Comprehensive Analysis of Recombinant Human Erythropoietin Glycoforms by Capillary Electrophoresis andNanoflow Liquid Chromatography Coupled with Middle-down Mass Spectrometry
R. Viner1, A. A. M. Heemskerk2, D. M. Horn1, J. Saba1, M. Bern3, D. Bush4, M. Santos5, H. Dewald5, A.R. Ivanov4, B. L. Karger4
1Thermo Fisher Scientific, San Jose, CA; 2Leiden University Medical Center, Leiden, The Netherlands; 3ProteinMetrics, Palo Alto, CA; 4Barnett Institute, Northeastern University, Boston, MA; 5 Sciex LLC, Brea, CA

Overview
Purpose: To develop a middle-down workflow for the characterization of humanrecombinant erythropoietin (rhEPO) glycoforms using a sheathless capillaryelectrophoresis (CESI) and nanoflow liquid chromatography (nLC) based separation
coupled to an Orbitrap mass spectrometer.
Methods: Reduced and alkylated rhEPO was digested with LysC. Digested rhEPO
was analyzed on Orbitrap-based instruments using CESI-MS or nLC/MS coupled withhigh resolution high mass accuracy (HR/AM) FT scanning of precursor and ETD orHCD MS2 fragment ions.
Results: To perform complete quantitative glycan site-specific mapping of rhEPO, weemployed limited Lys-C digest to yield one glycosylation site per peptide fragment of
3–10 kDa size ideal for middle-down analysis. This enabled more complete sitespecific sequencing of glycoforms compared with top-down analysis. The rhEPO
glycopeptides were well separated from non-glycosylated peptides by CESI. Glycoform
resolution was mostly based on differences in the number of sialic acid residues. nLCseparation provided complementary information and better separation of large doubly
glycosylated N- linked peptides.

Introduction
Erythropoietin is a glycoprotein that is synthesized mainly in the kidney and that
stimulates erythropoiesis. Recombinant human Erythropoietin (rhEPO) is used fortreatment of anemia resulting from chronic kidney disease. EPO has three
N-glycosylation sites and one O-glycosylation site, which makes glycoform profilingchallenging due to the high heterogeneity (Figure1). Multiple formulations of rhEPO
have been studied extensively using different approaches, including capillaryelectrophoresis and nanoflow liquid chromatography coupled to advanced mass
spectrometry (MS) detection1–2. Although CE-MS has been used to characterize intact
EPO glycoforms, quantitative mapping of each glycosylation site by CE-MS has notbeen previously accomplished. Here, we report that sheathless CE- and nLC-based
separations coupled to Orbitrap MS provide complementary information and allow forcomprehensive site-specific glycan mapping of rhEPO.

Methods
Sample Preparation
Reduced and alkylated rhEPO expressed in CHO or HEK cells (Erythropoietin-Alpha,ProSpec, NJ) was digested with LysC (enzyme:protein ratio of 1:200, for 2 hr at 37° Cin 20 mM ammonium acetate, pH 6.0), trypsin (enzyme:protein ratio of 1:100, for 4 hr
at 37° C in 20 mM ammonium bicarbonate, pH 8.0), or proteinase K (enzyme:proteinratio of 1:50, for 1 hr at 37° C in 20 mM ammonium acetate, pH 6.0). All enzymes werefrom Roche, IN.
FIGURE 1. Erythropoietin-Alpha structure (A) and sequence (B): green – signal
peptide; brown – N-terminus variant, light blue – EPO, dark blue – sequencevariant, red – glycosylation sites.

Liquid Chromatography

Characterization of O-glycoforms

The rhEPO tryptic or LysC digests were separated using the Thermo Scientific™
EASY-nLC™ 1000 HPLC system with a Magic C18 spray tip 20 cm x 75 μm I.D.
column (Michrom). Gradient elution was performed from 4–30% over 60 min and from30–85% over 10 min with ACN in 0.1% formic acid at flow rate of 300nL/min.

EPO alpha contains one O-glycosylation site at Ser126. Limited Lys-C digest yielded
peptides of different lengths containing Ser126 in a range of 3–9 kDa (Figure 3). All
O-linked glycopeptides can only be identified in the FT ETD experiments because theO-linked glycans are very labile and do not survive collisional activation (Figure 3). As
expected, CESI glycoform separation was mostly based on differences in the numberof sialic acid residues (Figure 4). The two predominant O-glycosylated peptides(N-acetylhexosamine-hexose with one or two sialic acids) migrated as completely
resolved peaks in CESI but not in nLC (Figure 4A vs. 4B) using similar analysis times.
The relative abundances of major CHO rhEPO O-glycoforms are shown in Table 1. We
detected several unmodified Ser126 peptides (total relative abundance 9%, Table 1),
which means that the site was only partial glycosylated in this sample. Additionally, weobserved partial O-glycosylation on Ser9 and Ser120 residues.

