High fidelity glycan sequencing using a combination of
capillary electrophoresis and exoglycosidase digestion
Andras Guttman
Senior Manager Applications, Separations
SCIEX, Brea, CA 92822
The
rapidly increasing
usage
of
glycoproteins
as
biopharmaceutical products has created a demand for fast,
efficient and reliable bioanalytical techniques for glycosylation
analysis [1]. One of the fastest growing groups of these new
generation drugs are monoclonal antibodies (MAbs). MAbs in
most instances possess a conserved N-linked glycosylation site
in each of the CH2 domains of the Fc portion of the heavy chain
of the molecule, but may also have additional attached sugar
structures on the Fab domains [2]. Increasing evidence shows
that the carbohydrate moieties of therapeutic antibodies play
important roles in their biological activity, physicochemical
properties, and effector functions [3]. Even minor changes in the
carbohydrate structures (linkage, position and site occupancy)
can influence the bioactivity of these products. The extremely
high diversity of glycosylation makes their structural elucidation
very difficult and in most instances only the combination of
various methods can provide the desired information [4]. The
most frequently used analytical methods for the structural
analysis of
complex carbohydrates include capillary
electrophoresis (CE), mass spectrometry (MS) and high
performance liquid chromatography (HPLC), often combined with
exoglycosidase digestion techniques.
Capillary electrophoresis with laser induced fluorescent detection
(CE-LIF) is a high resolution and high sensitivity separation
method applied for rapid profiling of complex carbohydrates [5].
CE-LIF is capable of discriminating between closely related
positional and linkage isomers. Individual structures
corresponding to peaks in the electropherogram can be
proposed based on their glucose unit (GU) values [6] published
in
publicly
available
databases
(https://glycobase.nibrt.ie/glycobase/about.action).
However,
there are instances when database-mediated structural
proposition is ambiguous, calling for more comprehensive
carbohydrate sequencing to achieve full structural elucidation.
Unlike the commonly sequenced linear biopolymers of DNA and
proteins, complex carbohydrates have branched structures and
two sugar units can be connected through various positions and
linkages making glycan sequencing a challenge.
The sequencing process begins with the release of the N-linked
carbohydrate moieties from the polypeptide backbone using an
appropriate endoglycosidase, in most instances PNGase F. The
released glycans are then labeled with a fluorophore (8aminopyrene-1,3,6-trisulfonic acid, APTS), imparting charge and
fluorescence
properties,
and
separated
by capillary
electrophoresis. The resulting CE trace represents the N-linked
glycan profile of the glycoprotein of interest. The sequencing
strategy involves consecutive use of specific exoglycosidase
enzymes to sequentially remove the sugar residues from the
non-reductive end of the glycan structures [7]. The resulting
glycan pools are analyzed by CE-LIF following each release step
and peak migration shifts are recorded. This step is repeated
using a series ofexoglycosidases in a consecutive manner,
usually starting with sialidase, followed by fucosidase,
galactosidase and hexoseaminidase until the characteristic
trimannosyl chitobiose core structure of all N-linked sugars is
reached. If antennary specification is needed, linkage specific
mannosidases (1-3 or 1-6) can be used to attain such
information [8].
This Technical Information Bulletin presents a high fidelity
carbohydrate sequencing strategy for samples where more than
one oligosaccharide is present in the released glycan pool. The
method works without the need to isolate any of the individual
structures. Published GU value data, or publicly available
databases can be used to access the mobility shifts and
accommodate structural elucidation.
