Analysis of Monoclonal Antibody
N-glycans by Fluorescence Detection
and Robust Mass Selective Detection
Using the Agilent LC/MSD XT
Application Note
Biotherapeutics & BiologicsAuthors

Introduction

Oscar Potter, Gregory Staples,
Jordy Hsaio, and Te-Wei Chu

N-glycosylation of therapeutic proteins is monitored during product development,
process development, and QC. Many analysts perform enzymatic N-glycan release
followed by labeling with a fluorescent tag to run HILIC-FLD analysis. The InstantPCtag from Prozyme Inc. allows manual sample prep within 1 hour, or alternatively,
allows high-throughput parallel processing with the Agilent AssayMap Bravo liquidhandling platform. High-resolution separation can be completed in short cycle times
using the Agilent AdvanceBio Glycan Mapping column (see 5991-4886EN).
While fluorescence detection is a popular detection technique, analysts running
HILIC-FLD workflows may face difficult challenges in the form of ambiguous peakassignment, particularly in the case of new sample types or unexpected peaks. In
such cases, analysts might resort to sending the samples through complementary
analyses such as LC-Q-TOF or MALDI-TOF, but this loses time, and incurs additionalexpenses.

Agilent LC/MSD XT

We previously demonstrated an enhanced workflow where a HILIC-FLD
system was hyphenated online to a high-resolution Q-TOF mass spectrometer(see 5991-6958EN). This approach couples the benefits of robust fluorescencebased quantitation with the powerful structural elucidation capabilities of mass
spectrometry. However, analysts who seek a routine cost-effective solution
may prefer to work with a rugged, small-footprint mass selective detector(MSD) based on single quadrupole technology. Therefore, we present a method
where a HILIC‑FLD system is coupled to a rugged, sensitive, and stackable
Agilent LC/MSD XT single quadrupole. The MSD data provide mass information tosolve ambiguous peak assignments and detect coeluting structures. The method
operates at less than 600 bar to maintain compatibility with a range of existing LCsystems.

InstantPC labeledmAb N-glycans

Agilent AdvanceBio
Glycan Mapping Column

Agilent 1260 Infinity II LC withan Agilent 1260 FLD and Agilent LC/MSD XT
Figure 1.

Flow chart of the sample preparation, LC column, and instruments used for identification and quantificationof InstantPC labeled N-glycans.

Experimental

Chromatography conditions

Sample preparation

Agilent 1260 Infinity II Binary Pump G7112B

Pump

Samples of monoclonal antibody (mAb) were expressed inour own lab (CHO mAb 1) or purchased from Sigma‑Aldrich(SiLu Lite P/N MSQC4) and from NIST (NIST mAb, Reference
Material 8671). All samples were adjusted to 1 µg/µL prior
to processing by GlykoPrep-plus Rapid N-Glycan Sample
Preparation with InstantPC (96-ct) from Prozyme Inc.
(GPPNG‑PC). The sample handling was automated using
the AssayMap Bravo Liquid Handling Platform (G5542A).
The final step of this protocol elutes the labeled glycans in
50 µL, so that each µL of this final sample solution represents
N-glycans released from 1 µg of mAb. Conveniently,
this workflow has the advantage of not requiring any
centrifugation or dry down steps.

Mobile phase A
50 mM Formic acid adjusted to pH 4.5 withammonium hydroxide
Mobile phase B
Acetonitrile
Sampler
Agilent 1260 Infinity II Multisampler (G7167A) withthermostat set at 11 °CColumns• AdvanceBio Glycan Mapping Column
1.8 µm, 2.1 × 100 mm used with method A• AdvanceBio Glycan Mapping Column
2.7 µm, 2.1 × 150 mm used with method B• AdvanceBio Glycan Mapping Column
1.8 µm, 2.1 × 150 mm used with method C2Column heater

Gradient tables

Agilent 1260 Infinity II G7116 thermostatic column
compartment with G7116-60015 solvent preheater set to40 °C

Method A (for 2.1 × 100 mm 1.8 µm column)
Timetable
Time (min) %A%B0.00
25.0 75.0
0.50
27.0 73.0
4.00
28.0 72.0
9.00
33.0 67.0
9.20
50.0 50.0
9.70
50.0 50.0
10.00
25.0 75.0
11.50
25.0 75.0
11.80
25.0 75.0
14.50
25.0 75.0
15.30
25.0 75.0
Stop time = 18 minutes

Columns were plumbed using 100 µm id SSTL tubing to an
Agilent 1260 Infinity Fluorescence Detector (G1321B) with a
8 µL flow cell (G1321-60005). The detector was set to λEx at
285 nm, λEm at 345 nm with PMT gain = 10.

