Drug discovery and development

Sensitive Glycoform Profiling of Interferon-beta-1a (Avonex)
and Recombinant Human Erythropoietin by CESI-TOF-MS
Sensitive and high resolution glycoform profiling of intact rhEPO and Interferon (rhIFN-β) using
CESI-MS with the Neutral OptiMS cartridge
Rob Haselberg,1 Gerhardus J. de Jong,2 and Govert W. Somsen1
1 Division of BioAnalytical Chemistry, VU University, The Netherlands
2 BioMolecular Analysis, Utrecht University, The Netherlands

Introduction
Today, 130 protein-based therapeutics are licensed. Withthe addition of anticipated new biotherapeutics, the market
is forecasted to exceed $140 billion USD in 2017. These
therapeutic drugs have changed and improved the qualityof life of millions of people around the world in the past few

coupled to a time-of-flight mass spectrometer (TOF-MS),presents sensitive high-resolution glycan profiling throughreduced ion suppression at nanoliter per minute flow ratesof 2 important biopharmaceutical molecules in intact mode:
recombinant human interferon-β-1a (rhIFN-β, Avonex) andrecombinant human erythropoietin (rhEPO).1decades. One of the most important quality attributes of aThe CESI-TOF-MS data revealed much improved results overbiopharmaceutical drug is its heterogeneity, which includes but

traditional sheath liquid CE-MS by enabling the identification

is not limited to glycosylation patterns. Glycosylation may vary inof 18 glycoform species, as well as deamidation, succinimide

degree and type, and is dependent on parameters such as celland oxidation products for rhIFN-β. The analysis of intact

line, host organism and cell culture conditions. During production,rhEPO using CESI-TOF-MS allowed for the identification ofthese variables can cause a broad spectrum of possible glycan

74 glycoforms in 60 min. In addition, oxidation and acetylation

species. Therefore, the comprehensive analytical characterization

products were identified, adding up to more than 250 differentof glycosylation heterogeneity is extremely important to ensureisoforms being detected.

the safety and efficacy of a biopharmaceutical drug.
Various analytical techniques (i.e., electrophoresis in either

Materials and methods

gel or capillary format as well as liquid chromatography) are

Chemicals: Acetic acid (99.8%) and ammonium hydroxide,

widely used in the structural characterization of glycans. Glycan
heterogeneity can be accessed by glycopeptide mapping,as well as released and labeled glycan analysis by capillaryelectrophoresis (CE) or liquid chromatography (LC). Even thoughthese are invaluable tools in the analysis of glycosylation, they all
involve some level of sample processing by enzymatic digestion.
Hence, it is very desirable to access glycosylation patterns withminimal to no sample manipulation to avoid sample preparation
artifacts in the analysis. This leads to intact protein analysis,
which will require not only a high-resolution separation technique(i.e., CE or LC), but also a suitable detection system.
In recent years, mass spectrometry has gained terrain as thedetection mode of choice due to its soft ionization mode (ESI)for sample introduction and mass-selective detection with highresolution and high mass accuracy.
In this technical note, the integration of capillary electrophoresis(CE) and electrospray ionization (ESI) into one process (CESI),

recombinant human interferon-β-1a (rhIFN-β, Avonex 6,000,000
IU, lot no. 060016A) from Biogen (Cambridge, MA) andrecombinant human erythropoietin (rhEPO, NeoRecormon
30,000 IU, lot no. H0002H01) from Roche (Mannheim, Germany)
were obtained as prefilled syringes. Insulin (from bovinepancreas), carbonic anhydrase II (from bovine erythrocytes),ribonuclease A (from bovine pancreas) and lysozyme (fromchicken egg white) were from Sigma-Aldrich (Steinheim,Germany). Protein test mixtures were prepared by diluting protein
stock solutions (1 mg/mL) to the appropriate concentration withdeionized water.
CE system: CE experiments were carried out using a CESI 8000
Plus High Performance Separation-ESI Module from SCIEX(Brea, CA). Unless otherwise stated, the applied separation
voltage was 30 kV with a supplemental forward pressure of0.5 psi. The capillary temperature was kept at 20 °C. The
capillary used in this study was the OptiMS cartridge withneutral surface (SCIEX, Brea, CA).
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Drug discovery and development
Prior to use, the capillary was rinsed with deionized water for