Figure 1: Glycan nomenclature. Left panel: graphical and alphabetic
representation of a core fucosylated, disialo biantennary glycan
with bisecting GlcNAc. Right panel: Symbols used for sugar units,
linkage positions and linkage types. [9]
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Experimental Design
Chemicals: The N-CHO Carbohydrate Analysis Assay Kit
(Beckman Coulter, sold through Sciex, Brea, CA; pn 477600)
was used in all experiments and contained all chemicals
necessary for glycan labeling and CE separation, including the
charged fluorophore (APTS), the N-CHO Carbohydrate
Separation Buffer and the maltooligosaccharide ladder. IgG was
purchased from Sigma Aldrich (St. Louis, MO). The PNGase F
enzyme and the exoglycosydase enzymes of Arthrobacter
ureafaciens sialidase (ABS), Bovine kidney fucosidase (BKF),
Jack bean galactosidase (JBG) and Jack Bean hexosaminidase
(JBH) were from ProZyme (Hayward, CA) and the reaction
mixtures were prepared following the manufacturer’s protocol.
The Agencourt CleanSEQ magnetic beads were from Beckman
Coulter (Brea, CA; pn A29151
Sample Preparation: Release of the N-linked IgG glycans was
accomplished by the addition of 2 U of recombinant PNGase F
(Prozyme) and incubation at 50°C for 1 hour. This was followed
by magnetic bead based partitioning of the released glycans
from the remaining polypeptide chains and the endoglycosidase
using 200 μL Agencourt CleanSEQ magnetic bead suspension
in 87.5% final acetonitrile concentration [10]. Following thorough
mixing, the tube was placed on a strong magnet for fast and
efficient partitioning. The supernatant was discarded and the
captured glycans were eluted from the beads into the same tube
by the addition of 21 μL of 40 mM APTS in 20 % acetic acid. The
elution step was immediately followed by initiating the reductive
amination reaction with the addition of 7 μL sodium
cyanoborohydrate (1 M in THF). The reaction mixture was
incubated at 37°C for 2 hours, then the excess labeling dye was
removed by the same Agencourt CleanSEQ magnetic beads,
which were used after the digestion reaction, again in 87.5% final
acetonitrile concentration. The captured APTS-labeled glycans
were eluted from the magnetic beads by adding 25 μL water and
then analyzed by CE-LIF.
Exoglycosidase
based
carbohydrate
sequencing:
Consecutive exoglycosidase digestions included Arthrobacter
ureafaciens sialidase (ABS) to remove α2-3, 6 and 8 linked sialic
acids; Bovine kidney fucosidase (BKF) to release α1-6 corelinked fucoses; Jack bean galactosidase (JBG) to remove β1-4
and 6 linked galactoses; and Jack bean hexosaminidase (JBH)
to remove the β1-2, 4 and 6 linked N-acetyl-glucosamines.
Briefly, the APTS-labeled released glycan pool was first
analyzed by CE-LIF, then sequentially digested using the above
listed enzymes (0.5 U each) or using an array of those at 37°C
overnight in 50 mM ammonium acetate buffer (pH 5.5). Samples
were dried in a centrifugal vacuum evaporator after each
digestion step for pre-concentration and to remove the salt
(ammonium acetate) content from the reaction mixture prior to
the next digestion step.
Capillary Electrophoresis: All capillary electrophoresis
separations were performed in a Beckman Coulter PA 800 plus
Pharmaceutical Analysis System (sold through SCIEX
Separations, a part of SCIEX), equipped with a solid state laser
and fluorescence detector (excitation: 488 nm, emission: 520
nm). The effective length of the N-CHO separation capillary was
50 cm (60 cm total length, 50 μm i.d.), filled with N-CHO
Carbohydrate Separation Buffer (both from SCIEX). The applied
electric field strength was 500 V/cm in reversed polarity mode
(cathode at the injection side). Samples were pressure injected
by applying 1 psi (6.89 kPa) for 5 sec. The 32 Karat version 9.1
software package (SCIEX) was used for data acquisition and
analysis. APTS labeled maltose (G2, lower bracketing standard)
and 2-aminoacridone (AMAC) labeled glucuronic acid (upper
bracketing standard) were co-injected with all samples for
migration time normalization [6]. The normalized migration times
were converted to glucose unit (GU) values by the application of
a fifth order polynomial time based standardization against the
maltooligosaccharide ladder. Preliminary identification of the
glycan structures corresponding to the peaks in the
electropherogram was aided by Glycobase ver 3.2 from NIBRT
(Dublin, Ireland) based on their GU values measured using
identical CE-LIF separation conditions. N-glycan nomenclature
and symbolic representations used the notation previously
described by Harvey et al [9].
