diagnostic ions including B2α (m/z 403.0647), B2 (m/z 485.1521) and B3/Z3α (m/z 629.1941). The B2α (or C2α ion at m/z 421.0753) ion can only be formed if the phosphate is located on the α-1,3 arm of the glycan. This is further confirmed by B3/Z3α ion showing no modification on the α-1,6 antenna. Fragment ion

2,4

A5/Y3 appearing at

m/z 828.2194 is generated by losing three hexoses via Y3 cleavage rendering the annotation of phosphorylation on the α-1,2 linked mannose of α-1,3 arm.

2,4

A4/Y3α ion at

m/z 707.2300 generated by Y-type glycosidic fragmentation and cross ring fragmentation on the GlcNAc under the central mannose contains four hexoses without phosphorylation. This also implies no phosphate is attached to α-1,6 arm. O-acetylation of sialic acids O-acetylation of sialic acids is the most common form of glycan acylation and 9-Oacetylated sialic acids are displayed on rhEPO glycans.46 Sialic acid O-acetylation on different rhEPO biosimilars indicates significant variations among the different biosimilars31 HPAE did not reveal O-acetylated sialic acids in glycans because acetylation is lost at high pH. O-acetylation of Nue5Ac residues are reported to increase the lifetime or decrease the turnover of the proteins in vivo.47 Derivatization of glycans with 2-AB did not lead to the loss of the acetyl group although its quantitation in 2-AB labeled glycans is biased due to the loss of sialic acids. Nevertheless, it is possible to gain qualitative information regarding sialic acid 24 ACS Paragon Plus Environment

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acetylation (e.g., number of acetyl groups attached to sialic acid, as well as composition and structure of the glycans harboring sialic acid O-acetylation). This information is especially important prior to glycopeptide mapping (see section on Site specific analysis of Lys-C digested rhEPO glycopeptides). In this report, several N-linked glycans bearing sialic acid O-acetylation were identified (Supporting Information Table S2). The majority of the acetylated glycans contained one O-acetyl-moiety on one sialic acid. Figure S6 in Supporting Information presents the distribution of

sialic acid O-acetylated glycan species reported in Supporting

Information Table S1, showing the most abundant N-glycans bearing one 9-O-acetyl neuraminic acid residue are F(6)A4G(4)4S(3)4 Ac (peaks 16 and 18) and F(4)A4G(4)4S(3)2 Ac (peaks 10 and 13). MS2 spectra of 2-AB labeled glycans modified with O-acetylated sialic acids showed a distinctive B1 ion at m/z 332.1062 (Supporting Information Figure S7) generated by the cleavage of O-acetylated sialic acid with the corresponding mass shift of 42 Da compared to sialic acid (m/z 290.0883).

Sulfation of N-linked glycans Sulfated N-linked glycans were reported on rhEPO, determined by NMR after exoglycosidase digestion with sialidase and galactosidase.48 Attachment of sulfate to highly sialylated glycans is expected to dramatically increase their retention by HPAE, and high pH acetate (up to 250 mM sodium acetate in 100 mM sodium hydroxide) was required for their elution. The HPAE chromatogram showing the sulfated glycan elution segment appears as Figure 2B. This represents the 70 to 84 minute segment of Figure 25 ACS Paragon Plus Environment


2A. The sulfated glycan structures depicted were annotated based on their HCD MS2 spectra. Some of the diagnostic ions are listed in Supporting Information Table S2. A

3,5

A type cross ring cleavage (in

3,5

A7/Z4α ion an m/z 1238.4348) occurring on the

mannose attached to chitobiose core (Figure 6) includes mannose carbons 4, 5 and 6, rendering this fragment to reveal that one of the two branches of α-1,6 antenna is extended by a single N-acetyllactoseamine (LacNAc) repeat, and is terminated by Neu5Ac. This ion confirms the absence of a second sialic acid on this branch. Additional evidence supporting the presence of LacNac extension is provided by 0,2A4 fragment ion at m/z 775.2636 on the galactose between β-1,2 and β-1,3 GlcNAcs. A characteristic, double glycosydic fragment ion (C8/Y3) appearing at m/z 827.2262 described the composition of the α-1,3 antenna, and in accordance with prior literature, assigned the location of sulfate to GlcNAc on that arm.48 The sulfated GlcNAc residue is further evidenced by the B1β ion at m/z 282.0291. Double glycosidic fragment B6/Z5 at m/z 1293.4418 provides information about the other branch of α-1,6 antenna. This branch contains two sialic acid residues and based on conclusions from structural interpretation of glycan species comprising of α-2,3 sialic acid linked to GlcNAc and Sialidase S digestion, the branch is assumed to bear α-2,3 sialic acid on GlcNAc. Such indirect evidence is delivered by 1,5A8/Y4α at m/z 1964.6428, its formation is accompanied by the removal of the branch which is considered to contain GlcNac linked α-2,3 sialic acid. Determination of the exact glycan topology requires a fragment showing the positions of two branches on α-1,6 antenna. Cross ring fragment 1,3A6 at m/z 1080.3891 takes place on α-1,6 arm mannose and annotates the branch with β-1,2 GlcNAc bearing LacNAc extension, adjacent to α-1,3 antenna. 26 ACS Paragon Plus Environment

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Sulfated 2-AB-labeled glycans were not observed in the UHPLC chromatograms. This may be due their poor solubility (as described for phosphorylated glycans). While the assumption of decreased solubility and consequent on-column “re-solubilization” during gradient formation was confirmed for the higher abundance phosphorylated glycans, this was not explicitly observed for the sulfated glycans.

N-glycolylneuraminic acid (Neu5Gc) Expression of rhEPO by CHO cells is known to produce a small fraction of glycans harboring Neu5Gc. The Neu5Gc content of the native glycans in this product was 2.1±0.1 pmol/ µg protein, or about 1% of the total sialic acid. The most abundant N-linked glycan harboring Neu5Gc and observed in both techniques was F(6)A4G(4)4Neu5Gc(3)1S(3)3, and the relative amounts found by HPAE-MS were 0.45± 6 % (of total glycan), as compared to 0.48 ± 3 % by UHPLC. The presence of the additional OH group on a Neu5Gc-containing glycan increases their retention times in both HPAE and UHPLC. Table 2 details the glycans observed by HPAE-MS, and reveals 6 different (minor) Neu5Gc-containing structures. Table 1 details the 2-AB-labeled glycans observed by UHPLC, and reveals 3 minor Neu5Gccontaining structures, one of which harbors O-acetylation. The loss of sialic acid upon derivatization may contribute to the different number of Neu5Gc glycans observed by the two methods.



Page 28 of 52

N-Acetyllactoseamine(LacNAc)repeats LacNAc repeats are known to be present on rhEPO.33 These repeats play an important role in glycoprotein clearance due to their recognition by galactose binding proteins on the hepatocyte surface, underscoring the importance of this motif in recombinant biosimilars.30 The most abundant glycans bearing LacNAc repeats include tetraantennary forms with each arm terminated by a sialic acid and harboring a single repeat; triantennary glycans bearing two repeats and a sialic acid terminating each arm; and tetraantennary glycans with one repeat and three arms terminated by sialic acid (Figure 1B, Scheme 1, structures 15, 18, 28 acquired by HPAE). Structures 15 and 18 are compositionally isomeric and without careful investigation of their MS2 spectra, this isomerism can easily be overlooked. In Table 3, the most diagnostic ions of the rhEPO glycans, including

those

harboring

LacNAc

repeats,

are

listed.

