Drug discovery and development

Comprehensive Analysis of Recombinant Human
Erythropoietin Glycoforms by Capillary Electrophoresis
and Nanoflow Liquid Chromatography Coupledwith Intact/Middle-Down Mass Spectrometry
Sensitive and high-resolution glycoform profiling of intact rhEPO using CESI-MS
with the Neutral OptiMS cartridge
M. Santos,1 R. Viner,2 D. Horn,2 J. Saba,2 M. Bern,2 D. Bush,4 H. Dewald,1 A.A.M. Heemskerk,1B.L. Karger3 and A.R. Ivanov3
1 SCIEX, Brea, CA
2 Thermo Fisher Scientific, San Jose, CA
3 Northeastern University, Boston, MA
4 Genedata, Lexington, MA

Introduction
Erythropoietin (EPO) is a naturally occurring red blood cellstimulating hormone produced in the kidney, and it was oneof the first therapeutic recombinant proteins. EPO is a heavily
glycosylated protein with a molecular weight of approximately
30 kDa, and 40% of its weight is due to glycosylation.1 It
contains 3 N-glycosylation sites on Asn24, Asn38 and Asn83,
and 1 O-glycosylation site on Ser126, which makes glycoform
profiling challenging due to the high heterogeneity (Figure 1).
Recombinant human EPO (rhEPO) has been studied extensively
using different approaches, including capillary electrophoresisand nanoflow liquid chromatography coupled to advanced
mass spectrometry (MS) detection.2-3 However, these analytical
approaches frequently involve some type of enzymatic digestion,
which can induce artifacts. Therefore, the characterizationof these biomolecules under intact conditions is important.
Capillary electrophoresis is a well-established analytical tool forthe analysis of intact native proteins. The recent developmentof CESI, which integrates capillary electrophoresis (CE) andFigure 1. Erythropoietin amino acid sequence and glycosylation sites.

electrospray ionization (ESI) into a single process within theMaterials and methods

same device, enabled the characterization of the glycosylation ofSample preparation

intact EPO at pH2. In this work, we show that by using CESI-MS
1

and a neutral coated capillary coupled to high-resolution, highmass accuracy mass spectrometry, we were able to identify andquantify multiple glycoforms of rhEPO under native conditions.
Additionally, we used a middle-down approach to assess
site-specific N- and O-glycosylation.

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 °C in 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:protein ratio of 1:50, for 1 hrat 37 °C in 20 mM ammonium acetate, pH 6.0). All enzymes
were from Roche, IN.
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Drug discovery and development
Liquid chromatography
The rhEPO tryptic or LysC digests were separated using theThermo Scientific EASY-nLC 1000 HPLC system with a Magic
C18 spray tip 20 cm x 75 μm I.D. column (Michrom). Gradientelution was performed from 4–30% over 60 min and from30–85% over 10 min with ACN in 0.1% formic acid at a flowrate of 300 nL/min.

CESI-MS conditions:Intact rhEPO was separated using a CESI 8000 Plus High
Performance Separation-ESI Module (SCIEX) equipped witha Neutral OptiMS cartridge consisting of a porous sprayer
operating in an ultra-low flow regime, and detected using an
Exactive Plus EMR (Thermo Fisher Scientific) at 35 K or
140 K FWHM resolution at m/z 200. The middle-down
experiments were carried out using the OptiMS Silica Surface
cartridge on a Thermo Scientific Orbitrap Elite mass spectrometer
and Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer

Figure 2. Base peak electropherograms for three constitutive runs ofrhEPO LysC digest (200 ng) and MS2 XIC for HexNac oxonium peak.

using FT/IT HCD, CID or ETD MS2 fragmentations in DDDTor HCDpdETD/CID methods. FT MS1 was acquired at
resolution settings of 60–120 K at m/z 200 and FTMS2
at resolution of 30–60 K at m/z 200.

good reproducibility with less than 10% RSD across runs
(Figure 2). Glycopeptides were well separated by CESI and
resolved within 20 min of a 50 min long run (Figure 2).

Important:Characterization of O-glycoforms• A separation current above 5 μA might cause permanent
damage to the separation capillary.