Capillary Electrophoresis
CESI-MS was performed using a CESI 8000 High Performance Separation System(AB Sciex) equipped with an OptiMS silica cartridge consisting of a porous sprayer
operating in an ultra-low flow regime (Figures 2A and 2B). The rhEPO digests wereseparated using a prototype neutral coated capillary (100 cm total length) or bare fusedsilica capillaries (90 cm total length) both with an inner diameter of 30 μm. The acidic
background electrolyte (BGE) of acetic acid was prepared by diluting glacial acetic acidto appropriate concentration.
Mass Spectrometry
Samples were analyzed on Thermo Scientific™ Orbitrap Elite™ mass spectrometer
and Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer using FT/IT
HCD, CID, or ETD MS2 fragmentations in DDDT or HCDpdETD/CID methods. FT MS1
was acquired at resolution settings of 60–120K at m/z 200 and FTMS2 at resolution of30–60K at m/z 200.

For the site Asn83, we did not observe as many N-C-ragged glycopeptides as for othersites, which simplified quantification of its glycoforms. The main glycopeptide was
R53–K97 (Figure 6). Due to the size of this peptide and the glycan-to-peptide ratio, thedifference of one sialic acid causes a smaller migration time shift (~0.4 min) than for
O-linked glycoforms (Figure 7 vs. Figure 4) and the difference in neutral HexHexNAc
residues contributes to migration shifts to even a lesser extent (Figure 7). Asexpected4, the main glycan compositions for Asn83 site were tetra-acidic
oligosaccharides (Figure 8).
FIGURE 5. Identification of rhEPO LysC double N-linked glycopeptide (A1–K45)
by Byonic node in Proteome Discoverer 2.0 software.

FIGURE 3. Identification of rhEPO LysC O-linked glycopeptides by Byonic nodein Proteome Discoverer 2.0 software (114–150, A) or by ProSightPC 3.0 software
using biomarker + delta m search ( 95–150, B).

Additionally, comparative glycopeptide profiling of this site for rhEPO expressed inCHO vs HEK cells was performed (Figure 8). It has been reported that oligosaccharide
structural features of recombinant proteins are cell line-, culture condition-, and speciespecific.5 Assuming equal detection response for all glycoforms, CESI-MS analysis
demonstrated significant differences in relative abundance of glycoforms expressed in
CHO vs. HEK cells. The main glycoform for this site was HexNAc6Hex7dHex1NeuAc4 in
HEK cells, and for CHO cells several larger tetra-sialylated species offset byHexNacHex units were dominant.

A114

150

Data Analysis
The Thermo Scientific™ ProSightPC 3.0, Protein Deconvolution 3.0, Pinpoint™ 1.4,and Proteome Discoverer™ 2.0 software with the Byonic™ search node (ProteinMetrics) were used for glycopeptide data analysis and glycoform quantification.
SimGlycan® 4.5 software (PREMIER Biosoft) was used for proteinase K digest
glycopeptide and glycan composition identification.

FIGURE 6. Identification of rhEPO LysC N-linked glycopeptide (R53–K97) by Byonicnode in Proteome Discoverer 2.0 software using HCD (A) or ETD (B) fragmentation.

BResultsConclusion

Capillary electrophoresis (CE) is well suited to the separation of glycopeptides due todifferences in charge states and Stokes radii3. Recent advances in electrospray ion
source technology have enabled sheathless coupling of CE to MS, eliminating thedilution effects of prior sheath flow techniques (Figures 2A and 2B), increasing
sensitivity and reproducibility of glycoprotein analysis. We were able to identify andquantify multiple EPO glycopeptides using 200 ng of sample with excellent S/N.
Migration times and peak areas demonstrated good reproducibility with less than 10%
RSD across runs (Figure 2C). Glycopeptides were well separated by CESI andresolved within 20 min of a 50 min long run (Figure 2C).
FIGURE 2. CESI-MS of rhEPO. Schematic of a CESI sprayer (A); A Thermo
Scientific™ nanospray source with an OptiMS sprayer adaptor (B); Base peakelectropherograms for three constitutive runs of rhEPO LysC digest (200 ng)
and MS2 XIC for HexNac oxonium peak (C).

 CESI-MS technique is reproducible (RSD <10%) and sensitive, obtaining thesame sequence coverage and number of glycopeptides with five times lower
amount of sample than for nLC-MS experiments.
 CESI separation of glycoforms is clearly based on differences in the number ofsialic acid residues (that is, difference in charge) and the peptide to glycan massratios.

