Application Note
Biotherapeutics andBiosimilars

A Comprehensive Approach for
Monoclonal Antibody N-linked Glycan
Analysis from Sample Preparation toData AnalysisAuthors

Introduction

David L. Wong, Oscar Potter,Jordy Hsiao, and Te-Wei Chu
Agilent Technologies, Inc.
Santa Clara, CA, USA

Monoclonal antibodies (mAbs) and their derivatives comprise a very importantclass of biopharmaceutical molecules with a wide range of applications. Due to theheterogeneous nature of these mAbs, comprehensive analytical characterization isrequired. These analyses include determining the complete amino acid sequencesof the mAbs and their variants, as well as characterization of post-translational
modifications (PTMs) including glycosylation, oxidation, and deamidation.
Glycosylation plays an important role in many biological processes. It also affects
the therapeutics’ efficacy, stability, pharmacokinetics, and immunogenicity1.
Glycan characterization usually involves techniques such as NMR, HPLC, or mass
spectrometry (MS). Since glycans are very diverse in composition/structures and arepoorly ionized by electrospray, the MS-based approach for glycan characterization
has been challenging. InstantPC is a novel fluorescence tag from ProZyme Inc.
(Figure 1) that has been developed to improve MS ionization efficiency, andsensitivity for N-glycan molecules.
OOInstantPCNOONOO

NH

Glycan -NH2OGlycanOO
NHNH

NO+NO

Figure 1. Diagram of InstantPC-labeled N-glycans
released from an mAb.

OH

The traditional method of glycan analysis is laborious, andinvolves many steps, starting with enzymatic glycan release
by PNGaseF (overnight), followed by sample cleanup,labeling with a fluorescence tag by reductive amination (2-ABor InstantPC), and finally cleanup of the released labeled
N-glycans prior to LC-FLD or LC/MS analysis2,3. Despite thesignificant improvement of MS sensitivity using fluorescenttags, the labor intensiveness of manual sample preparation,
low reproducibility, and limitation to scale‑up on sample
processing have been major issues for the biopharmaceuticalindustry.

Experimental

This study demonstrates how to increase sample
throughput for glycan characterization workflows using
the Agilent AssayMAP Bravo liquid handling platform. Thesolution incorporates the Agilent 1290 Infinity II LC system,
Agilent AdvanceBio Glycan Mapping column, Agilent
highly sensitive fluorescence detection (FLD), and theAgilent 6545XT AdvanceBio LC/Q-TOF. The Q-TOF data are
analyzed automatically with Agilent MassHunter BioConfirm
B.09.00 software (Figure 2). This solution dramatically
improves productivity by allowing convenient sample
preparation, streamlined data acquisition, and data analysis.
This solution provides the flexibility to perform quantitation
based on FLD or MS signals with accurate mass peakassignment from an N-glycan mass database.

Sample prep

Separation

Agilent AssayMAP Bravo

Agilent 1290
Infinity II LC

GlykoPrep Rapid N-Glycan
preparation with InstantPC

Agilent AdvanceBio
Glycan Mapping column

Figure 2. mAb Glycan characterization workflow.

2Detection

Agilent 6545XT
AdvanceBio LC/Q-TOF

Sample preparation
Four monoclonal antibody (mAb) samples were used in thisstudy:•The monoclonal antibody standard, RM 8671, was from
National Institute of Standards & Technology (NIST)
A.K.A. NISTmAb.

•Formulated Herceptin (Trastuzumab) was from
Genentech (So. San Francisco, California, USA).

•Sigma SiLu mAb was purchased from Sigma-Aldrich(SiLu Lite, P/N: MSQC4).

•CHO mAb1 was expressed and purified from theAgilent R&D lab.

All mAb samples were diluted with DI water to 1.0 µg/µL
prior to sample preparation using the AssayMAP Bravo liquidhandling system (G5542A) with the GlykoPrep-plus Rapid
N-Glycan Sample Preparation with InstantPC (96‑ct) fromProZyme Inc. A detailed procedure for the sample preparation
is described in ProZyme’s application note (productcode: GPPNG-PC). After the final cleanup step, the eluted,
released, labeled N-glycans had a final volume of 50 µL, sothat each 1 µL of the prepared sample contained N-glycans
from 1 µg of mAb.

