2
Novel Glycan Column Technology for the LC-MS Analysis of Labeled and Native
N
-Glycans Released from Proteins and Antibodies
Overview
Purpose:
Development of novel high-performance liquid chromatography (HPLC)
and ultra high-performance liquid chromatography (UHPLC)
columns for high-
resolution separation and structural characterization of native and fluorescently
labeled
N
-glycans released from various proteins, including antibodies.
Methods:
UHPLC with fluorescence detection (FLD) for the chromatographic analysis
of labeled
N
-glycans. LC with mass spectrometry (MS) and LC-MS/MS analysis for
structural characterization of both labeled and native
N
-glycans from proteins by MS
detection.
Results:
We have developed a high-performance, silica-based HPLC/UHPLC column
(Thermo Scientific
™
GlycanPac
™
AXH-1) specifically designed for simultaneous
separation of glycans by charge, size, and polarity. It is designed for high-resolution,
and high-throughput analysis, with unique selectivity for biologically relevant glycans,
either labeled or native, by LC-FLD and LC-MS methods.
Introduction
Glycans are widely distributed in biological systems in ‘free state’ and conjugated
forms such as glycoproteins, glycolipids, and proteoglycans. They are involved in a
wide range of biological and physiological processes.
1
The functions, including
efficacy and safety of protein-based drugs such as recombinant proteins and
monoclonal antibodies (MAbs), are often dependent on the structure and types of
glycans attached to the proteins.
2
The structures of glycans are quite diverse,
complex, and heterogeneous due to post-translational modifications and physiological
conditions. The structural characterization and quantitative estimation of glycans is
highly essential in biotherapeutics and biopharmaceutical projects.
3
However, it is
tremendously challenging to comprehensively characterize glycan profiles and
determine the structures of glycans.
The GlycanPac AXH-1 columns are high-performance HPLC/UHPLC columns
specifically designed for structural, qualitative, and quantitative analysis of glycans.
They are designed for high-resolution and high-throughput analysis, with unique
selectivity for biologically relevant glycans, including glycans from antibodies
—
either
labeled or native
—
by LC-fluorescence or LC-MS methods. Because glycans are
highly hydrophilic and polar substances,
hydrophilic interaction liquid chromatography
(HILIC)
columns based on amide, amine, or zwitterion packing materials are often
used for glycan analysis. These HILIC columns separate glycans mainly by hydrogen
bonding, resulting in size- and composition-based separation. However, identification
of the glycan charge state is not possible with these types of columns because
glycans of different charge states are intermingled in the separation envelope, making
this approach limited. The GlycanPac AXH-1 column, which is based on advanced
mixed-mode chromatography technology, overcomes these limitations and can
separate glycans based on charge, size, and polarity configuration. In addition, each
glycan charge state can be quantified. The GlycanPac AXH-1 column provides both
greater selectivity and higher resolution, along with faster quantitative analysis.
Methods
Sample Preparation
Release native glycans from glycoproteins with PNGase F enzyme. Conjugate the
released glycans with 2-amino benzamide (2AB) label group using the reported
procedure of Bigge et al.
4
Here, 2-AB A1 (P/N GKSB 311), 2-AB A2 (P/N GKSB 312),
and 2-AB A3 (P/N GKSB 314) were purchased from Prozyme
®
(Hayward, CA). Prior
to analysis, dissolve samples in 100% buffer (100 mM ammonium formate, pH = 4.4)
and dilute further with acetonitrile to make 30% buffer and 70% acetonitrile.
Liquid Chromatography
All the glycans were separated using a Thermo Scientific
™
Dionex
™
UltiMate
™
3000
BioRS system and either a Thermo Scientific
™
Dionex
™
UltiMate
™
FLD-3400RS Rapid Separation Fluorescence Detector or MS detector.
Mass Spectrometry
MS analysis was performed with a Thermo Scientific
™
Q Exactive
™
Benchtop
LC-MS/MS in negative ion mode at the following settings: MS scan range 380
–
2000.
FT-MS was acquired at 70,000 resolution at
m/z
200 with AGC target of 1e
6
; and DDA
MS2 acquired at 17,500 resolution at
m/z
200 with AGC target of 2e
5
.
Data Analysis
SimGlycan
®
software (PREMIER Biosoft) was used for glycan identification and
structural elucidation data analysis.
5
SimGlycan software accepts raw data files from
Thermo Scientific
™
mass spectrometers and elucidates the associated glycan
structure by database searching and scoring techniques.
Results
Separation of Labeled Gl
The GlycanPac AXH-1 colu
analysis as well as charact
in proteins. Figure 1 shows
column using fluorescence
charge: the neutral glycans
N
-glycans from monosialyla
pentasialylated species. Gl
their size and polarity. The r
using 2AB labeled glycan st
based on charge, size, and
information. The chromatog
fluorescence detection, pro
The structure of glycans pr
using the GlycanPac AXH-1
FIGURE 1. Separation of
and polarity.
FIGURE 2. Comparison
from fetuin.
LC-MS and LC-MS/MS An
Column
The coupling of the Glycan
attractive as MS, with it’s a
of complex glycans. 2AB la
GlycanPac AXH-1 column
dependant MS/MS spectra
software was used for glyc
analysis is shown in Figure
data further validated the a
based on charge, size, and
(Figure 4) is common with