FIGURE 4. CESI-MS (A) and nLC-MS (B)separation of O-linked E114–K150
glycopeptide.
AA

CB

 In the present study, we used neutral or bare fused silica capillaries which
demonstrated high adsorption for glycopeptides with high glycan to peptide ratios.
New MS compatible coatings with moderate to low reversed electroosmotic flowwould be highly desirable.

TABLE 1. Peptide Quantification ofmajor rhEPO glycoforms for Ser 126site. Each glycoform was calculated
as a sum of all detected peptides.

Glycoform The primary O-linked glycoforms of ProSpec’s CHO rhEPO are HexNacHex+1(2)
NeuAc with the relative abundance of unglycosylated Ser 126 of approximately 9%.

RelativeAbundance (%)

 The primary N-linked glycoforms of ProSpec’s CHO rhEPO for all three sites are
tetra-sialylated species with average glycan composition ofHexNAc6-8Hex7-9dHex1NeuAc4.

42.5FIGURE 7. CESI-MS separationof Asn83 glycoforms (R53–K97).

37
6.5

RT: 25.4842 - 46.2705 SM: 13G
32.1103
8398.9131
1004—B

60
400Characterization of rhEPO N-linked Glycopeptides
several LysC peptides containing Asn24 and Asn38 sites in nLC-MS runs but not in
CESI-MS experiments, probably due to the nature of the capillaries used, large
glycopeptide size (>8 kDa), and relative ratio of glycan to peptide. The major peptide
was A1–K45 with sum glycan composition for both sites of HexNAc12Hex14dHex2NeuAc8(Figure 5 and Table 2). ETD spectra of this peptide were of insufficient quality for
unambiguous assignment of glycan composition for each site. For this reason, Table 2
presents the glycan composition as a sum of different glycan composition for two sites
on this peptide.

36.7532
8398.8770

80

33.1504
8690.0156

60
40
20010033.5604
8689.9668
31.7532
36.84678689.9844
8689.9619
33.21619055.1260

42.79538690.0215

80
60
40
20034.0145
9055.1074
32.54289055.1006
30

37.11889055.1748
35Time (min)Glycoform

RelativeAbundance (%)

NL:
1.40E7m/z=8689.88208690.0373 F: msMS
EPO_HEKlysC_3_50min_FTETD_x
tract

HexNAc12Hex14dHex2NeuAc8

35HexNAc13Hex15dHex2NeuAc8

18HexNAc14Hex16dHex2NeuAc817

NL:
6.24E6m/z=9055.07419055.1889 F: msMS
EPO_HEKlysC_3_50min_FTETD_x
tract

HexNAc12Hex14dHex2NeuAc7

17HexNAc6Hex7dHex1NeuAc41242.8039
8398.9209

32.92538689.9971

100

Relative Abundance

EPO alpha has three N-glycosylation sites: Asn24, Asn38, and Asn83. We detected33.1504
8398.8760

31.65158398.9170

TABLE 2. Peptide quantification of major rhEPOglycoforms for Asn 23/38 sites. Each glycoform
was calculated as a sum of all detected peptides,
including acetylated and sodiated species.

NL:
3.76E6m/z=8398.82008398.9900 F: msMS
EPO_HEKlysC_3_50min_FTETD_x
tract

80920

MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

The rhEPO samples used in the current study were not of pharmaceutical grade and,as a result, we observed not only high heterogeneity of glycoforms and oxidation
products, but also different additional proteoforms including altered primary structuresof N-termini and some other sequence variants for residues S34–K45 (Figure 1B). Using
HR/AM ETD and HCD fragmentation in combination with data analysis tools, we wereable to identify these sequence variants and additional partial glycosylation sites, but it
significantly complicated our study.

Reproducibility of CESI-MSA

B

FIGURE 8. Comparison of CHO and HEK rhEPO Asn83 N-glycoforms using theXtract deconvolution algorithm in Protein Deconvolution 3.0 software.

40

42.92030.000045

 Comparative glycoprofiling of Asn83 site for rhEPO expressed in CHO vs. HEK
using CESI-HRAM middle-down demonstrates clear differences in glycoform
abundances and validates the utility of this approach for in-depth characterizationof glycoproteins.

References
1. Haselberg, R.; de Jong, G. J.; Somsen, G. W. Anal. Chem. 2013, 85 (4), 2289–2296.
2. Balaguer, E.; Demelbauer, U.; Pelzing, M.; Sanz-Nebot, V.; Barbosa. J.; Neusüss, C.;
Electrophoresis 2006, 13, 2638–2650.
3. Moini, M. Anal. Chem. 2007, 79, 4241–4246.
4. Kolarich, D.; Jensen, P. H.; Altmann, F.; Packer, N. H. Nat. Protoc. 2012, 7 (7), 1285–1298.
5. Higgins, E. Glycoconjugate J. 2010, 27, 211–225.
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