LC/MSD XT Parameters
Parameter

Value

Ion source
SourceAgilent Jet Stream in positive mode

Sheath gas

300 °C at 10.0 L/min

Dry gas temperature

150 °C at 9.0 L/min

Nebulizer pressure

35 psig

VCap

2,500 V
Nozzle: 500 V

Method B (for 2.1 × 150 mm 2.7 µm column)
Timetable
Time (min) %A%B
0.00
27.0 73.0
1.00
28.5 71.5
9.00
29.5 70.5
22.00
41.0 59.0
22.50
50.0 50.0
23.50
50.0 50.0
23.70
27.0 73.0
25.00
27.0 73.0
25.50
27.0 73.0
27.50
27.0 73.0
28.00
27.0 73.0
Stop time = 30 minutes

Acquisition settings
Mass range

500–1,400 m/z

Fragmentor

100 V

Gain EMV

1.0

Step size

0.10

Peak width

0.2

Flow (mL/min)
0.70
0.70
0.70
0.70
0.70
0.70
0.70
0.70
0.80
0.90
0.70

Software
OpenLab CDS Chemstation Edition Rev C.01.07 SR3Reagents

Flow (mL/min)
0.50
0.50
0.50
0.50
0.80
0.70
0.70
0.70
0.80
0.90
0.50

Method C (for 2.1 × 150 mm 1.8 µm column)

All reagents and solvents used were of the highest purity
available.

Time (min) %A%B
0.00
22.0 78.0
0.50
26.0 74.0
13.00
27.5 72.5
28.00
39.0 61.0
28.50
50.0 50.0
28.60
50.0 50.0
28.80
22.0 78.0
31.00
22.0 78.0
31.5022.0 78.0
33.50
22.0 78.0
Stop time = 37 minutes

3Flow (mL/min)
0.60
0.60
0.60
0.60
0.50
0.40
0.40
0.50
0.55
0.60

Results and Discussion

LU

CHO mAb 1Agilent AdvanceBio Glycan Mapping column
2.1 × 100, 1.8 µm
Method A

25Separation
We optimized the separation of typical therapeutic mAb
glycan profiles on three different columns, targeting various
cycle times. The methods begin with a shallow gradient thatmaximizes resolution of the common neutral glycans thatappear in most therapeutic mAb samples. The gradient slope
was increased midway through the run to elute any larger,
acidic glycan structures more efficiently. These methods
were all designed to stay well under 600 bar to maximize
robustness, as well as allowing for compatibility with a wide
range of LC instruments. While all three separation methods
are shown in Figure 2, we chose to use Method C for theremainder of the experiments because it was found to givethe best overall performance for our three mAb N-glycan
samples.

201510518 min

0024

68

1012

1416min

LU
30
NIST mAb
Agilent AdvanceBio Glycan Mapping column
2.1 × 150, 2.7 µm
Method B

25201510530 min0LU
80051015702025min

Sigma SiLu mAbAgilent AdvanceBio Glycan Mapping column
2.1 × 150, 1.8 µm
Method C

60
504030201037 min

00Figure 2.

45101520253035 min

Three separation methods using different column formats withthree mAb samples.

LC/MSD XT Sensitivity

We optimized the MSD parameters to maximize sensitivity.
InstantPC-labeled N-glycans were observed almost
exclusively as doubly charged ions of the forms [M+2H]2+,
with some [M+NH4+H]2+, and [M+H+Na]2+. Increasing
the dry gas temperature or fragmentor voltage can cause
collision‑induced dissociation of the N-glycans, offering
powerful structural elucidation strategies similar to thoseachievable with a Q-TOF (see 5991‑5253EN).

Traditionally, analysts have avoided using single quadrupole
mass spectrometers for LC/MS analysis of N-glycans
due to concerns about sensitivity. However, two technicalinnovations have led to dramatic improvements in the limitof detection for N-glycans. The first is the availability of ahighly sensitive ion source, Agilent Jet Stream, which uses asuper‑heated sheath gas flow around the electrospray plumeto dramatically improve ionization. The second innovationis the availability of a InstantPC tag from Prozyme Inc. thatradically increases ionization efficiency versus traditional
fluorescent labels.

