1. Figure 3: Two hours of reaction yielded [glycopeptide+38] as main products, with
lactonization in both sialylated glycoforms, as observed before esterification of glycans 5,7,8.
As the EG2‐hFc antibody was produced in CHO cells2, 9 that do not express 2,6
sialyltransferase6, lactone formation is consistent with an 2,3 linkage.
Integration values in Table 1 clearly show that MALDI-MS of esterified species provides a
higher assessment of the sialylation content than when performed on unreacted
glycopeptides.
Esterification of non mutant and mutant Tratzumab Eg2 glycopeptides. The (*) symbol
indicates the metastable peaks in Figures 4 and 5. After ethyl esterification, neutralized
sialylated glycoforms contributed to decrease the metastable fragmentation rate. The inset
in Figure 5 (bottom) shows the difference in peak shape between adjacent regular (m/z
2997) and metastable (m/z 3049) ion peaks. The mutant samples produced more of these
metastable ions that the nonmutants, due to higher contents in sialic acid.
Upon esterification, nonsialylated glycoforms saw their masses increase by 10 (+28, ‐18).
For sialylated glycoforms, an extra addition of 28 per sialic acid residue was observed.
Figure 4 shows these results for non‐mutant TZM, and Figure 5 shows comparable results
for the mutant variant of this mAb. Table 2 highlights the relative abundance comparison of
mutant TMZ2 mAb glycopeptides before and after ethyl esterification.
Spectra of the esterified species showed two forms of detection of sialic acid. In Figures 4-5,
peaks at m/z 3326 correspond to the addition of an ethyl esterified sialic acid residue to
core-fucosylated complex type bi-antennary N-glycan (G2F), suggesting a 2,6 linkage of
sialic acid to galactose. This is expected, as these mAbs were produced in the presence of
2,6 sialyltransferase3. On the other hand, m/z 3280 peaks represent an addition of 273 to
G2F, i.e. lactonization of sialic acid, indicating a 2,3 linkage. This is also expected as the
mAbs were produced in CHO cells6.
Conclusion
 Esterification of mAb glycopeptides leads to one main product per glycoform, and
enabled to differentiate 2,3 from 2,6 sialyl linkages
 This fast and simple method is efficient for the analysis of sialylated glycopeptides, as it
neutralizes sialic acids and decrease the metastable fragmentation rate
 MALDI-MS ion abundances give a better estimate of sialylation levels for esterified that
for unreacted glycopeptides by MALDI-TOF-MS reflectron positive mode.
 Antibody esterified ion patterns are simple and predictable
References
1. J. M. Hayes et al., in M. Daeron and F. Nimmerjahn (eds.), Curr. Topics Microbiol. Immunol. 382,
2014, doi: 10.1007/978-3-319-07911-0_8, Springer Intl Publishing.
2. J. Zhang et al. Protein Expr Purif 2009, 65, 77.
3. C. Raymond, et al., MAbs 2015, 7, 571.
4. P. Lopez et al., submitted.
5. K. R. Reiding et al., Anal Chem 2014, 86, 5784.
6. M. Butler, M. Spearman, Curr Opin Biotechnol 2014, 30, 107.
7. X. Liu et al., Anal Chem 2007, 79, 3894.
8. D. J. Harvey, J Am Soc Mass Spectrom 2005, 16, 647.
9. E. Bodnar et al., Can J Chem, Epub doi: 10.1139/cjc-2015-0061.
Acknowledgements
• Members of MabNet including Dr. Mike Butler’s laboratory (Microbiology, University of
Manitoba) for providing monoclonal antibodies for analysis
• Members of the Perreault research group for their help and discussion.
Second Level Head
Body text.
2013
Overview
A fast, simple and convenient glycopeptide esterification method is demonstrated here. Monoclonal
antibody (mAb) glycopeptides isolated from tryptic digests were ethyl esterified, allowing for better
estimation of sialylation levels and for differentiation of terminal sialic acid linkages by MALDI-TOF-
MS.