Limited LysC digest yielded peptides of different lengths• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

range of 3–9 kDa (Figure 3). All O-linked glycopeptides canData analysis

containing Ser126 (where the O-glycan site is located) in a
only be identified in the FT ETD experiments because theO-linked glycans are very labile and do not survive collisionalactivation (Figure 3). As expected, CESI glycoform separation

The Thermo Scientific ProSightPC 3.0, Protein Deconvolution

was mostly based on differences in the number of sialic acid

3.0, Pinpoint 1.4, and Proteome Discoverer 2.0 software with theresidues (Figure 4). The 2 predominant O-glycosylated peptides

Byonic search node (Protein Metrics) were used for glycopeptide(N-acetylhexosamine-hexose with 1 or 2 sialic acids) migrated

data analysis and glycoform quantification. SimGlycan 4.5

as baseline resolved peaks in CESI but not in nLC (Figure 4A vs.

software (PREMIER Biosoft) was used for proteinase K digest

Figure 4B) using similar analysis times. The relative abundances

glycopeptide and glycan composition identification.

of major rhEPO O-glycoforms are shown in Table 1. We detected
several unmodified Ser126 peptides (total relative abundance

Results9%, Table 1), which means that the site was only partially

Reproducibility of middle-down analysis

O-glycosylation on Ser9 and Ser120 residues.

glycosylated in this sample. Additionally, we observed partial

CESI-MS is a sensitive and reproducible analytical technique

Looking at the N-glycosylation of EPO, we found the mostemployed for the separation of glycopeptides due to differences

abundant glycopeptide containing Asn24 and Asn38 wasin charge states and Stokes radii.4 We were able to identify andA1–K45 with sum glycan composition for both sites ofquantify multiple EPO glycopeptides using 200 ng of sample with

HexNAc12Hex14dHex2NeuAc8 (Figure 5 and Table 2). We wereexcellent S/N. Migration times and peak areas demonstrated

not able to unambiguously assign the glycan composition forp2

Drug discovery and development
each individual site. For this reason, Table 2 presents the glycan
composition as a sum of different glycan composition for bothsites on this particularly large peptide. The most abundant
glycopeptide containing the Asn83 site was R53–K97 (Figure6). It is worth noting that the difference of a single sialic acidcauses a small migration time shift (~0.4 min) due to a change
in mobility of the peptide as a result of a change in the overall
negative charge. To a lesser extent, differences in the number of
HexHexNAc residues also affect the mobility due to a possible
change in the Stokes radii, but not on the overall charge ofFigure 5. Identification of rhEPO LysC double N-linked glycopeptide(A1–K45) by Byonic node in Proteome Discoverer 2.0 software.

the peptide as this species is neutral (Figure 7). As expected,5
the main glycan compositions for Asn83 site were tetra-acidic

Glycoform

oligosaccharides (Figure 8).

Relative abundance (%)
42.5
37
6.54—9Table 1. Peptide quantification of major rhEPO glycoforms for Ser126
site. Each glycoform was calculated as a sum of all detected peptides.

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

Glycoform

Relative abundance (%)

HexNAc12Hex14dHex2NeuAc835HexNAc13Hex15dHex2NeuAc818HexNAc14Hex16dHex2NeuAc817HexNAc12Hex14dHex2NeuAc717HexNAc6Hex7dHex1NeuAc412Table 2. Peptide quantification of major rhEPO glycoforms for Asn23
and Asn28 sites. Each glycoform was calculated as a sum of all
detected peptides, including acetylated and sodiated species.

We also performed a comparative glycopeptide profiling of
this site for rhEPO expressed in both CHO and HEK cells(Figure 8). It has been reported that oligosaccharide structuralfeatures of recombinant proteins are specific to cell line, culture
condition and species.6 Assuming equal detection response
for all glycoforms, CESI-MS analysis demonstrated significant
differences in relative abundance of glycoforms expressedin 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 by HexNacHex
units were dominant.
Figure 4. CESI-MS (A) and nLC-MS (B) separation of O-linked
E114–K150 glycopeptide.
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Characterization of rhEPO undernative conditions.
For the analysis of rhEPO under native conditions, we useda neutral coated capillary, which prevented the protein fromsticking to the capillary surface, enabling a better separation ofthe isoforms. The CESI 8000 Plus System was coupled to an
Exactive Plus EMR system at different resolution settings. Figure
9A shows a separation of EPO using 40 mM ammonium acetate(pH 7.5) as the background electrolyte and a corresponding ion
density map on Figure 9B. The analysis of the deconvolutedspectra (Figure 10B) of each peak (Figure10A) revealed thepresence of 167 proteoforms at a resolution of 140 K (at m/z200) and 428 glycoforms at a resolution of 35 K (at m/z 200)with only 2.25 ng of sample injected in both cases, with the main
form being HexNAc19Hex22dHex3NeuAc13 migrating in peak 4 ata resolution of 35 K. These results also show that the different
glycoforms of rhEPO separate mostly based on differences in thenumber of sialic acid residues (Figure 10) correlating well withresults from the middle-down approach (Table 3). Due to the highheterogeneity, the separation is not baseline resolved. However,in combination with HRAM-MS, the CESI-MS of native rhEPO
still provided better dynamic range and more accuratequantification than direct infusion.

