2
A Complete Workflow Solution for Monoclonal Antibody Glycoform Characterization Combininga Novel Glycan Column Technology and
Bench-Top Orbitrap LC-MS/MS
Overview
Purpose:
To develop a complete workflow solution for monoclonal antibody (mAb)
glycoform characterization using a unique glycan column technology and a Thermo
Scientific™ bench-top Orbitrap™ LC-MS/MS.
Methods:
Glycans are separated using a recently developed high-performance
HPLC/UHPLC column, a Thermo Scientific™ GlycanPac™ AXH-1 column. A data-
dependent high-energy collision dissociation (HCD) method was performed in negative
ion mode to analyze the glycans.
Results:
The GlycanPac AXH-1 column separates glycans with unique selectivity
based on charge, size and polarity. A complete workflow solution was developed for
glycan profiling combining the unique column technology and a bench-top Orbitrap LC-
MS/MS (Figure 1). This workflow was applied to antibody glycoform characterization.
Confident identification and structural confirmation were achieved for released glycans
from a standard glycoprotein and a monoclonal antibody.
Introduction
Because glycosylation is critical to the efficacy of antibody therapeutics, the FDA
requires that a consistent human-type glycosylation be maintained for recombinant
monoclonal antibodies (mAb), irrespective of the system in which they are produced.
The complex branching and isomeric nature of glycans pose significant analytical
challenges for their identification and characterization. Liquid chromatography (LC)
coupled to mass spectrometry (MS) has emerged as one of the most powerful tools for
the structural characterization of glycans.
The recently developed GlycanPac AXH-1 column is a high-performance
HPLC/UHPLC column specifically designed for structural, qualitative and quantitative
analysis of glycans. It has a unique selectivity for biologically relevant glycans
including glycans from antibodies, either labeled or native and is designed for high-
resolution, high-throughput analysis by LC-fluorescence or LC-MS methods. Because
glycans are very hydrophilic and polar,
hydrophilic interaction liquid chromatography
(HILIC)
columns based on amide, amine or zwitterionic packing materials are often
used for their analysis. HILIC columns separate glycans mainly by hydrogen bonding,
resulting in size and composition-based separation. Identification of the glycan charge
state is not possible by HILIC. The GlycanPac AXH-1 column overcomes these
limitations and can separate glycans based on charge, size and polarity configuration.
It provides both greater selectivity and higher resolution. In this study, we
characterized N-linked glycans released from a glycoprotein standard and a
monoclonal antibody by LC-MS/MS methods using the new column technology and
high-resolution Orbitrap mass spectrometry.
Methods
Sample preparation
Native glycans are released from glycoproteins or mAb with PNGase F enzyme. The
released glycans are conjugated with 2-amino benzamide (2-AB) label group with
reported procedure of Bigge
et. al.
1
Liquid chromatography
All the glycans are separated using a recently developed high-performance
HPLC/UHPLC column, GlycanPac AXH-1, on a Thermo Scientific™ Dionex™ Ultimate
3000 UHPLC with either s fluorescence or MS detector.
For intact antibody, a Thermo Scientific™ ProSwift RP-10R monolithic column (1 x 50
mm) was used for desalting. LC solvents are 0.1% formic acid in H
2
O (Solvent A) and
0.1% formic acid in acetonitrile (Solvent B). Column was heated to 80 °C during
analysis. Flow rate was 60 µL/min. After injection of 1 µg mAb, a 15 min gradient was
used to elute mAbs from the column (0.0 min, 20%B; 1.0 min, 35%B; 3.0 min, 55%B;
4.0 min, 98%B; 7.0 min, 98% B; 7.1 min, 20%B; 15.0 min, 20%B).
Mass spectrometry
A data-dependent high-energy collision dissociation (HCD) method was performed in
negative ion mode to analyze the glycans. The following MS and MS/MS settings were
used: MS scan range 380-2000
m/z
. FT-MS was acquired at 70,000 resolution at
m/z
200 with AGC target of 1x10
6
and DDA MS2 acquired at 17,500 resolution at
m/z
200
with AGC target of 2x10
5
. Intact mAbs were analyzed by ESI-MS for intact molecular
mass. The spray voltage was 4kV. Sheath gas flow rate was set at 10. Auxiliary gas
flow rate was set at 5. Capillary temperature was 275 °C. S-lens level was set at 55.
In-source CID was set at 45 eV. For full MS, resolution was 17,500 for intact mAb. The
AGC target was set at 3x10
6
. Maximum IT was set at 250 ms.
Figure 1. A complete LC-MS/MS w
glycan profiling
Results
Separation of Glycans Based on C
The GlycanPac AXH-1 column can be
analysis and characterization of unch
proteins. The separation and elution o
glycans elute first, followed by the sep
di-sialylated, tri-sialylated, tetra-sialyla
of each charge state are further separ
study, the structure of glycans present
resolution LC-MS/MS. As shown in Fi
obtained from the MS/MS data validat
separate labeled N-glycans based on
different charge state glycans is com
column as shown in Figure 3.
Figure 2. LC-MS analysis of 2-AB la
GlycanPac AXH-1 (1.9 µm) column
Data analysis
SimGlycan® software from PREMIE
structural elucidation
2
. SimGlycan s
Scientific mass spectrometers and e
database searching and scoring tec
Full MS spectra of mAb were analyz
Deconvolution™ 2.0 software. Mass
averaging spectra across the most a
A minimum of at least 8 consecutive
used to produce a deconvoluted pea
compared to the expected masses o
glycoforms
N-Acetyl-Glucosamine
(GlcNAc),
Mannose
(Man)
,
Galactose
(G
N-AcetylNeuraminicAcid
(Neu5Ac)
,
N-Glycolyl-NueraminicAcid
(N
Native
SimGlycan
software
The Q Exacti
LC-MS
analysis
PNGase F
digestion