Under these electrophoretic conditions, the peak shape of all

30 min at 50 psi and stored overnight filled with water. After use,

4 proteins were narrow, symmetrical and baseline resolved

the capillary was rinsed for 10 min with deionized water andwith theoretical plates between 80,000 and 130,000, indicating

air at 50 psi, respectively, and subsequently stored at +4 °C.

excellent separation conditions. The neutral coated capillary

Before each run, the capillary was flushed with fresh backgroundenabled the increase of separation resolution by a factor of 2 andelectrolyte (BGE) for 3 min at 50 psi. BGEs of acetic acid wereincreased the migration time window by 4 times compared to aprepared by diluting the appropriate amount of glacial acetic acidpositively coated capillary.2 The quality of the spectra was good,

to 30 mL with deionized water and adjusting the pH with 0.1 M

which allowed for the correct molecular mass assignment after

ammonium hydroxide (50 mM acetic acid, pH 3.0). The sample

deconvolution.

was injected for 10 s at 5 psi equaling 1% of the capillary volume.
Mass spectrometry: CESI-MS experiments were performed
using a micrOTOF orthogonal-acceleration time-of-flight (TOF)
mass spectrometer (Bruker Daltonics, Bremen, Germany)

Glycoform Profiling of Intact PharmaceuticalProteins:
Recombinant human interferon-β-1a (rhIFN-β)

equipped with a nanospray endplate and gas diverter to allow

For over 20 years, the FDA-approved, disease-modifying agent

nano electrospray ionization (nanoESI). The mass accuracy

rhIFN-β-1a (Avonex) has been used for the treatment of relapsingwas <5 ppm within a single day of measurements, and theforms of multiple sclerosis (MS) and to slow the physicalresolution was about 20,000 in the range above m/z 1250, as

disability suffered by those affected by MS.3 This molecule iswas determined experimentally by infusion of the ESI tuning mix(Agilent Technologies, Waldbronn, Germany). Optimized spray
conditions for low-flow infusion and CESI-MS experiments wereas follows: dry gas temperature, 180 °C; dry gas nitrogen flow,
1.2 L/min; nebulizer pressure, 0.0 bar. Electrospray in positive
ionization mode was achieved using an ESI voltage of 0.9 kV.

Important:• A separation current above 5 μA might cause permanent damage to the separation capillary.
• Generally, please do not apply >2000V to generate electrospray as it may result in capillary damage.

Results
Assessing the analytical performance of thelow-flow CESI-MS system for analysis of
intact proteins
The neutral coated capillary is very attractive for the analysis of
intact proteins because the coating acts as a barrier preventing
the interaction with the surface. The neutral coating also keeps
the electroosmotic flow (EOF) to a minimum. The EOF is criticalfor a stable spray, thus a supplemental pressure of 0.5 psicorresponding to a 5 μL/min was applied along with the voltage
during the separations. To evaluate the analytical performance ofthe neutral coated capillary, a mixture of lysozyme, ribonucleaseA, carbonic anhydrase II and insulin (5μg/mL each) was chosen.
This mixture was analyzed using 50 mM acetic acid (pH 3.0)
as the BGE.

Figure 1. (A) BPE obtained with CESI-MS of rhIFN-β (45 μg/mL) employing a neutrally coated capillary. (B) Deconvoluted mass spectra obtainedin the apexes of the peaks migrating at 30.1 min (*) and 30.5 min (**).
(C) EIEs for 10 selected rhIFN-β glycoforms, including their deamidated
products at the indicated m/z value (±0.3 Da; 11+ charge state). Assigned
glycan structures are shown as insets. Symbols: green circle/yellow
circle, hexose (mannose/ galactose); red triangle, fucose; blue square,
N-acetylhexosamine; purple diamond, sialic acid. Conditions: BGE, 50
mM acetic acid (pH 3.0); CE voltage, 15 kV; tip-to-endplate distance,
1.0 mm. Further conditions: see the materials and methods section.
From Haselberg et al. (Anal. Chem. 2013, 85, 2289–2296), reproducedwith permission.*p2Drug discovery and developmentapproximately a 23 kDa glycoprotein with the glycosylation siteof high-resolution glycan separation only obtained with a neutral