One

example,

F(6)A3G(4)3Lac2S(3)3 carries the one LacNAc repeat on each branch of the α-1,6 antenna. A Y-type glyosidic cleavage (in

3,5

X1γ/Y3 double charged fragment ion at m/z

745.2516) of the α-1,6 linkage isolated the 3 antenna with the fucosylated chitobiose core. Also, a

0,2

A6 cross ring cleavage on the α-1,6 arm mannose (and either Z or Y

type glycosydic cleavages of terminal sialic acids in

0,2

A6/Z8α and

0,2

A6/Y8α fragment

ions) revealed the LacNAc repeats on that arm. The relative peak area distribution of the three structures represents 15 % of the total glycan pool when calculated from HPAE data and 19 % using UHPLC results. Tetraantennary glycans carrying two LacNAc units present 3.03 % (calculated from HPAE data) and 3.07 % (calculated from


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UHPLC data) of the total glycan pool. Those having three LacNAc repeats are present at 0.25 % based on HPAE and 0.1 % according to data calculated from UHPLC.

Site specific analyses of Lys-C digested rhEPO glycopeptides Several LysC peptides containing both Asn24 and Asn38 sites were detected, the major peptide was E21-K45. Asn24 contains mostly hybrid and high-mannose structures including phosphorylated Man5 and Man6. The most abundant O-acetylated glycan (Supporting information Figure S6 peaks 16 & 18, and Table 2 peak 27) also resides on Asn24. Asn38 contains large tetraantennary highly branched structures with or without LacNAc repeats. This can be attributed to geometric isolation of this glycosylation site. For the site Asn83, not as many semi-enzymatic glycopeptides were observed as for other sites. The main glycopeptide was R53-K97 (Fig 7). As expected30, the main glycan compositions for Asn83 site were tetrasialylated oligosaccharides with or without acetylation.

As shown in Figure 7, the glycan attached to Asn83 bears two LacNAc

repeats with one of the four sialic acids being O-acetylated. The EThcD spectrum enabled unambiguous determination of the site of N-glycosylation as Asn.83 This site contains mostly tetraantennary glycans with or without LacNAc repeats, terminated by different number of sialic acids (Supporting Information Table S4). Exceptions include two glycans for the same site containing F(6)A2G(4)2S(3)2 or F(6)A2G(4)2S(3)3. HCD analysis of this glycan revealed that the third sialic acid was attached to the GlcNAc via the α-1,3 arm.



CONCLUSIONS This study compared the native and 2-AB labeled N-glycome of rhEPO, investigated by HPAE-MS and UHPLC-MS, respectively. N-linked glycans released from rhEPO include glycans that harbor phosphate, sulfate, LacNAc repeats, Neu5Gc, and sialic acid Oacetylation, each of which challenges current analytical platforms. HPAE fractionates glycans according to charge classes and separates glycans and their isomers according to subtle differences in structure. The effects of branch and linkage differences as well as other glycan features on retention are reported21 and knowledge of these effects assists glycan annotation. However, the high pH eluents remove Oacetyl groups from sialic acid, therefore this feature is not determined by high pH HPAE, and the basic eluent induces detectable epimerization. Because HPAE examines native glycans, sialylated glycan profiles can be more accurately quantified by this technique. In addition, phosphorylated and sulfated glycans do not precipitate under HPAE conditions, and the additional charge contributed by the inorganic anion further facilitates their separation on the anion exchange resin. Retention by HILIC is governed by hydrodynamic glycan volume. Because reductive glycan amination exposes glycans to heat in acid, highly sialylated rhEPO glycans undergo significant desialylation, altering the glycan distribution. In addition, artifact dehydrated glycans are generated. This approach employs eluents in which phosphorylated and sulfated glycans are poorly soluble, rendering characterization of these glycans problematic


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HPAE separated three phosphorylated glycans, eluting and annotated at the end of the di- and tri-sialylated charge clusters. Sulfated glycans, like phosphorylated glycans, were well separated by HPAE even from highly sialylated glycans. Sulfated glycans eluted at high salt concentration, which was efficiently removed by electrolytic desalting. This produced information rich HCD fragmentation of these minor peaks. One interesting motif, α-2,3 linked sialic acid attached to GlcNAc, was also found on minor rhEPO glycan species by both platforms, and both supported effective annotation of glycans harboring this motif. The relative levels of glycan with 2,3-linked Neu5Ac to GlcNAc were lower in the 2-AB labeled glycan pool than in the native glycan pool, implying this linkage, like α-2,3 linked sialic acid, is susceptible to decomposition during reductive amination. The labeling reaction does not lead to the loss of acetyl groups of sialic acids, structures bearing O-acetyl-sialic acid can be annotated from the 2-AB labeled glycan pool, but accurate quantification may be problematic due loss of sialic acids during labeling. Glycans from rhEPO harbor LacNAc repeats. Their distribution in the current study reveals glycans with up to three LacNAc repeats, each on a separate branch of the tri and tetra-antennary glycans, also recognized by both platforms and allowing effective annotation of this motif. Both platforms also reveal approximately 1% of the total rhEPO glycan sialic acid occurs as Neu5Gc. Site specific rhEPO glycopeptide analysis after partial digestion with Lys-C confirmed the presence and site specific location of all the glycans observed by both chromatographic platforms used to characterize the glycome. This confirms the



complementarity of the HPAE-MS and UHPLC-MS approaches, and reveals significant strengths for each. HILIC of 2-AB glycans exhibited good recovery of sialic acid Oacetylation, but questionable quantification of sialylated glycan profiles. HPAE retains fully sialylated-, phosphorylated, and sulfated glycans without the need for derivatization, but does not reveal the presence of sialic acid O-acetylation. These observations indicate these workflows are complementary.

ASSOCIATED CONTENT Supporting information Dehydration of 2-AB labeled glycans (Figure S1); Pulsed Amperometric Detection (PAD of native and 2-AB labeled rhEPO glycans (Figure S2); based peak chromatogram of 2AB labeled glycan pool of rhEPO and XIC chromatograms of Man5P and Man6P (Figure S3); HCD MS2 spectrum of phosphorylated Man5 (Figure S4A and S4B); conclusion of structural interpretation of Man5P (Figure S4C); HCD MS2 spectrum of phosphorylated Man6 (Figure S5); normalized extracted ion chromatograms of 2-AB labeled glycans terminated by O-acetylated sialic acids (Figure S6), HCD MS2 spectrum of 2-AB labeled F(6)A4G(4)4S(3)31Ac (Figure S7); peak area distribution and its reproducibility (% CV) of major glycans detected by HPAE and UHPLC (Table S1); glycans terminated by O-acetyl-sialic acids found in 2-AB labeled rhEPO glycan pool (Table S2); diagnostic fragments of sulfated rhEPO glycans identified by HPAE-MS (Table S3); site specific identification of glycans (Table S4).