100863.0

FA2Comprising 43.8%of profile

Max: 457,487

Diagnostic signals were obtained for glycans making up as
little as 0.1 % of the profile. When comparing this sensitivity
with alternative workflows, remember that these results
were achieved without adding a preconcentrating step to theend of the sample preparation workflow, therefore saving
considerable time. Sensitivity could be boosted even further
by drying the samples and reconstituting them in a lower
volume of sample matrix.

100

80

80

60

60
874.0

40

0

800

20

850

900

0950 m/z

Max: 58,698

Man5Comprising 3.9%of profile

80

800

850

100

FA2G2Ga1
Comprising 1.2%of profile

900

1,000 m/z

Max: 10,573

1,114.6
1,117.3

749.0

40

950

1,106.1

80

60

Max: 13,801

881.9

757.0

100

871.0

40871.5

20A2[3]G1
Comprising 0.35%of profile

60759.94020200700

Figure 3.

725

750

775

800

0825 m/z

1,050

1,100

1,150

1,200m/z

Mass spectra of four representative glycans from 3 µg of mAb at various levels of relative abundance. The signal for A2[3]G1, which makes up just
0.35 % of the glycan profile, is clearly observable with a high signal-to-noise ratio.

5

Peak assignment

Table 1 was constructed showing the composition of theglycans that were detected across the three samples. Glycan
compositions are shown in the form: HxNxFxSgx + Core
(where H = Galactose or Mannose, N = N-acetylglucosamine,
F = Fucose, Sg = N-glycolylneuraminic acid, andCore = trimannosyl, a core common to all N-glycans). In manycases, these compositions allowed us to propose glycan
structures by supplementing basic knowledge of therapeuticmAb glycosylation patterns and HILIC retention order. Since
most therapeutic mAbs contain a similar, limited set ofcommon N-glycan structures, Table 1 can serve as a usefulreference for assigning N-glycans in this workflow.

The data from all three samples (using method C) wereintegrated, and the peaks were assigned to glycan
compositions using an approach similar to that shown inapplication note 5991-5253EN.
Note: InstantPC labeling causes a mass increment of261.1477 m/z versus the free reducing-end form of theglycans.

LU
NIST mAb
14

11

7

12

12
10

178
6

4

14,642

132

19 20

50
5.0
7.5
LU
CHOmAb
114

10.0

12.5

10516 25

15.0

173

6 8

40
5.0
7.5
LU
14 SiLu mAb

910.0

21,1312.5

717.5

1217

63205.0Figure 4.

5

10

20.0

22.5

51025min31
27.5 min25.0

152025min

25.0

27.5 min

LU
90
80
70
60
50
40
30
20
1000

15

2025

min

8
94
7.5

22.5

20

26 27

15.0

82

29

20.0

LU
90
80
70
60
50
40
30
20
1000

18

104

15

115

12

1024 30 28

22

17.5

42 1
2

5
23

18

12

10

60

11

7128

LU
90
80
70
60
50
40
30
20
100

10.0

21,13
12.5

15.0

26 27 15

18
17.5

20.0

32
22.5

25.0

27.5 minZoomed FLD chromatograms of the three mAb N-glycan samples showing detection of major andminor glycans for method C. Insets show the zoomed-out data. Annotations refer to Table 1.

6

Table 1.
ID

Observed[M+2H]2+

Proposedcomposition

Theoretical[M+2H]2+

Proposed structure
OxfordAlternative

1659.9

F1+Core659.774

F1M3

G0F-2GlcNAc

2688.4

N1+Core688.284

A1G1-GlcNAc

3748.9

H2+Core748.798

M5Man54761.5

N1F1+Core761.313

FA1G0F-GlcNAc

5790.0N2+Core789.824

A2G06842.4

H1N1F1+Core842.340

FA1G1

G1F-GlcNAc

7863.0N2F1+Core862.853

FA2G0F8871.0H1N2+Core870.851

A2[6]G1G19871.0

H1N2+Core870.851

A2[3]G1G1'10923.6

H2N1F1+Core923.366--11944.0

H1N2F1+Core943.879

FA2[6]G1G1F

12944.0

H1N2F1+Core943.879

FA2[3]G1

G1F'13952.0

H2N2+Core951.877

A2G2G214964.7

N3F1+Core964.393

FA2B

G0FB

15991.9

H5+Core991.877

M8Man816996.1

H1N1Sg1F1+Core995.885

FA1G1Sg1

G1Sg1F-GlcNAc

171025.1

H2N2F1+Core1024.906

FA2G2G2F

181025.1

H2N2F1+Core1024.906

FA2G1Ga1

G1F+αGal

191045.6

H1N3F1+Core1045.419

FA2[6]B1G1

G1FB

201045.7

H1N3F1+Core1045.419

FA2[3]B1G1

G1FB'