Figure 7. CESI-MS separation of Asn83 glycoforms (R53–K97).

Figure 8. Comparison of CHO and HEK rhEPO Asn83 N-glycoforms
using the Xtract deconvolution algorithm in Protein Deconvolution
3.0 software.
Figure 6. Identification of rhEPO LysC N-linked glycopeptide (R53–K97) byByonic node in Proteome Discoverer 2.0 software using HCD (A) or ETD(B) fragmentation.

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Drug discovery and development

Figure 10. EPO profiling under native conditions, pH 7.5 (A) andcorresponding deconvoluted spectra (B) at 140 K @ m/z 200 resolution.

Figure 9. Base ion electropherogram of EPO under native conditions(A) and corresponding ion density map (B).

EPO

Relative
abund- Ser 126
ance,%

Relative
abund- Asn 23/38
ance,%

Relative
abund- Asn 83
ance,%

Relative
abundance,%

HexNAc19Hex22dHex3NeuAc13

44.79

HexNAcHexNeuAc

42.5

HexNAc12Hex14dHex2NeuAc835HexNAc6Hex7dHex1NeuAc446.7

HexNAc19Hex22dHex3NeuAc1418HexNAcHexNeuAc237HexNAc13Hex15dHex2NeuAc818HexNAc7Hex8dHex1NeuAc428HexNAc19Hex22dHex3NeuAc1217HexNAc2NeuAc

6.5

HexNAc12Hex14dHex2NeuAc717HexNAc6Hex7dHex1NeuAc3

14HexNAc19Hex22dHex3NeuAc1017HexNAc2Hex4HexNAc13Hex15dHex2NeuAc817HexNAc5Hex7dHex1NeuAc3

11.6

HexNAc6Hex7dHex1NeuAc412—9HexNAc6Hex7dHex1NeuAc412HexNAc6Hex7dHex1NeuAc34.7

Table 3. Relative quantification of major hEPO glycoforms using intact and middle-down approaches. Each glycoform was calculated as a sum of all
detected, including acetylated and sodiated species.
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Drug discovery and development
Conclusions

References• The CESI-MS technique is reproducible (RSD <10%) andsensitive, obtaining the same sequence coverage and numberof glycopeptides with 5 times lower amount of sample than for
nLC-MS experiments.

1. Haselberg, R.; de Jong, G.J.; Somsen, G.W. Anal. Chem.
2013, 85 (4), 2289–2296.

• CESI separation of glycoforms is clearly based on differencesin the number of sialic acid residues (that is, difference incharge) and the peptide to glycan mass ratios.
• The primary O-linked glycoforms of CHO rhEPO are
HexNacHex+1(2)NeuAc with the relative abundance of
unglycosylated Ser126 of approximately 9%.
• Comparative glycoprofiling of Asn83 site for rhEPO
expressed in CHO vs. HEK using CESI-HRAM middle-down
demonstrates clear differences in glycoform abundancesand validates the utility of this approach for in-depthcharacterization of glycoproteins.

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.
6. Rush, R.S.; Derby, P.L.; Smith, D.M.; Merry, C.; Rogers, G.;
Rohde, M.F.; Katta, V. Anal. Chem. 1995, 67, 1442−1452.

• Comparative glycoprofiling of rhEPO using CESI-HRAM
middle-down and intact strategies demonstrates remarkablecorrelation in glycoform abundances and validates theutility of this combined approach for in-depth characterizationof glycoproteins with multiple glycosites.

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