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Wuhrer, M. Glycomics using mass spectrometry. Glycoconj J 2013, 30 (1), 11-

(8) Oh, M. J.; Hua, S.; Kim, U.; Kim, H. J.; Lee, J.; Kim, J. H.; An, H. J. Analytical detection and characterization of biopharmaceutical glycosylation by MS. Bioanalysis 2016, 8 (7), 711-27. (9) Szabo, Z.; Guttman, A.; Bones, J.; Shand, R. L.; Meh, D.; Karger, B. L. Ultrasensitive capillary electrophoretic analysis of potentially immunogenic carbohydrate residues in biologics: galactose-alpha-1,3-galactose containing oligosaccharides. Mol Pharm 2012, 9 (6), 1612-9. (10) Rudd, P. M.; Dwek, R. A. Rapid, sensitive sequencing of oligosaccharides from glycoproteins. Curr Opin Biotechnol 1997, 8 (4), 488-97. (11) Mittermayr, S.; Bones, J.; Guttman, A. Unraveling the glyco-puzzle: glycan structure identification by capillary electrophoresis. Anal Chem 2013, 85 (9), 4228-38. (12) Reusch, D.; Haberger, M.; Kailich, T.; Heidenreich, A. K.; Kampe, M.; Bulau, P.; Wuhrer, M. High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer. MAbs 2014, 6 (1), 185-96. (13) Ahn, J.; Bones, J.; Yu, Y. Q.; Rudd, P. M.; Gilar, M. Separation of 2aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent. J Chromatogr B Analyt Technol Biomed Life Sci 2010, 878 (3-4), 403-8. 33 ACS Paragon Plus Environment


(14) Zauner, G.; Deelder, A. M.; Wuhrer, M. Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics. Electrophoresis 2011, 32 (24), 3456-66. (15) Ikegami, T.; Tomomatsu, K.; Takubo, H.; Horie, K.; Tanaka, N. Separation efficiencies in hydrophilic interaction chromatography. J Chromatogr A 2008, 1184 (1-2), 474-503. (16) Kozak, R. P.; Tortosa, C. B.; Fernandes, D. L.; Spencer, D. I. Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionizationmass spectrometry. Anal Biochem 2015, 486, 38-40. (17) Lauber, M. A.; Yu, Y. Q.; Brousmiche, D. W.; Hua, Z.; Koza, S. M.; Magnelli, P.; Guthrie, E.; Taron, C. H.; Fountain, K. J. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Anal Chem 2015, 87 (10), 5401-9. (18) Reusch, D.; Haberger, M.; Maier, B.; Maier, M.; Kloseck, R.; Zimmermann, B.; Hook, M.; Szabo, Z.; Tep, S.; Wegstein, J.; Alt, N.; Bulau, P.; Wuhrer, M. Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1: separation-based methods. MAbs 2015, 7 (1), 167-79. (19) Szabo, Z.; Thayer, J. R.; Agroskin, Y.; Lin, S.; Liu, Y.; Srinivasan, K.; Saba, J.; Viner, R.; Huhmer, A.; Rohrer, J.; Reusch, D.; Harfouche, R.; Khan, S. H.; Pohl, C. Indepth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry. Anal Bioanal Chem 2017, 409 (12), 3089-3101. (20) Maier, M.; Reusch, D.; Bruggink, C.; Bulau, P.; Wuhrer, M.; Molhoj, M. Applying mini-bore HPAEC-MS/MS for the characterization and quantification of Fc N-glycans from heterogeneously glycosylated IgGs. J Chromatogr B Analyt Technol Biomed Life Sci 2016, 1033-1034, 342-352. (21) Rohrer, J. S. Separation of asparagine-linked oligosaccharides by high-pH anionexchange chromatography with pulsed amperometric detection: empirical relationships between oligosaccharide structure and chromatographic retention. Glycobiology 1995, 5 (4), 359-60. (22) Natowicz, M.; Hallett, D. W.; Frier, C.; Chi, M. Schlesinger, P. H.; Baenziger, J. U., Recognition and receptor-mediated uptake of phosphorylated high mannose-type oligosaccharides by cultured human fibroblasts. J Cell Biol 1983, 96 (3), 915-9.


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(23) Harvey, D. J. Fragmentation of negative ions from carbohydrates: part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates. J Am Soc Mass Spectrom 2005, 16 (5), 622-30. (24) Harvey, D. J. Fragmentation of negative ions from carbohydrates: part 2. Fragmentation of high-mannose N-linked glycans. J Am Soc Mass Spectrom 2005, 16 (5), 631-46. (25) Harvey, D. J. Fragmentation of negative ions from carbohydrates: part 3. Fragmentation of hybrid and complex N-linked glycans. J Am Soc Mass Spectrom 2005, 16 (5), 647-59. (26) McAlister, G. C.; Russell, J. D.; Rumachik, N. G.; Hebert, A. S.; Syka, J. E.; Geer, L. Y.; Westphall, M. S.; Pagliarini, D. J.; Coon, J. J. Analysis of the acidic proteome with negative electron-transfer dissociation mass spectrometry. Anal Chem 2012, 84 (6), 2875-82. (27) Ni, W.; Bones, J.; Karger, B. L. In-depth characterization of N-linked oligosaccharides using fluoride-mediated negative ion microfluidic chip LC-MS. Anal Chem 2013, 85 (6), 3127-35. (28) Skibeli, V.; Nissen-Lie, G.; Torjesen, P. Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin. Blood 2001, 98 (13), 3626-34. (29) Takeuchi, M.; Takasaki, S.; Miyazaki, H.; Kato, T.; Hoshi, S.; Kochibe, N.; Kobata, A., Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem 1988, 263 (8), 3657-63. (30) Bones, J.; McLoughlin, N.; Hilliard, M.; Wynne, K.; Karger, B. L.; Rudd, P. M. 2DLC analysis of BRP 3 erythropoietin N-glycosylation using anion exchange fractionation and hydrophilic interaction UPLC reveals long poly-N-acetyl lactosamine extensions. Anal Chem 2011, 83 (11), 4154-62. (31) Shahrokh, Z.; Royle, L.; Saldova, R.; Bones, J.; Abrahams, J. L.; Artemenko, N. V.; Flatman, S.; Davies, M.; Baycroft, A.; Sehgal, S.; Heartlein, M. W.; Harvey, D. J.; Rudd, P. M. Erythropoietin produced in a human cell line (Dynepo) has significant differences in glycosylation compared with erythropoietins produced in CHO cell lines. Mol Pharm 2011, 8 (1), 286-96. (32) Falck, D.; Haberger, M.; Plomp, R.; Hook, M.; Bulau, P.; Wuhrer, M.; Reusch, D. Affinity purification of erythropoietin from cell culture supernatant combined with MALDITOF-MS analysis of erythropoietin N-glycosylation. Sci Rep 2017, 7 (1), 5324.