Introduction
Glycosylation has become an indispensable parameter to control in the production of
biopharmaceuticals. This type of post translation modification has been demonstrated to control the
efficacy of proteins by changing solubility, folding, and receptor binding activity1. Mammalian
antibody N-glycans often exhibit terminal sialic acids that are important to cellular communication,
protein half-life1 as well as providing anti-inflammatory properties.This study presents an
ethyl‐esterification method adapted to tryptic glycopeptides isolated from mAb proteolytic digestion
mixtures. Glycopeptides were ethyl esterified using 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide
(EDC) and 1-hydroxybenzotriazole (HOBt) as activators in ethanolic solution5. This method allows
quantification of neutral esterified sialylated glycoforms and direct distinction of 2,3 and 2,6 sialic
acid linkages by MALDI-TOF-MS reflectron positive mode.
Materials
Esterification reagents 1-hydroxybenzotriazole (HOBt) hydrate and 1-ethyl-3-(3
dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma. Synthetic EEQYNSTYR
was prepared by solid phase peptide synthesis. Eg2-hFc antibody2 was obtained through the
Monoclonal Antibody NSERC Strategic Network (MabNet, www.mabnet.ca). Six sialylated
monoclonal antibody (mAb) samples were prepared for analysis also through MabNet3. Their mAb
framework was TrastuzumabTM (TMZ), corresponding to that of human IgG1. EEQFNSTYR and
EAQFNSTYR were obtained from tryptic digests of porcine IgG4.
Methods
High-performance liquid chromatography reverse phase: Isolation of glycopeptides was performed
using the following gradient: 95:5 water (H2O): acetonitrile (ACN) + 0.1% Trifluoroacetic acid (TFA)
held for 5 min, then to 90:10 H2O:ACN + 0.1%TFA over 5 min, and finally to 70:30 H2O:ACN +
0.1%TFA over 10 min. Fractions were collected manually, pooled, and evaporated to dryness.
Esterification: The method was adapted from conditions reported by Reiding et al. for free glycans5.
Briefly, 10 µg samples of tryptic mAb glycopeptides were reacted with EDC and HOBt, both 0.25 M
in ethanol (10 µL). The reaction proceeded at 37oC for 1 to 3 h depending on completion.
Esterification was stopped by the addition of an equal volume of acetonitrile. Samples were then
stored at -20oC.
Cleanup: Samples were eluted on C18 cartridges with 3 x 1 mL of H20 + 0.1%TFA, then 5 x 500 µl of
50:50 H20-ACN + 0.1% TFA.
Alkylation of synthetic EEQYNSTYR: To 20 g of the peptide, 10 L of 500 mM 2-Iodoacetamide
(IA) solution were added, and the mixture was left to react in the dark at 37oC for 3-4 days. Cleanup
of the alkylated sample was achieved using solid-phase extraction on C18 medium.
Mass Spectrometric Analysis: MALDI-MS and MS/MS analyses were performed using
UltrafleXtreme (Bruker Daltonics, Germany) in both positive and negative ion modes with 2,5-
dihydroxybenzoic acid (DHB) matrix.
Systematic Study of Glycopeptide Esterification for the Determination of Sialylation Levels in Antibodies
Andrey Oliveira1, Paul Lopez1, Rini Roy1, Céline Raymond2, Edward Bodnar1, Yves Durocher2 and Hélène Perreault1
University of Manitoba, Winnipeg, Canada1; Human Health Therapeutics Portfolio, National Research Council Canada, Montréal, Canada2
Discussion
MALDI MS has been widely used to determine the chemical structure of glycans, however the lability of
sialic acid residues has resulted in inaccurate quantification of sialylation levels. Positive mode ionization
tends to underestimate the amount of sialylated glycoforms due to their inherent negative charge(s).
Metastable ions are often observed in the reflector positive mode, but these cannot be used for quantitation
due to their distorted peak shapes. Negative mode tends to overestimate the levels of sialylation.
In order to overcome this MS limitation, neutralization of sialylated N-linked glycopeptides was achieved
using ethyl-esterification5.
SYNTHETIC EEQYNSTYR: As esterification targets carboxyl groups, the peptide was expected to have an
increment mass of 84 Da (28 x 3). Instead, an increase of 38 Da was observed for the main product, as
shown in Figure 1A (bottom spectrum) by ions at m/z 1227.7. This corresponds to the esterification of two
COOH groups in the peptide, along with the loss of a water molecule. The investigation of the esterified
product's structure was pursued by MS/MS, and Figure 1B compares the results obtained for precursor ions
at m/z 1189.5 (top) and 1227.7 (bottom). A suggested structure for the synthetic peptide after ethyl-
esterification is shown in Figure 1A. Most MS/MS matched this structure, especially those at m/z 1070.