at the Asn80. As described in the introduction, it is expected thatcoated surface and the high sensitivity of the nanoflow regime

variation in glycosylation patterns of rhIFN-β-1a may affect theoffered by CESI-MS and high-resolution mass spectrometry.

biological activity, requiring great scrutiny from quality control
of produced batches.4
The analysis by CESI-MS of rhIFN-β revealed a uniqueseparation profile consisting of multiple peaks nearly baseline
resolved (Figure 1A). The deconvoluted mass spectra wereconstructed across the peak profile at 12 s intervals. The mass
with highest intensity (22,375.4 Da) corresponds to the molecularmass of rhIFN-β with a fucosylated disialylated biantennary

Recombinant human erythropoietin (rhEPO)
EPO is a hormone essential to red blood cell production. rhEPO
is an approximately 30 kDa glycoprotein where up to 40% ofits molecular weight is due to glycosylation.6 EPO contains
3 N-glycosylation sites on Asn24, Asn38 and Asn83, and1 O-glycosylation site on Ser126. EPO is well known for its highheterogeneity, making the glycoform profiling very challenging.

glycan structure comprising 4 hexose and 5 N-acetylhexosamine

Figure 2A shows the base peak electropherogram (BPE) ofunits. This glycoform has been described as the most abundantrhEPO, obtained using CESI-MS with a neutral coated capillary.

present in rhIFN-β3. Another peak at 22,358.4 Da was observed

The separation profile spans over a 20 min window with a highand assigned to a succinimide intermediate, which is formedsignal-to-noise ratio. The deconvoluted spectrum in Figure 2B

during deamidation. The deamidated form with a mass ofcorresponds to the peak at 38 min with a main mass of 29,597

22,376.4 Da migrates at 30.5 mi. It is worth noting that while theDa. Minor peaks corresponding to oxidation and acetylation

deamidated variant carries a mass increase of only 1 Da, it also

products were also observed.

increases the overall negative charge, resulting in an increasein mobility, which is why the deamidated form is observed at30.5 min, 0.4 min after the parent peak. Also observed wereminor peaks of +16 Da relative to the main peak, which after
deconvolution corresponds to a mass of 22,391.1 Da, assigned
to an oxidation product. Other interferon standard samples wereinvestigated under these same conditions and no oxidation wasfound, indicating that the oxidation is likely to be present in thesample and not an artifact of ESI.
From deconvoluted spectra, 18 different glycoform masses wereextracted and are listed in Table 1. Deamidation, oxidation andsuccinimide products of these glycoforms were also observed,
giving an overall estimate of 80 different isoforms of rhINF-βdetected in a single run. Figure 1C shows 10 of the glycoforms
observed with corresponding deamidated forms. The resolutionof glycoforms was mainly due to the differences in sialylation andto a lesser extent in number of HexHexNAc residues. It is worth
noting that in previous results from direct infusion ESI-MS,1 only

The deconvoluted spectra were obtained every 12 s acrossthe entire peak profile, and the masses of the most intense
protein peaks can be found in Table 2. Overall, 74 distinct
glycoforms were detected, varying in sialic acid (SiA) andhexose N-acetyl-hexosamine (HexHexNAc) residues. Almost
all glycans were detected with corresponding oxidized andacetylated variants, which when combined, resulted in morethan 250 isoforms detected.7
Taking into account the glycans observed, the contour map from
Figure 2C reveals the powerful glycan separation obtained. Take
the glycoforms with a charge state of +14 as an example (clearhighlighted area). In this figure, it is clear that the difference ofa single sialic acid results in a shift of 2 min in migration time
(Figure 2C2). Now, if one considers the group of sialoforms,
the neutral HexHexNAc residue contributes to a difference of
0.5 min, even though these species are not charged(Figure 2C3).