(33) Thomson, R. I.; Gardner, R. A.; Strohfeldt, K.; Fernandes, D. L.; Stafford, G. P.; Spencer, D. I. R.; Osborn, H. M. I. Analysis of Three Epoetin Alpha Products by LC and LC-MS Indicates Differences in Glycosylation Critical Quality Attributes, Including Sialic Acid Content. Anal Chem 2017, 89 (12), 6455-6462. (34) Angata, T.; Varki, A. Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev 2002, 102 (2), 439-69. (35) Amon, R.; Reuven, E. M.; Leviatan Ben-Arye, S.; Padler-Karavani, V. Glycans in immune recognition and response. Carbohydr Res 2014, 389, 115-22. (36) Ghaderi, D.; Taylor, R. E.; Padler-Karavani, V.; Diaz, S.; Varki, A. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol 2010, 28 (8), 863-7. (37) Noguchi, A.; Mukuria, C. J.; Suzuki, E.; Naiki, M. Failure of human immunoresponse to N-glycolylneuraminic acid epitope contained in recombinant human erythropoietin. Nephron 1996, 72 (4), 599-603. (38) Tangvoranuntakul, P.; Gagneux, P.; Diaz, S.; Bardor, M.; Varki, N.; Varki, A.; Muchmore, E. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proc Natl Acad Sci U S A 2003, 100 (21), 12045-50. (39) Noguchi, A.; Mukuria, C. J.; Suzuki, E.; Naiki, M. Immunogenicity of Nglycolylneuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. J Biochem 1995, 117 (1), 5962. (40) Takashiba, M.; Chiba, Y.; Jigami, Y. Identification of phosphorylation sites in Nlinked glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem 2006, 78 (14), 5208-13. (41) Olson, L. J.; Zhang, J.; Lee, Y. C.; Dahms, N. M.; Kim, J. J. Structural basis for recognition of phosphorylated high mannose oligosaccharides by the cation-dependent mannose 6-phosphate receptor. J Biol Chem 1999, 274 (42), 29889-96. (42) Hemmerich, S.; Rosen, S. D. Carbohydrate sulfotransferases in lymphocyte homing. Glycobiology 2000, 10 (9), 849-56. (43) Fiete, D.; Srivastava, V.; Hindsgaul, O.; Baenziger, J. U. A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha that mediates rapid clearance of lutropin. Cell 1991, 67 (6), 1103-10. (44) Aich, U.; Hurum, D. C.; Basumallick, L.; Rao, S.; Pohl, C.; Rohrer, J. S.; Kandzia, S. Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides. Anal Biochem 2014, 458, 27-36. 36 ACS Paragon Plus Environment

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(45) Green, E. D.; Adelt, G.; Baenziger, J. U.; Wilson, S.; Van Halbeek, H. The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanasereleased oligosaccharides by 500-megahertz 1H NMR spectroscopy. J Biol Chem 1988, 263 (34), 18253-68. (46) Balaguer, E.; Neususs, C. Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry. Anal Chem 2006, 78 (15), 5384-93. (47)

Schauer, R. Analysis of sialic acids. Methods Enzymol 1987, 138, 132-61.

(48) Kawasaki, N.; Haishima, Y.; Ohta, M.; Itoh, S.; Hyuga, M.; Hyuga, S.; Hayakawa, T. Structural analysis of sulfated N-linked oligosaccharides in erythropoietin. Glycobiology 2001, 11 (12), 1043-9.

ACKNOWLEDGEMENT Roche Diagnostics GmbH is thanked for providing rhEPO. The authors also thank Dr. Julian Saba for his help during data evaluation. Dr. Rania Harfouche is also thanked for proofreading the manuscript. CONFLICT OF INTEREST The authors declare that they have no conflict of financial interest LEGENDS Figure 1. A) Base peak chromatogram of 2-AB labeled rhEPO glycans. Peaks annotated in green are artifact peaks caused by removal of water during the labeling reaction B) Percent distribution of peak areas of glycans calculated from extracted ion chromatogram (XIC) of glycans from both UHPLC and HPAE traces. Three replicates of



each chromatographic platform were evaluated. Peak areas of isomeric structures were summed.

Figure 2. A) Base peak HPAE chromatogram of native rhEPO glycans. Insets are enlarged trisialylated and tetrasialylated charge clusters. Peaks denoted with asterisk are artifact peaks (epimers). B) Enlarged part of the chromatogram from 70 to 85 min is showing minor sulfated glycan peaks; their structures were annotated based on HCD MS2 spectra.

Figure 3. A) Extracted ion chromatogram (XIC) of two biantennary isomers terminated harboring α-2,3 linked sialic acids linked to GlcNAc. Peaks denoted with red asterisks are epimers. B) HCD MS2 spectrum of the isomer bearing the GlcNAc linked α-2,3 sialic acids on the α-1,6 antenna position. Peaks denoted with green asterisks are doubly charged fragment ions.

Figure 4. A) Base peak chromatogram of native rhEPO glycans acquired by HPAE. B) XIC (from trace A) of Man5P. C) XIC (from trace A) of Man6P. D) XIC (from trace A) of hybrid phosphorylated glycan. E) Base peak chromatogram of Sialydase A digested rhEPO native glycans. F) XIC (from trace E) of Man5P. G) XIC (from trace E) of Man6P. F) XIC (from trace E) of the hybrid glycan shows that the glycan elutes earlier after loss of sialic acid. Peaks denoted with asterisks are epimer artifacts.


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Figure 5. HCD MS2 spectrum of the hybrid phosphorylated glycan. The spectrum, rich in diagnostic ions, allows annotation of the phosphate on the α-1,3 linked mannose on α-1,6 antenna position. Peaks denoted with green asterisks are doubly charged fragment ions.

Figure 6. HCD MS2 spectrum of a sulfated glycan eluting at 78.52 min (Figure 2). The spectrum accounts for the location of sulfate on the α-1,3 antenna. Structural motifs such as α-2,3 sialic acid linked to GlcNAc and LacNAc repeat are also identified. Peaks denoted with green asterisks are doubly charged, denoted with red asterisks are triply charged fragment ions.

Figure 7. EThcD spectrum of a large glycopeptide bearing O-acetylated sialic acid. Fragmentation of the peptide backbone allows for accurate calculation of the molecular weight of glycan attached to Asparagine 83.

Table 1. Glycans observed by UHPLC-MS. In addition to the derivatized glycans, the table lists artifact peaks (-H2O) Abbreviation “Ac” indicates O-acetylation on sialic acids.

Table 2. Glycans observed by HPAE-MS.



Table 3. Glycans observed by HPAE-MS and their diagnostic ions generated by MS2. Peaks are numbered based on Table 2.

Scheme 1. Glycan structures recognized by both platforms. Numbers in red refer to UHPLC peak numbers (Table 1), numbers in black refer to peaks annotated by HPAE (Table 2).


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Page 41 of 52

34

Relative Abundance

100

29

A) 22

50

20 8

1

0

30 % Peak Area

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6

2

3 4

6 7 5

10

21

15 19 11 17 14 10 16 13 18 9 12

14

28 27

30

26 23 25 24

33

35

38 37

31

18 Time (min)

40

36

32

41 45 42 4344 39 46

22

26

30

B) HPAE UHPLC

15

0

Glycans Figure 1. A) Base peak chromatogram of 2-AB labeled rhEPO glycans. Peaks annotated in green are artifact peaks caused by removal of water during the labeling reaction B) Percent distribution of peak areas of glycans calculated from extracted ion chromatogram (XIC) of glycans from both UHPLC and HPAE traces. Three replicates of each chromatographic platform were evaluated. Peak areas of isomeric structures were summed.

