Agilent AdvanceBio Sialic Acid Profiling and Quantitation Kit User Manual
Agilent AdvanceBio Sialic Acid Profiling and Quantitation Kit
User Manual
Notices
CAUTION
WARNING
© Agilent Technologies, Inc. 2021
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Manual Part Number
5994-2800EN
GS24-SAP
Rev. AA beta
Edition
Second edition, January 2021
Printed in USA
Agilent Technologies, Inc.
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Contents
Introduction 5
Kit Workflow 6
Kit Components 7
Equipment and Reagents Provided By User 8
Sample Considerations 9
Kit Capacity 10
Protocol 11
Getting started 11
Performing the assay 11
Sample Analysis by UHPLC or HPLC 14
Suggested MS conditions for DMB Labeled Sialic Acids 15
FAQs 16
References 18
Technical Assistance 18
Agilent AdvanceBio Sialic Acid Profiling and Quantation Kit User Manual 3
4 Agilent AdvanceBio Sialic Acid Profiling and Quantation Kit User Manual
Introduction
Glycans are carbohydrates composed of monosaccharides arranged into many different possible
oligosaccharide structures based on composition and linkage position. Sialic acid capping at the
nonreducing terminal of N- or O-glycans can serve a key role biological processes and in
mediating the effectiveness of therapeutic glycoproteins.1 The composition of glycans present on
biotherapeutic glycoproteins can affect immunogenicity, pharmacokinetics, and
pharmacodynamics.1 Depending on the molecule and the application, terminal sialic acid may
reduce the rate of clearance, reduce antibody-dependent cellular cytotoxicity (ADCC) activity, or
can be anti-inflammatory.3
N-acetylneuraminic acid (NANA or Neu5Ac) and N-glycolylneuraminic acid (NGNA or Neu5Gc).
Neu5Ac is usually the predominant species, while Neu5Gc is not synthesized by humans and its
presence on biotherapeutics can potentially be immunogenic. Because of this, it is essential to
monitor not only the absolute quantity of sialic acid, but also the levels of different sialic acid
species present in therapeutic glycoproteins.
The AdvanceBio Sialic Acid profiling and quantitation kit represents a sensitive, high-throughput
approach to sialic acid quantitation. Sialic acids are released from glycoproteins using acid
hydrolysis, followed by derivatization with the fluorophore 1,2-diamino
4,5-methylenedioxybenzene (DMB), allowing for separation by reversed-phase (RP) liquid
chromatography with fluorescence detection (FLD).
The workflow offers both qualitative characterization of sialic acid species using a sialic acid
reference panel (SARP), as well as quantitation with picomolar level sensitivity using included
NANA and NGNA quantitative standards. The workflow enables reliable and reproducible
high-throughput profiling and quantitation of sialic acids, providing a broad detection range and
improved sensitivity for molecules with low levels of sialylation versus traditional DMB labeling
workflows such as our GKK-407 kit.
-
5 Two sialic acid species commonly found in biotherapeutics are
Sialic acid species comparison may be made by labeling the included qualitative sialic acid
reference panel (SARP, p/n GKRP-2503), which includes the following sialic acid species: Neu5Gc
(NGNA); Neu5Ac (NANA); Neu5,7Ac2; Neu5Gc,9Ac; Neu5,9Ac2; Neu5,7(8)9Ac3.
Use of the AdvanceBio Sialic Acid profiling and quantitation kit offers several advantages:
• 96-well format
• Broad range of detection of sialic acid levels, from 1 to 2,000 pmol
• Picomolar sensitivity for proteins with low levels of sialylation
• Quantitative NANA and NGNA standards are included
• Workflow may be completed in five hours including incubation periods
• Automation-friendly: please contact us for assistance, we are happy to provide guidance on
how the kit can be implemented on your automation platform of choice.
Agilent AdvanceBio Sialic Acid Profiling and Quantation Kit User Manual 5
Kit Workflow
Step 1: Release of sialic acid using acid hydrolysis (2 hours)
Sialylated Glycoprotein + acid Sialic Acid + Glycoprotein
Step 2: Labeling of released sialic acid (3 hours)
Samples are now ready for separation by reversed-phase liquid chromatography.
Figure 1. Sialic acid release and DMB labeling workflow.
Neu5Gc
OH
HO
HN
HO
O
OH
OH
O
HO
COOH
Ring
opening
HO
OH
HO
HN
O
O
OH
HO
OH
OH
O
Figure 2. DMB labeling mechanism of sialic acid Neu5Gc.
DMB-labeled
DMB
H
N
2
+
H2N
2 H2O
O
O
Neu5Gc
OH
OH
HO
HN
HO
HO
O
O
O
N
N
OH
OH
6 Agilent AdvanceBio Sialic Acid Profiling and Quantation Kit User Manual
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