Data processing
and report

Agilent MassHunter
BioConfirm
B.09.00 Software

LC/MS analysis
LC/MS analyses were conducted on a 1290 Infinity II LC
system equipped with an Agilent 1260 Infinity Fluorescence
Detector (G1321B), coupled with a 6545XT AdvanceBio
LC/Q-TOF system with a Dual Agilent Jet Stream source.
The detector was set to λEx = 285 nm, λEm = 345 nm, with
PMT gain = 10. Glycans were chromatographically separatedwith an AdvanceBio Glycan Mapping column (2.1 × 100 mm,1.8 µm). Tables 1 and 2 list the LC/MS parameters used.
Approximately 1–2 µL of each N-glycan sample were injectedfor LC/MS analysis.

Data processing
The InstantPC-labeled released N-glycans were analyzed
using the Released Glycans Workflow of MassHunter
BioConfirm B.09.00 software. This analytical workflow uses
the Agilent Personal Compound Database (PCD) glycan
database. The PCD glycan database provides accurate glycan
identification and confirmation. Finally, a summarized reportof the analyses was generated in PDF format using theReport Builder program in BioConfirm B.09.00.

Table 1. Liquid chromatography parameters.
Agilent 1290 Infinity II LC SystemColumn

Agilent AdvanceBio Glycan Mapping,
2.1 × 100 mm, 1.8 µm

Thermostat

4 °C

Solvent A

50 mM formic acid adjusted to pH 4.5 with ammoniumhydroxide

Solvent B

Acetonitrile

Gradient

0–0.5 minutes, 75–71 %B
0.5–16 minutes, 71–67.5 %B
1–22 minutes, 67.5–60 %B
22–22.5 minutes, 60–40 %B
22.5–23.5 minutes, 40 %B (0.7 mL/min)
23.5–24 minutes, 40–75 %B (0.7 mL/min)
24–30 minutes, 75 %B (0.9 mL/min)

Column temperature

40 °C

Flow rate

0.4 mL/min

Injection volume

2.0 µL

Agilent 1260 Infinity Fluorescence Detector (G1321B) was used. The detector
was set to λEx = 285 nm, λEm = 345 nm, with PMT gain = 10.

Table 2. MS Acquisition parameters.
Agilent 6545XT AdvanceBio LC/Q-TOF system
Gas temperature

150 °C

Drying gas

9 L/min

Nebulizer (psig)

35

Sheath gas temperature

300 °C

Sheath gas flow

10 L/min

VCap

3,000 VNozzle voltage

500 VFragmentor

120 V

Skimmer65 VQuad AMU

95

Acquisition modeLow mass range, HiRes (4 GHz)

Mass range

m/z 300–1,700

Acquisition rate

2 spectra/sec

3

Results and Discussion

Figure 3 shows the representative chromatograms ofN-glycans (FLD and MS EIC) from the NISTmAb. The FLD
chromatogram (Figure 3 top, zoom in) reveals that more than
15 glycan peaks were detected. The glycosylation pattern ofthe major abundant glycans, such as the G0F, G1F isoforms,
and G2F was comparable between the fluorescent and MS
data (Figure 7).

LC-FLD analysis of released labeled glycans is one of themost widely used approaches to determining therapeuticprotein glycosylation. We have previously publishedapplication notes showing optimized separation of several
mAb glycan profiles using various column dimensions
and run conditions4,5. The separation method in this report
represents the best overall performance with maximum peak
resolution and excellent robustness for the different mAb
N-glycan samples in this study.

×102
1.1

G0F
FLD

7
6

0.9

5

Response

0.8

Response

1.0

G1F

0.7
0.5

10.4

45

H5N4F2

6789Acquisition time (min)

0×101

G0F
Counts (%)

EIC

1.2Counts (%)

H6N4F1
H5N4Sg1

H4N3S1
H4N5F1

101112G2F

0.1

1.4

H4N4F1Sg1

H3N2F1

3G1F’

0.2

H4N3F1G2F

G1
Man532G1F G1F’

G0F

40.6

0.3

G0Zoom

G1F

1.0
0.8

G1F’

0.6

2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2

G0ZoomG1F G1F’

G0F

H3N2F1

4

H6N4F1
H5N4Sg1

H4N3S1
H4N5F1
H5N4F256789
Acquisition time (min)G2F0.4

H4N4F1Sg1

H4N3F1G1Man53G2F

1011120.2012345678910111213 14 15 16 17Acquisition time (min)1819202122

Figure 3. FLD chromatogram and mass spectra (EIC) of InstantPC-labeled N-glycans from NISTmAb.