ACS Paragon Plus Environment


30

100

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

A)

90

15

23

24

39 40 41 42 43 44 45 46 29 30

70 60

8

50 40

10

19+20 17+18 21+22 16

14

80

30 20

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7 6 45

2 1 3 25

26+27

21+22 31 25 28 19+20 29 31* 16 28 47 49 51 53 55 57 59 3334 9 12+13 23 Time (min) 10 25 32 11 15 * * 24 Sulfated glycans 35 36 * 14 * * 35

45

55

65

85

Time (min)

B)

S

80.32

S

S

S

78.52 71.69

78.21

72.94

70

75

72

74

76

S

79.53

79.37 78.97

78

S

80

81.48

82

84

Time (min) Figure 2. A) Base peak HPAE chromatogram of native rhEPO glycans. Insets are enlarged trisialylated and tetrasialylated charge clusters. Peaks denoted with asterisk are artifact peaks (epimers). B) Enlarged part of the chromatogram from 70 to 85 min is showing minor sulfated glycan peaks; their structures were annotated based on HCD MS2 spectra. ACS Paragon Plus Environment

28.04 29.76

100

A ) *

0

25

27 Time (min)

29

* 31

33

35

B ) 100

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Relative Abundance

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0,2A

7* 1,3 ( X1)

C6/Z4 2,4A /Y 1051.8684 7 5 5/Y3α*(B6/Y4) 1,3X 1,5A /Y ) ( 4 Z1γ/Z5 0,2A /Z 6 3α B6/Z6 1,3X /Y *3,5X /C 5 3 0,2A /Y ) 5β 4 5 3α ( 1813.6375 7 6 1,3A (0,4X / ) 2,4A /Y ) (B6/Z5β) (1,3X3α/Y5)(951.3301 2 5 0,4 5 7 4 Y6 [Precursor ‐2H]2‐1,5 (2,4A5) 2,4A6/Y3 A5/Y5 0,2A /Y 1,5 1,3 A /Y ( A4/Y5β) 5 5β 6 6 (Y5β) A5 1,5A /Y ) 1914.6841 ( 2,4 1316.4636 3,5X /B 5 6 ( A7/Y6) 1607.5549 6 3 2,4 3,5 ( A7/Z3) A6/Y5 Z2 C6/Y6 1529.5521 C4α 551.0516 1547.5469 0,2 0,2A * B6* 1102.9056 A7/Z6 2,4A 6 620.7104 1256.4373 7 424.1461 1898.6686 0,2 877.2938 744.2653 1,5A * ( 1795.6392 A7/Z5β) 1464.5044 4 918..8125 1565.5682 1445.5090 771.2635 B 715.2442 1570.5611 4α 586.2028 364.1278 382.1368 2,4A /Z 891.3159 539.6808 7 6 1589.5477 1,5A

1386.4915

466.1598

0

C6/Z5β (B6/Y6)

400

600

800

1000

1200

1400

1600

1800

2000

m/z

Figure 3. A) Extracted ion chromatogram (XIC) of two biantennary isomers terminated harboring α‐2,3 linked sialic acids linked to GlcNAc. Peaks denoted with red asterisks are epimers. B) HCD MS2 spectrum of the isomer bearing the GlcNAc linked α‐2,3 sialic acids on the α‐1,6 antenna position. Peaks denoted with green asterisks are doubly charged fragment ions. ACS Paragon Plus Environment


Sialidase A treated

Total glycan pool 100

100

A) 20 10

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

33.40

33.47

50

70

10

30

P 100

B)

20 50

33.40 100

70

P

C) 36.33*

20 10

30

45.30

D)

100

70

P

48.13*

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30

50

70

P

G) 36.39+ 30

50

70

P

32.95

H) 35.52+

20 70

50

33.47

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70

P

30

20 50

50

38

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35

F)

20 30

32

33.27

36.33* 10

29

20

30

33.47

32.95

E)

32.95

45.30

33.23

100

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10

30

Time (min)

50

70

Time (min)

Figure 4. A) Base peak chromatogram of native rhEPO glycans acquired by HPAE. B) XIC (from trace A) of Man5P. C) XIC (from trace A) of Man6P. D) XIC (from trace A) of hybrid phosphorylated glycan. E) Base peak chromatogram of Sialidase A digested rhEPO native glycans. F) XIC (from trace E) of Man5P. G) XIC (from trace E) of Man6P. F) XIC (from trace E) of the hybrid glycan shows that the glycan elutes earlier after loss of sialic acid. Peaks denoted with asterisks are epimer artifacts. ACS Paragon Plus Environment

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100

290.0883 P P

P

P P P

P

P

2,4A

B1

7*

P P

2,4X * 6

P

2,4X /C * 6 6

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


C6/Y6 P

Y3r/Z5 P X

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1637.5132

6/Y4 1679.5133 2,4A /Y 1599.5460 1138.3622 954.7898 7 5 1866.5948 B5/Z3 913.2721984.8002 2,4A /Y1517.45591619.4915 6 6 Z2 0,4 871.2162 1476.39661536.4624 1,5A 1078.84562,4 2 551.0518 X2/Y3 A /Y 6 1314.3608 5 671.2525 1830.6213 424.1454

889.2249

0 200

600

976.3028

1000

1400

Y6 Z6

2,4A 1,5A

7/Y6

1800

1968.63422,4X

6rP

2096.7241 2200

m/z

Figure 5. HCD MS2 spectrum of the hybrid phosphorylated glycan. The spectrum, rich in diagnostic ions, allows annotation of the phosphate on the rr1,3 linked mannose on rr1,6 antenna position. Peaks denoted with green asterisks are doubly charged fragment ions. ACS Paragon Plus Environment


S

S

S

S 100

S

S

290.0883 S

1,3A

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B6/Z5

6

C8/Y8*

1,5A

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C8/Y3

801.2852

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Z5α/Y8‐S*

Z8* (Z5α')

1,4X /B ‐S 3 8

998.6710

0,3A

* 9/Y8*

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9/Y6*

1293.4418

1004.6638

888.2881

S

S

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7/Y8

*

Z5α/Y8*

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S 1,5A

S

9/Y4α 3,5X /B ‐S 6 8

Y5α/Y8* 1238.4348 3,5X /C 1311.4326 4 8 1199.3905 3,5X /C 2109.6931 S Z4/Y4α‐S 4 8 2415.7922 C3/Z8 1178.3801 1361.9569 2065.6577 0,2A 1240.4200 970.9881 2332.7881 4 364.1209 1,5A /Y 1080.3891 775.2636 8 4α 1208.3876 2313.7642 Z 282.0291 424.1480 2 2,5A /Z ‐S 2433.8135 1143.1581 827.2262 7 4 2126.6821 551.0516 1435.4994 1721.6282 1964.6428 671.2585 1,5A

0

S

S

Y5α'*

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 46 of 52

400

0,2A

2

800

1200

m/z

1600

2000

2400

Figure 6. HCD MS2 spectrum of a sulfated glycan eluting at 78.52 min (Figure 2). The spectrum accounts for the location of sulfate on the α‐1,3 antenna. Structural motifs such as α‐2,3 sialic acid linked to GlcNAc and LacNAc repeat are also identified. Peaks denoted with green asterisks are doubly charged, denoted with red asterisks are triply charged fragment ions ACS Paragon Plus Environment