211089.5

H1N1F1S1+Core

1089.427

FA1G1S1G1S1F-GlcNAc

221097.7

H1N2F1Sg1+Core

1097.425

FA2G1Sg1

G1Sg1F

231106.1

H3N2F1+Core

1105.932

FA2G2Ga1G2F+αGal

241106.1

H3N2F1+Core

1105.932

FA2G2Ga1 isoG2F+αGal'

25

1126.5

H2N3F1+Core1126.446

FA2BG2G2FB261170.4

H2N2F1S1+Core1170.454

FA2G2S1A1F

271170.4

H2N2F1S1+Core1170.454

FA2G2S1 iso

A1F iso281178.7

H2N2F1Sg1 + Core1178.451

FA2G2Sg1

Ag1F

291187.1

H4N2F1+Core1186.959

FA2G2Ga2

G2F+(αGal)2301207.7

H3N3F1+Core1207.472

FA2BG2Ga1

G2FB+αGal311259.4

H3N2F1Sg1+Core1259.477

FA2G2Sg1Gal1

Ag1F+αGal

321316.2

H2N2F1S2+Core1316.001

FA2G2S2

A2F

7Result tables

Some of the profiles contained peaks representing coeluting
glycan structures. These coelutions involved minor glycans,and quantitation based on FLD alone would report the peakarea as the combined contribution of two structures.

Peaks in the FLD chromatograms were reported as relative
sum % of the total glycan profiles. Figure 5 shows the results.

A50

NIST mAb
CHO mAb 1SiLu mAb

45Rel. Sum %40353025
20
15105
F- G2G 2F
G1 lcN-G Ac
lcN
A
G0 M cF- an
Gl 5cNAc
G1F- GGl 0cN
AcG1H2
N1 G
F1 1’+Core
G2G0G1Sg M FB1F a-G n8lc
G1 NA
F+ cαGaG1 l
FBG1S1 G1F- FBGl ’cN
G1 Ac
G2 Sg1F F
G2 +αGF+ alαGal
G2 ’
FBA
A1 1FFisG2 A o
F+ g1( FG2 αGa
FB l)2Ag +αG
1F al+αGa
l
A2F

F

F’G0G1G1G0F0B
6NIST mAb
CHO mAb 1SiLu mAb

Rel. Sum %54321Figure 5.

G2G0FBG1Sg M
1F an-G 8lcG1 NAcF+αGalG1FB
G1S1 G1F
F- B’GlcNAG1 cSgG2 1FF+G2 αGaF+ l
αGal’G2FBA1A1 FFisoG2 Ag
F+ 1F(αG2 GalFB )2+Ag αG
1F al+αGalA2FG1H2N1 G
F1 1’+CoreG0F-2Gl
G1 cNA-G clcN
AcG0 MaF- n5GlcNAc
G1F- G0GlcNAc0A) Relative abundance of N-glycans in the three mAb samples. B) Zoomed-in chart showing only the minor components.
Minor glycans were detected easily at less than 1 %.

8For analysts who need accurate quantitation of these lowabundance coeluting structures, we propose using theLC/MSD XT data to supplement the FLD. The mass spectrum
can be integrated across the time period corresponding to thecoeluting FLD peaks. The FLD area can then be apportioned
to the different structures based on the combined relative
intensity of the doubly charged N-glycan ions in thisspectrum. We have previously shown that the MS signals for
InstantPC labels closely correspond to their true abundance
as defined by fluorescence intensity (see 5991-6958EN).

Conclusion
The Agilent LC/MSD XT based on single quad technology canbe hyphenated online to a typical UHPLC HILIC-FLD system
to provide the option of mass spec-based identification forevery peak in every sample. Using an Agilent Jet Stream
ion source in combination with the InstantPC glycan tag
provides ample sensitivity to detect MS signals for bothmajor and minor peaks in typical antibody samples without
needing to concentrate the sample. For typical monoclonal
antibody samples, this allows confident assignment ofglycan structures. While this application note used thetraditional approach of basing relative quantitation on robust
fluorescence detection, the MS data can be used to assist
quantitation in the case of coeluting peaks.

9For More Information
These data represent typical results. For more information on
our products and services, visit our Web site atwww.agilent.com/chem.

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Agilent shall not be liable for errors contained herein or for incidental or consequential
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Information, descriptions, and specifications in this publication are subject to change
without notice.
© Agilent Technologies, Inc., 2017
Printed in the USA
May 22, 20175991-8071EN