Esterification of N-glycopeptides from porcine IgG. Figure 2 shows the comparison between N-glycosylated
glycoforms prior (on top) and after (on bottom) ethyl esterification on different peptide backbones. An
increase of 38 m/z was observed for EEQYNSTYR whereas for EAQFNSTYR the increase was of 10 m/z.
In glycosylated EEQFNSTYR, MS/MS (not shown) indicates that the mass shift correspond to (+56, -18):
addition of ethyl groups on the second glutamic acid and C-terminal arginine along with a loss of H20 from
N-terminal glutamic acid due to cyclization.
In EAQFNSTYR, the increase of 10 m/z corresponds to (+28, -10): addition of ethyl group on the C-terminal
arginine, and dehydration of N-terminal glutamic acid through cyclization. Suggested structures for both
esterified peptides are shown in Figure 2.
Esterification of N-glycopeptides from Eg2-hFc. Figure 3 shows the products of ethyl esterification reaction
on Eg2 (antibody) EEQYNSTYR glycopeptides over time. For this sample, partial lactonization of both sialic
acids had occurred prior to esterification, which indicates α2,3 binding of sialic acid to galactose, as typically
encountered in CHO cells6. After 1 h of reaction time, an increase of 10 m/z was observed for nonsialylated
glycoform products. For sialylated glycoforms lactonization took place. These species were detected at
increments of 273 m/z relative to G2F (+291, ‐18). Ions at m/z 2616.2, 2778.3, 2940.3, 3213.4 and 3486.5
correspond to [glycopeptide+28] products having lost an ethanol molecule. They appear as minor products
but could also result from in‐source fragmentation.
Glycoform FG0 FG1 FG2 FG2S FG2S2
Unreacted + 58.3 100 83 11.4 4
Esterified + 55 100 83.6 20.8 9.4
Unreacted - 54.8 100 88.4 29.4 12.7
TABLE 1: Relative abundances of ions corresponding to glycoforms of EEQYNSTYR from EG2-hFc obtained
by MALDI-MS for unreacted and esterified (2 h) samples. The experimental error is within +/- 10% of the
reported values.
TABLE 2: Relative abundances of ions corresponding to glycoforms of EEQYNSTYR mutant TMZ2 sample
analyzed by MALDI-MS. The experimental error is within +/- 10% of the reported values.
Glycoform FG0 FG1 FG2 FG2S FG2S2
Unreacted + 3.6 11.7 100 67.7 12.7
Esterified + 0 16.2 100 258 112
Unreacted - 0 18.2 100 667.2 182.2
2997.348
3049.380
3067.2453010.105
3037.104
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.[a.u.]
2990 3000 3010 3020 3030 3040 3050 3060
m/z
A B
FIGURE 1: A) Positive, reflective mode MALDI-TOF-MS spectra of synthetic peptide EEQYNSTYR.
Top: before and bottom: after esterification. B) MALDI-TOF MS/MS spectra of [M+H]+ ions of top:
synthetic peptide EEQYNSTYR and bottom: ethyl-esterified EEQYNSTYR.
FIGURE 2: Positive, reflective mode MALDI-TOF-MS spectra of complex glycoforms of EEQFNSTYR and
EAQFNSTYR porcine IgG4. Top: unreacted glycopeptides and bottom: esterified glycopeptides.
FIGURE 3: Positive, reflective mode MALDI-TOFMS spectra of lightly sialylated glycoforms of EEQYNSTYR
from EG2-hfc obtained for top: unreacted glycopeptides and bottom 3: esterified glycopeptides.
FIGURE 4: Reflective more MALDI-TOFMS spectra of non-mutant TMZ2 mAb EEQYNSTYR glycoforms. Top:
positive mode, unreacted; middle: positive mode, esterified; bottom: negative mode, unreacted.
FIGURE 5: Reflective mode MALDI-TOFMS spectra of mutant TMZ2 mAb EEQYNSTYR glycoforms. Top:
positive mode, unreacted; middle: positive mode, esterified; bottom: negative mode, unreacted. Inset:
comparison of peak shapes for regular (left) and metastable (right) ions generated by the in-flight loss of sialic
acid.