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Ac

┌ ┌┌

┌

┌

┌

┌┌

RM˩ E˩ V˩G˩Q˩ Q˩AV˩ EVW QG

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LAL LS EA VLR˩ GQA˩LLVN*S˩S˩QPWE˩PLQLH˩V˩DK

100 2208.6296

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y+426

1577.2895

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Peptide + Glycan 315.3880

c +4 533.2864

c+

2

c +3

434.2180

y+528

718.3365

c +5

590.2864

1016.5306 200

600

1692.7510

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1000

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b+543

1840.2239

b+542

1820.4102

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c+227

c +9 0

y+536

1513.4559 1572.8969

c +7 846.4251

305.1744

y+543 1835.0220

y+421

1569.8948

c +6

b+537 1702.7425

1699.4557

z+1+531

1841.5390

2190.2280

y+424

1469.2900

b+332

z+1+333

2664.4290 2677.5131

2628.1442

1791.5191

c+633

b+24

y+439

1331.9189

Peptide + Glycan 485.4938 Peptide + Glycan 613.5824 Peptide + Glycan 741.6325 Peptide + Glycan 798.6343 Peptide + Glycan 879.7009 Peptide + Glycan 1026.7415

1400

m/z

1800

2200 Peptide + Glycan 824.2051

2600

3000

Peptide + Glycan 36.2848

Peptide + Glycan 844.0188 Peptide + Glycan 137.3712

Peptide + Glycan 961.6865 Peptide + Glycan 1087.1395

b - ions

c - ions

Figure 7. EThcD spectrum of a large glycopeptide bearing O-acetylated sialic acid. Fragmentation of the peptide backbone allows for accurate calculation of the molecular weight of glycan attached to Asparagine 83.


Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1,5

2,11

3,18

10,16

11,24

14,41

19,30

21,22

25,46

4,6

5,7

7,23

15,37

16,29

26,44

28,40

Page 48 of 52

8,8

9,15

17,42

18,36

30,34

35,38

Scheme 1. Glycan structures recognized by both platforms. Numbers in red refer to UHPLC peak numbers (Table 1), numbers in black refer to peaks annotated by HPAE (Table 2).


Page 49 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Journal of Proteome Research Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Retention time (min) 8.01 8.83 9.13 9.35 9.93 10.29 10.46 12.20 13.09 13.45 13.53 14.16 14.71 15.09 15.31 15.60 16.19 16.47 16.77 16.95 17.28 17.55 18.12 18.43 18.70 18.88 19.46 19.64 20.02 20.42 20.76 21.01 21.40 21.85 22.32 22.63 22.98 23.02 23.42 24.16 24.64 24.86 25.34 26.19 26.90 28.04

Charge ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐3 ‐3 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐2 ‐3 ‐3 ‐3 ‐3 ‐3 ‐3 ‐3 ‐2 ‐3 ‐3 ‐3 ‐3 ‐3 ‐3 ‐2 ‐3 ‐3 ‐3 ‐3 ‐3 ‐3 ‐3

Observed m/z 952.3472 1234.4451 1234.4400 1264.4568 1097.9019 1162.4249 1162.4250 1243.4518 1280.4677 1280.4678 1280.4683 1562.5502 1061.3754 1061.3742 1426.0172 1426.0170 1463.0342 1463.0356 1745.1254 1745.1252 1571.5651 1571.5630 1608.5834 1608.5824 1260.1046 1260.1046 1280.1219 1280.1218 1169.0862 1169.0872 1280.1212 1791.1486 1381.8242 1266.1176 1290.7968 1290.7979 1290.7970 1271.4497 1973.7157 1387.8301 1412.5083 1412.5084 1393.1600 1509.5406 1534.2086 1631.2493

Calculated mass (Da) Theoretical mass (Da) Accuracy (ppm) Glycan 1906.7102 1906.7138 ‐1.89 F(6)A2G(4)2 2470.9060 2470.8944 4.69 F(6)A2G(4)2S(3)2‐H2O 2470.8958 2470.8944 0.57 F(6)A2G(4)2S(3)2‐H2O 2530.9294 2530.9202 3.64 F(6)A2G(4)2S(3)2(Ac) 2197.8196 2197.8142 2.46 F(6)A2G(4)2S(3)1 2326.8656 2326.8568 3.78 F(6)A2G(4)1S(3)2 2326.8658 2326.8568 3.87 F(6)A2G(4)1S(3)2 2488.9194 2488.9097 3.90 F(6)A2G(4)2S(3)2 2562.9512 2562.9464 1.87 F(6)A3G(4)3S(3)1 2562.9514 2562.9464 1.95 F(6)A3G(4)3S(3)1 2562.9524 2562.9464 2.34 F(6)A3G(4)3S(3)1 3127.1162 3127.1221 ‐1.89 F(6)A3G(4)3S(3)3‐H2O 3187.1499 3187.1478 0.66 F(6)A3G(4)3S(3)3 (Ac) 3187.1463 3187.1478 ‐0.47 F(6)A3G(4)3S(3)3 (Ac) 2854.0502 2854.0419 2.91 F(6)A3G(4)3S(3)2 2854.0498 2854.0419 2.77 F(6)A3G(4)3S(3)2 2928.0842 2928.0786 1.91 F(6)A4G(4)4S(3)1 2928.0870 2928.0786 2.87 F(6)A4G(4)4S(3)1 3492.2666 3492.2543 3.52 F(6)A4G(4)4S(3)3‐H2O 3492.2662 3492.2543 3.41 F(6)A4G(4)4S(3)3‐H2O 3145.1460 3145.1373 2.77 F(6)A3G(4)3S(3)3 3145.1418 3145.1373 1.43 F(6)A3G(4)3S(3)3 3219.1826 3219.1741 2.64 F(6)A4G(4)4S(3)2 3219.1806 3219.1741 2.02 F(6)A4G(4)4S(3)2 3783.3375 3783.3497 ‐3.22 F(6)A4G(4)4S(3)4‐H2O 3783.3375 3783.3497 ‐3.22 F(6)A4G(4)4S(3)4‐H2O 3843.3894 3843.3755 3.62 F(6)A4G(4)4S(3)4 (Ac) 3843.3891 3843.3755 3.54 F(6)A4G(4)4S(3)4 (Ac) 3510.2823 3510.2695 3.65 F(6)A4G(4)4S(3)3 3510.2853 3510.2695 4.50 F(6)A4G(4)3S(3)3 3843.3873 3843.3755 3.07 F(6)A4G(4)4S(3)4 (Ac) 3584.3130 3584.3062 1.90 F(6)A4G(4)4Lac1S(3)2 4148.4963 4148.4819 3.47 F(6)A4G(4)4Lac1S(3)4‐H2O 3801.3765 3801.3649 3.05 F(6)4G(4)4S(3)4 3875.4141 3875.4017 3.20 F(6)A4G(4)4Lac1S(3)3 3875.4174 3875.4017 4.05 F(6)A4G(4)4Lac1S(3)3 3875.4147 3875.4017 3.35 F(6)A4G(4)4Lac1S(3)3 3817.3728 3817.3598 3.41 F(6)A4G(4)4Neu5Gc(3)1S(3)3 3949.4472 3949.4384 2.23 F(6)A3G(4)3Lac3S(3)2 4166.5140 4166.4971 4.06 F(6)A4G(4)4Lac1S(3)4 4240.5486 4240.5339 3.47 F(6)A4G(4)4Lac2S(3)3 4240.5489 4240.5339 3.54 F(6)A4G(4)4Lac2S(3)3 4182.5037 4182.4920 2.80 F(6)A4G(4)4Lac1Neu5Gc(3)1S(3)3 4531.6455 4531.6293 3.57 F(6)A4G(4)4Lac2S(3)4 4605.6495 4605.6297 4.30 F(6)A4G(4)4Lac2Neu5Gc(3)2S(3)2 (Ac 4896.7716 4896.7615 2.06 F(6)A4G(4)4Lac3S(3)4