4232425

26

272829While fluorescence detection does not allow for direct
structure elucidation, MS analysis of mAb glycans can beused to determine glycan monosaccharide composition.
In the case of many mAb N-glycans, this composition issufficient to achieve a high-confidence structural assignment.
The combination of the positively charged InstantPCtag and sensitive Agilent Jet Stream (AJS) electrosprayionization (ESI) source technology dramatically increases
MS detection sensitivity for N-glycans. In addition, we have
optimized the MS parameters to maximize the sensitivity of
the InstantPC‑labeled N-glycans while minimizing in-source
×105

fragmentation of these fragile molecules. The optimized
conditions have significantly improved the MS spectrum
quality, leading to accurate N-glycans identification andrelative quantification results. Figure 4 shows the MSspectrum of an InstantPC-labeled N-glycan (G2F) where only
the doubly charged ions of its protonated form, [M+2H]2+,
as well as its adducts [M+H+Na]2+ and [M+H+K]2+ wereobserved (Note: InstantPC tag causes a mass increment of261.1477 Da compared to the free reducing end form of theglycan).

[M+2H]+2
1,024.9058

1.2
1.1
1.0G2F

0.9

Counts

0.8
0.7
0.6
0.5
0.4

[M+H+Na]+2
1,035.8964

0.3
0.2

[M+H+K]+2
1,043.8809

0.1
01,024

1,026

1,028

1,0301,032

1,034 1,036 1,038
Mass-to-charge (m/z)

1,040

1,042

1,044

1,046

1,048

1,050

Figure 4. Representative spectrum of an InstantPC-labeled N-glycan (G2F). Excellent isotopic fidelity of thecharge states of the InstantPC-labeled G2F glycan and its adducts. The red boxes represent the theoreticalisotopic pattern, and the blue lines represent the actual raw MS spectrum.

5

We have introduced a workflow in MassHunter BioConfirm
software for released glycan profiling. This workflow enables
the easy setup of sample batch analysis. The software canaccommodate many commercial or customized fluorescenttags. A Personal Compound Database (PCD) containing
accurate mass and structural information of glycans is usedfor identification using the Agilent proprietary Find by Formulaalgorithm. Subsequently, a summary analytical report can becreated in a customer-defined report format. Figure 5 showsthe extracted ion chromatograms (EICs) of the identified
glycans.
The Biomolecules results table (Figure 6) in BioConfirm allows
quick review of detailed glycans information including names,
mass, retention time, peak area, composition, and databasematching score. Multiple IDs are displayed for glycans with×106

5.0

3.4

4.5

3.2

4.0
Counts (%)

G0F

3.0

G1F

2.8
2.6

InstantPC-labeled glycans were previously shown to give
similar relative quantitation results for MS and FLD analysis4.
The FLD chromatogram for the CHO mAb1 sample wasintegrated using the Agilent MassHunter Qualitative Analysis
software. The relative sum abundance of the top sevenmost abundant N-glycans was calculated and comparedagainst the same data from the MS analysis (Figure 7). To
get equivalent results, do not saturate the MS detector. Anideal quantity for this workflow would be to inject N-glycans
released from approximately 0.5 µg of mAb.

Zoom
H4N3F1S1

3.5

H4N3F1Sg1

3.0
2.5
2.0

H4N5F1

H4N3S1

1.5

2.4

1.0

2.2

0.5

2.0
Countspossible isoform structures. It also allows users to review theTIC of the sample as well as the individual glycan MS spectra.
In addition, multiple data files can be processed and analyzed
in batch mode. The user may use peak areas of the selectedglycans in the results table for relative quantitative analysis.

7.7

1.8

7.8

7.9

8.0
8.1
8.2
Acquisition time (min)

8.3

8.4

8.5

G1F’

1.6
1.4
1.2

G1/G1’

H4N3F1

1.0

H6N5F1G2F

H5N4Sg2

0.8
0.6
H6N4F1

H4N4F1Sg1

0.2
0Man5G00.4

H5N4F2

H3N2F1123456

7891011
Acquisition time (min)H7N4F1

H5N4Sg1

1213

H5N4F1Sg1

14151617181920Figure 5. Extracted ion chromatograms of the identified glycans from NISTmAb. Inset: zoom of EICs of identified glycans eluted in the retention time
range of 7.6–8.6 minutes.