Results and Discussion
Sequencing of complex glycan pools usually preceded by
monosaccharide composition analysis to gain information about
the type of sugar building blocks aiding to pick the relevant
exoglycosidases to be used [11]. Commonly occurring
monosaccharides in IgG molecules include sialic acid, galactose,
N-acetylglucosamine, fucose and mannose.
As the first step, the N-linked glycans were enzymatically
removed from the sample by PNGase F digestion, labeled by
APTS, purified with magnetic bead technology and analyzed by
CE-LIF as shown in Figure 1, trace A. This step was followed by
consecutive application of the corresponding exoglycosidases to
begin sequencing of the APTS labeled glycan. As the first step,
the sample was treated with sialidase to remove all sialic acids
from the non-reductive end of the sugar structures (Figure 1B).
The arrows between trace A and B depict the shift of peaks from
the higher mobility regime (GU = 4 – 6, sialylated glycans) to the
neutral regime (GU = 7 – 10). Please note that cutting off the
sialic acids decreased the hydrodynamic volume of the
structures, but also removed the extra charges the sialic acids
p2
held. After sialidase treatment, the desialylated glycan pool was
treated using fucosidase to remove the core fucose residues
(Figure 1 C). This step again resulted in a mobility shift, however,
towards the higher mobility region as shown by the arrows
between traces B and C. In this instance, the total charge of the
glycans did not change but their hydrodynamic volume
decreased. The next step of the sequencing process was the
removal of the galactose residues (Figure 1 D). Finally,
hexosaminidase was added to the reaction mixture to remove all
antennary and bisecting GlcNAc residues, resulting in only one
large remaining peak in trace E, corresponding the N-linked core
of trimannosil chitobiose.
respectively. The shift for the digalactosylated A2G2 structure
was the sum of these two (2.44 GU).
A
B
Figure 2. Bottom-up identification of the IgG N-glycan structures.
C
D
E
Figure 1. CE-LIF traces of top-down sequential digestion of the
APTS labeled IgG glycan pool. Symbols: sialidase:
; galactosidase:
and hexosaminidase:
; fucosidase:
.
At this stage, identification of the structures started in a bottom
up fashion as shown in Figure 2, considering the GU values and
GU shifts of the peaks identified in Figure 1. Here we start with
trace E, the trimannosil chitobiose structure with GU=4.83. The
GU values of the two peaks in trace D were 6.67 and 7.18,
corresponding to the shift caused by the removal of two
antennary GlcNAc residues (0.92 each) and an additional
bisecting GlcNac (0.51).
Trace C shows the glycan structure profile after sialidase and
fucosidase treatment featuring several additional major peaks in
the neutral region possessing GU values of 7.74, 8.04 and 9.11.
Based on the GU shifts corresponding the galactose residues,
these structures were identified as monogalactosylated
biantennary structures with 1-6 (GU=7.4) and 1-3 (GU=8.04)
linkages as well as a digalacto biantennary structure (GE=9.11).
Interesting to note that the galactoses on the 1-6 and 1-3
antennas contribute different GU shifts values of 1.07 and 1.37,
Trace B shows the electropherogram of the reaction mixture
before fucosidase treatment with peaks corresponding to GU
values of 7.63, 8.72, 9.06 and 10.10 matching database entries
for core fucosylated agalacto- (FA2G2), monogalacto- with 1-6
(FA2(6)G1) and 1-3 (FA2(3)G1) linkages and digalactobiantennary (FA2G2) structures, respectively. The average
contribution of the core fucose residue to the total GU values
was 0.99. Finally, the GU shifts between the original IgG glycan
pool were compared with the sialidase treated traces suggesting
the presence of several monosialo and disialo structures with GU
values between 4 and 5, as well as between 6 and 7
representing the core fucosylated biantennary structures with
one or two sialic acid residues containing 2-3 or 2-6 linkages.