Table 1. Glycans observed by UHPLC‐MS. In addition to the derivatized glycans, the table lists artifact peaks (‐H2O) Abbreviation “Ac” indicates O‐acetylation on sialic acids. ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Retention time (min) 19.77 21.22 21.65 28.04 29.76 30.20 30.59 30.90 31.23 31.71 32.10 33.49 33.66 40.77 40.83 41.05 41.39 41.44 41.71 41.72 42.10 42.24 44.29 45.50 44.91 50.26 50.26 50.51 51.11 51.49 53.66 58.76 59.31 59.99 61.01 64.42

Charge r2 r2 r2 r2 r2 r2 r2 r2 r2 r2 r2 r1 r1 r3 r3 r3 r3 r3 r3 r3 r3 r3 r2 r2 r3 r3 r3 r3 r3 r3 r3 r3 r3 r3 r3 r2

Table 2. Glycans observed by HPAE/MS

Observed m/z 1037.8668 1220.4319 1402.9963 1102.3873 1102.3880 1365.9790 1548.5449 1183.4142 1365.9797 1365.9795 1548.5459 1313.3931 1475.4457 1372.4803 1250.7716 1129.0618 1372.4817 1250.7711 1129.0623 1075.0442 1007.3515 1075.0424 1328.9606 1138.3630 1591.2240 1469.5142 1134.3903 1347.8037 1347.8044 1226.0941 1177.4082 1474.8474 1353.1335 1353.1350 1231.4245 1985.1867

Calculated m/z 2077.7494 2442.8796 2808.0084 2206.7904 2206.7918 2733.9738 3099.1056 2368.8442 2733.9752 2733.9752 3099.1076 1314.4010 1476.4536 4120.4646 3755.3385 3390.2091 4120.4688 3755.3370 3390.2106 3228.1563 3025.0782 3228.1509 2659.9370 2278.7418 4776.6957 4411.5663 3406.1946 4046.4348 4046.4369 3681.3060 3535.2483 4427.5659 4062.4242 4062.4287 3697.2972 3972.3892

Theoretical m/z Accuracy (ppm) 2077.7454 1.93 2442.8776 0.82 2808.0098 r0.50 2206.7881 1.04 2206.7881 1.68 2733.9730 0.29 3099.1052 0.13 2368.8409 1.39 2733.9730 0.80 2733.9730 0.66 3099.1053 0.74 1314.3998 0.91 1476.4526 0.68 4120.4651 r0.12 3755.3329 1.49 3390.2077 0.41 4120.4651 0.90 3755.3329 1.09 3390.2007 2.92 3228.1479 2.60 3025.0685 3.21 3228.1479 0.93 2659.9363 0.26 2278.7381 1.62 4776.6927 0.63 4411.5605 1.31 3406.1956 r0.29 4046.4283 1.61 4046.4283 2.13 3681.2961 2.69 3535.2382 2.86 4427.5554 2.37 4062.4232 0.25 4062.4232 1.35 3697.2911 1.65 3972.3916 r0.60


Page 50 of 52

Glycan F(6)A2G(4)2S(3)1 F(6)A3G(4)3S(3)1 F(6)A4G3(4)S(3)1 F(6)A2G(4)1S(3)2 F(6)A2G(4)1S(3)2 F(6)A3G(4)3S(3)2 F(6)A4G(4)4S(3)2 F(6)A2G(4)2S(3)2 F(6)A3G(4)3S(3)2 F(6)A3G(4)3S(3)2 F(6)A4G(4)4S(3)2 Man5rP Man6rP F(6)A4G(4)4Lac2S(3)3 F(6)A3G(4)3Lac2S(3)3 F(6)A4G(4)4S(3)3 F(6)A4G(4)4Lac2S(3)3 F(6)A4G(4)4Lac1S(3)3 F(6)A4G(4)4S(3)3 F(6)A4G(4)3S(3)3 F(6)A3G(4)3S(3)3 F(6)A4G(4)3S(3)3 F(6)A2G(4)2S(3)3 hybridMan6S1F1rP F(6)A4G(4)4Lac3S(3)4 F(6)A4G(4)4Lac2S(3)4 F(6)A4G(4)4Neu5Gc(3)1S(3)2 F(6)A4G(4)4Lac1S(3)4 F(6)A4G(4)4Lac1S(3)4 F(6)A4G(4)4S(3)4 F(6)A4G(4)3Ne5Gc(3)1S(3)3 F(6)A4G(4)4Lac2Neu5Gc(3)1S(3)3 F(6)A4G(4)4Lac1Neu5Gc(3)1S(3)3 F(6)A4G(4)4Lac1Neu5Gc(3)1S(3)3 F(6)A4G(4)4Neu5Gc(3)1S(3)3 F(6)A4G(4)4S(3)5

Page 51 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Peak number

Glycan

1

F(6)A2G(4)2S(3)1

2

F(6)A3G(4)3S(3)1

Diagnostic HCD fragments m/z

Fragment symbol

586.2099 (1), 688.2383 (1), 961.3071 (1), 1122.4554 (1)

1,3

715.2487 (1), 1215.2711 (1), 1257.8012 (1), 1288.7123 (1)

1,3

2,4

A5 ( A5), C5/Z3 (B5/Y3), B5/Y3r, 0,2

2,4

A4 ( X3/C4), C5/Z4 (B5/Y4),

3

F(6)A4G3(4)S(3)1

933.3228 (1), 965.3020 (1), 1242.5038 (1), 1520.5133 (1)

4

F(6)A2G(4)1S(3)2

382.1328 (1), 891.3159 (1), 1570.5611 (1)

C4r, C6/Z4, Z1v/Z5

5

F(6)A2G(4)1S(3)2

526.2111 (1), 1096.4139(2), 1369.4959 (1)

C5/Z3 , Y4, Z 5 /Y3r

6

F(6)A3G(4)3S(3)2

586.1882 (1), 1224.7247 (1), 1783.6344 (1), 1797.0231 (1)

2,5

A5 ( A5),

1,3

0,4

A5/Z6 ( A5/Z6r ),

1,3

A5 ( A5),

3,5

A5, B5/Z3r

X6r /C 4r (

7

F(6)A4G(4)4S(3)2

951.3370 (1), 1547.1306 (1), 1617.3955 (1)