6Figure 6. Screenshot of Agilent MassHunter BioConfirm B.09.00 software with representative glycan profiling results.

50 %

MSFLD

Relative sum% of major glycans in CHO mAb1

45 %
40 %
35 %
30 %
25 %
20 %
15 %
10 %
5%
0%

G0

G0F

Man5G1G1F

G1F'G2F

Figure 7. Relative sum % of the major N-glycans in the CHO mAb1
(0.5 µg), comparing results from MS-based quantitation (blue) with
FLD‑based quantitation (orange).

7

To summarize and compare the MS results, the top five mostabundant N-glycans for each mAb sample were used tocalculate relative sum %. Figure 8 presents the data.
Sigma mAb

45 %

45 %

40 %

40 %

35 %

35 %

30 %

30 %

25 %

25 %

20 %

20 %

15 %

15 %

10 %

10 %

5%

5%

0%

G0F

G1F

G1F'

G2F

G0Herceptin

60 %

0%

45 %

NISTmAb

G0F

G1F

G1F'

G1Ga1F

G1F-GlcNAc

G1F

G1F'

G2F

G0CHO mAb

40 %

50 %

35 %
30 %

40 %25 %30 %

20 %
15 %

20 %

10 %

10 %

5%0%

G0FG1FG1F'G0Man50%

G0F

Figure 8. Relative sum % of the top five N-glycans in each of the four mAb samples. Note: The NISTmAb contained a structuresuspected to be G1F with an additional alpha-1,3-galactose, and this was labeled as G1Ga1F.

8

The BioConfirm B.09.00 software allows users to generate
their own glycan profile reports using the Report Builder
program. Figure 9 shows an example of a released glycan
report. In the Report Builder, users can customize the reportsections with information such as Sample Information,Sample Chromatogram, Biomolecule Summary, andBiomolecule Details. The corresponding glycan structures aredisplayed along with the identified glycans.

Figure 9. Agilent MassHunter BioConfirm B.09.00 Software – Released Glycan Report.

9Conclusion

References

This study demonstrated the performance of theAgilent AssayMap Bravo, 6545XT AdvanceBio LC/Q-TOF,
and MassHunter BioConfirm software, when used as anintegrated solution for released glycan analysis.

1.

Rademacher, T. W; Williams, P; DwekMark, R. A.
“Agalactosyl glycoforms of IgG autoantibodies arepathogenic” P. Natl. Acad. Sci. 1994, 91, 6123-6127.

2.

Anumula, K. R. “Advances in fluorescence derivatization
methods for high-performance liquid chromatographic
analysis of glycoprotein carbohydrates” Anal. Biochem.
2006, 350, 1-23.

•This workflow combines high-throughput sample
preparation with excellent chromatographic separation
using the Agilent AdvanceBio Glycan Mapping column.

•The easy setup and use of the glycan database included
with BioConfirm B.09.00 provided the ability to accuratelyprofile, identify, and perform relative quantification.

3.

N-Glycan Analysis of mAbs and Other Glycoproteins withUHPLC and Fluorescence Detection, Agilent Technologies,
publication number 5991-5253EN.

•The 6545XT-based glycan analysis generated similar
quantitative results to that of fluorescence analysis,
making it possible to compare different N-glycans acrossdifferent mAb samples.

4.

Comparison of Relative Quantification of Monoclonal
Antibody N-glycans Using Fluorescence and MS
Detection, Agilent Technologies, publication number5991‑6958EN.

•The Report Builder function in BioConfirm B.09.00
provides the ability to create custom reports.

5.

Analysis of Monoclonal Antibody N-glycans
by Fluorescence Detection and Robust Mass
Selective Detection Using the Agilent LC/MSD XT,Agilent Technologies, publication number 5991-8071EN.

In conclusion, the Agilent solution automated the entire
process of N-linked glycan analysis from sample preparation
to data analysis with high precision. This approach provided
high sensitivity and best quantitation for glycan analysis
using fluorescence and additional identification by mass
spectrometric detection.

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For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2017
Printed in the USA, October 11, 20175991-8550EN

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