6 glycoforms were observed. Additionally, with sheath-liquid

Table 2 displays the glycoforms detected and their relative

CE-MS, only 10 glycoforms were observed using a sample

intensity, assuming equal detection response. The relative

10 times more concentrated.5 From the extracted-ionintensity spans more than 3 orders of magnitude between

electropherograms (EIEs), peak areas for all glycoforms could be

the most abundant SiA13Hex22HexNAc13Fuc3 to the least

derived (Table 1). Based on these peak areas and the injected

abundant SiA14Hex32HexNAc29Fuc3. Applying the relative

amount, glycoform concentrations were estimated to be between

intensities of the glycoforms detected to the injected sample

0.5 and 1000 nM, demonstrating the excellent sensitivity andamount, a glycosylation concentration can be estimated ranging

dynamic range of the system.

from 0.35 to 630 nM, once again illustrating the superiorThis work clearly demonstrated that a much improved glycan
coverage of intact rhINF-β can be achieved with the combination

sensitivity of CESI-MS. No non-glycosylated species were
found during these analyses.
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Drug discovery and development

Figure 2. CESI-MS of rhEPO (200 μg/mL) employing a neutrally coatedcapillary. (A) BPE; (B) deconvoluted mass spectrum obtained in the apexof the peak migrating at 38.0 min; (C1) contour plot with zooms of (C2)
the 14+ charge state of the glycoforms and (C3) the SiA13 sialoformsof the 14+ glycoforms. Conditions: CE voltage, 30 kV; BGE, 2 M acetic
acid (pH 2.1). For further conditions, see Figure 1. From Haselberg et al.
(Anal. Chem. 2013, 85, 2289–2296), reproduced with permission.*

Table 1. Symbols: green circle/yellow circle, hexose (mannose/galactose); red triangle, fucose; blue square, N-acetylhexosamine;
purple diamond, sialic acid. From Haselberg et al. (Anal. Chem.
2013, 85, 2289–2296), reproduced with permission.*
p4Drug discovery and development

Table 1. Glycan composition and molecular mass of the rhEPO glycoforms observed with CESI-MS employing a neutrally coated capillary. The relative
intensity of the different glycoforms is indicated by the color intensity (log scale). From Haselberg et al. (Anal. Chem. 2013, 85, 2289–2296), reproducedwith permission.*p5

Drug discovery and development
Conclusions

References

In this technical note, the glycosylation profiling of 2 intact
proteins of therapeutic interest was demonstrated thanks to thecombination of 3 powerful analytical tools. The high-resolution
separation was accomplished thanks to a neutral coated capillary
combined with the high sensitivity provided by the nanoflow
regime of CESI-MS and high-resolution mass spectrometry.

1. Haselberg, R.; Jong, G.J.; Somsem, G.W. Anal. Chem. 2013,85, 2289–2296.
2. Haselberg, R.; Ratnayake, C.K.; de Jong, G.J.; Somsen, G. W.
J.Chromatogr. A 2010, 1217, 7605−7611.
3. http://www.avonex.com/.
4. Orrù, S.; Amoresano, A.; Siciliano, R.; Napoleoni, R.;
Finocchiaro, O.; Datola, A.; De Luca, E.; Sirna, A.; Pucci, P.
Biol. Chem. 2000, 381, 7−17.
5. Haselberg, R.; Brinks, V.; Hawe, A.; De Jong, G.J.; Somsen,G.W. Anal. Bioanal. Chem. 2011, 400, 295−303.7.
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.
7. Balaguer, E.; Demelbauer, U.; Pelzing, M.; Sanz-Nebot,V.; Barbosa, J.; Neusüß, C. Electrophoresis 2006, 27,2638−2650.

* This article is based on results previously published byHaselberg et al. (Anal. Chem. 2013, 85, 2289–2296),
used with permission.

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