8

F(6)A2G(4)2S(3)2

612.2126 (1), 655.2222 (1), 877.3018 (1)

1,5

9

F(6)A3G(4)3S(3)2

10

F(6)A3G(4)3S(3)2

A5/Z6 ( A5/Z6),

1,5

0,2

X4/B4,

586.1968 (1), 702.2354 (1), 620.7111 (1)

0,4

A5,

609.2177 (1), 701.7426 (1), 961.3204 (1), 981.3388 (1), 1020.3671(1)

F(6)A4G(4)4S(3)2

586.1997 (1), 745.2598 (2), 951.3350 (1)

12

Man5rP

241.0119 (1) ,625.1400 (1), 464.6221 (2)

B1,

13

Man6rP

403.0647 (1), 503.1622 (1), 828.2194 (1)

B2r, C2,

1224.7286 (1), 1357.5454 (1), 1369.2887 (1), 1411.4933 (1)

0,4

F(6)A4G(4)4Lac2S(3)3

1,5

1,3

A5 ( A5),

3,5

0,2

0,4

2,4

A5/Y6,

A5/Y3

0,4

A7/Z4 ( A7/Z4t),

A6 /Y5 (1,5 A6/Y5t), 3,5X4r /Z3 , 0,2X3r/Z3

745.2516 (2), 831.2962 (1), 849.3029 (1) 508.1692 (1), 745.2545 (2), 817.2729 (2), 909.6541 (1), 933.3264 (1) no informative MS2 spectrum was obtained

B4r/Z 4r, C4 , C 5/Z 3r (B5/Y3r), C 4r/Y6r,

544.1929 (1), 697.2336 (1), 831.2922 (1),

C3 ,

X1v/Y3 ,

0,2

1,5

F(6)A4G(4)4S(3)3 F(6)A4G(4)4Lac2S(3)3 F(6)A4G(4)4Lac1S(3)3

A6/Z8r,

X3r /B5r,

2,4

817.2736 (2), 933.3184 (1), 951.3461 (1)

C5/Z3r (B5/Y3r),

20 21

F(6)A4G(4)3S(3)3 F(6)A3G(4)3S(3)3

no informative MS2 spectrum was obtained 771.2693 (1), 773.2714 (1), 803.2772 (2)

1,5

A4/Z4 ( A4/Z4t),

22

F(6)A4G(4)3S(3)3

no informative MS2 spectrum was obtained

23

F(6)A2G(4)2S(3)3

891.3174 (1), 1309.8828 (1), 1325.6896 (1), 1327.2146 (1)

3,5

A5,

1,5

X4/Z3r , 1,5

24

hybridMan6S1F1rP

889.2249 (1), 976.3028 (2), 1268.1974 (1)

C5/Z3, Y4r ,

25

F(6)A4G(4)4Lac3S(3)4

927.8170 (2), 1238.6274 (1)

C6r,

26 27

F(6)A4G(4)4Lac2S(3)4

831.2980 (1), 927.8262 (2) no informative MS2 spectrum was obtained

0,2

508.1684 (1), 539.6834 (1), 609.210 (1), 745.2513 (2)

C2 ,

A5/Y8,

A4r /Y6r' (

3,5

1,5

F(6)A4G(4)4Lac1S(3)4

586.2018 (1) ,646.2240 (1), 745.2516 (2), 799.2654 (1),

30 31

F(6)A4G(4)4S(3)4 F(6)A4G(4)3S(3)3NeuGc(3)1 F(6)A4G(4)4Lac2S(3)3NeuGc(3)1

484.1669 (1), 697.2315 (1), 745.2542 (2), no informative MS2 spectrum was obtained

0,2

1513.5317 (1), 1760 (2), 1905.1782 (2)

Z3/Z4r ',

628.2124 (1), 745.2567 (1), 1463.4932 (1), 2000.6831 (2)

0,2

1369.4485 (1), 1548.0494 (2), 1692.6046 (1), 2804.9583 (1)

0,4

F(6)A4G(4)4Lac1S(3)3NeuGc(3)1

A5 /Z6r

1,3

A5/Z6r ( A5/Z6r), 1,3

X6r /B4r,

1,3

2,4

A5/Y6r ( A5/Y6r)

1,5

A5/Y3r

X2/Z 1v (2,4 X2/Z 1v), 2,4X4 /Y3r

X3r/Z6 ,

A7 /Z4 (3,5A7 /Z 4t)

0,4

A5/Y7,

29

34

2,4

A6/Z8t, C 8 /Y3

2,4

F(6)A4G(4)4Lac1S(3)3NeuGc(3)1

A6/Y8r

X3/B5,

1,3

F(6)A4G(4)4S(3)3

2,4

0,2

0,4

19

33

A5/Y6r )

2,4

F(6)A3G(4)3Lac2S(3)3

32

2,4

A5

16 17

28

X2/C6

A3, C4/Z4 (B4/Y4)

15

F(6)A4G(4)4S(3)2NeuGc(3)1 F(6)A4G(4)4Lac1S(3)4

1,3

X6/C4, C4/Z5t (B4/Y5t)

1,3

A5/Y4r ( A5/Y4r'), C4,

0,2

1,3

A6/Y5r ,

3,5

18

X2/C5

A5/Z4 ( X3/B5)

3,5

A5/Y6 ( A5/Y6r ,

0,4

0,4

1,3

0,4

A4/Y5, B5/Z3,

X3/C5,

11

A 5,

2,4

X6 /C 4 ), B3r (B3), 1,3A5 ( 2,4A5, 0,4A5)

0,2

0,4

14

1,5

3,5

X2/B6

A6/Z4,

1,3

2,4

2,4

1,3

0,2

A5/Z8,

0,2

A5/Y8, C6r, 0,2

1,5

A6/Z7,

X7/B5, C6r

0,2

X8/B5,

A4/Y6t), 0,2X3r/B4r (0,2X3/B4), C4 (C4r )

A7 /Y6r', Y1v/Y5r' (2,4A9/Y6r')

3,5

A5,

1,3

X6r'/C 6r (

0,4

1,3

X6r/C6r), C 7/Z 3

X6r'/B6r ( X6r/B6r), Y1v/Y5,

35

F(6)A4G(4)4S(3)3NeuGc(3)1

671.2523 (1), 1170.1840 (1), 1180.1240 (1)

B3t,

36

F(6)A4G(4)4S(3)5

929.6541 (3), 944.9918 (3), 1617.5479 (1)

1,5

1,5

2,4

2,4

X4/Y3r ( X4r/Y3),

1,5

X6r/Y4

A4 /Y6 , B4 /Z6

A6/Y6, C6/Y6, B6/Z3

Table 3. Glycans observed by HPAErMS and their diagnostic ions generated by MS2. Peaks are numbered based on Table 2. ACS Paragon Plus Environment


Page 52 of 52

For TOC only

HPAE

Ac P

Na+ OHNa+ CH3COO-

S

P Desalter MS

S

Native N-linked Glycans of rhEPO Ac P

S

HILIC NH4+ HCOOCH3CN

2-AB labeled N-linked Glycans of rhEPO


MS

Ac

High Performance Anion Exchange and Hydrophilic Interaction Liquid

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