Figure 3. Identified IgG glycan structures using the combination of
CE-LIF and consecutive exoglycosidase digestions.
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Following the above explained scheme, the structures of all IgG
glycans in Figure 1, trace A was identified as shown in Figure 3.
For structural elucidation, GU values listed in Table 1 were used.
Table 1. Abbreviated names, structures and CE glucose unit (GU)
values of IgG N-glycans.
Conclusions
A high fidelity glycan sequencing scheme was demonstrated
using capillary electrophoresis-laser induced fluorescence
detection in combination with exoglycosidase digestion steps.
This method allowed multi-structure glycan sequencing without
the necessity for isolation of the individual components for
analysis. The PNGase F released and APTS labeled N-glycan
pool was subject to sequential exoglycosidase digestion using
sialidase, fucosidase, galactosidase and finally hexosaminidase.
Following enzymatic cleavage, the resulting reaction products
were analyzed using CE-LIF and corresponding GU values and
shifts were determined. Through the analysis of all cleavage
data, bottom up identification of all individual structures was
accomplished using the corresponding GU database values.
Capillary electrophoresis provided excellent resolving power to
separate positional and linkage isomers, making sequence
determination easier. The high sensitivity of laser induced
fluorescence (LIF) detection enabled trace level analysis, i.e.,
even minor glycan structures were detected and structurally
elucidated.
Acknowledgement
The authors also acknowledge the support of the MTA-PE
Translation Glycomics project (#97101).
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References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Guttman, A., Bioanalytical Tools for the Characterization of Biologics and Biosimilars. LC.GC Magazine, 2012. 30 p. 412-421.
Varki, A., R. Cummings, J. Esko, H. Freeze, P. Stanley, C. Bertozzi, G.W. Hart, and M. Etzler, Essentials of Glycobiology. 2nd
ed2009, Plainview, NY: Cold Spring Harbor Laboratory Press.
Hayes, J.M., E.F. Cosgrave, W.B. Struwe, M. Wormald, G.P. Davey, R. Jefferis, and P.M. Rudd, Glycosylation and Fc
receptors. Curr Top Microbiol Immunol, 2014. 382: p. 165-99.
Mittermayr, S., J. Bones, and A. Guttman, Unraveling the glyco-puzzle: glycan structure identification by capillary
electrophoresis. Anal Chem, 2013. 85(9): p. 4228-38.
Guttman, A., High-resolution carbohydrate profiling by capillary gel electrophoresis. Nature, 1996. 380(6573): p. 461-2.
Mittermayr, S. and A. Guttman, Influence of molecular configuration and conformation on the electromigration of
oligosaccharides in narrow bore capillaries. Electrophoresis, 2012. 33(6): p. 1000-7.
Guttman, A., Multistructure sequencing of N-linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion.
Electrophoresis, 1997. 18(7): p. 1136-41.
Szabo, Z., A. Guttman, J. Bones, R.L. Shand, D. Meh, and B.L. Karger, Ultrasensitive capillary electrophoretic analysis of
potentially immunogenic carbohydrate residues in biologics: galactose-alpha-1,3-galactose containing oligosaccharides. Mol
Pharm, 2012. 9(6): p. 1612-9.
Harvey, D.J., A.H. Merry, L. Royle, M.P. Campbell, R.A. Dwek, and P.M. Rudd, Proposal for a standard system for drawing
structural diagrams of N- and O-linked carbohydrates and related compounds. Proteomics, 2009. 9(15): p. 3796-801.
Varadi, C., C. Lew, and A. Guttman, Rapid magnetic bead based sample preparation for automated and high throughput Nglycan analysis of therapeutic antibodies. Anal Chem, 2014. 86(12): p. 5682-7.
Guttman, A., Analysis of monosaccharide composition by capillary electrophoresis. J Chromatogr A, 1997. 763(1-2): p. 271-7.
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