development of a rapid 2-ab sample preparation workflow ... · preparation workflow for n-glycan...
TRANSCRIPT
Application Note
Pharm & Biopharma
AuthorsVaishali Sharma, Andres Guerrero, John Yan, Aled Jones, Michael Kimzey, Emily Dale, Ted Haxo, and Sergey Vlasenko Agilent Technologies, Inc.
AbstractThe characterization of N-glycans is essential to the development of biotherapeutics. Typically, enzymatically released N-glycans are derivatized with a tag to allow for fluorescence (FLR) and mass spectrometry (MS) detection by HILIC UHPLC-FLR and UHPLC/MS. N-glycan sample preparation often requires numerous hours or days to complete. Although newer fluorescent tags such as Agilent InstantPC (formerly ProZyme) provide high FLR and MS sensitivity, 2-AB (2-aminobenzamide) is a tag that has been used to generate N-glycan data for more than 20 years and is well established in many laboratories. Presented herein is the development and application of a rapid N-glycan sample preparation workflow using a five-minute in solution deglycosylation step followed by direct on-matrix 2-AB labeling and cleanup without the need for a dry down step, samples are ready for analysis in approximately 2 hours.
We present a comparison study consisting of two monoclonal antibodies (MabThera, NISTmAb) and one Fc fusion protein (Enbrel), using two different N-glycan sample preparation workflows offered by Agilent: 1) Agilent GlykoPrep Rapid N-Glycan Prep with 2-AB; 2) Agilent AdvanceBio Gly-X N-Glycan Prep with 2-AB Express (formerly ProZyme). Agilent AdvanceBio Gly-X technology and 2-AB Express labeling offers much-improved time to results compared to traditional sample preparation methods.
Development of a Rapid 2-AB Sample Preparation Workflow for N-Glycan Release and Labeling
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IntroductionThe structure of N-linked glycans can play a critical role in the pharmacology of therapeutic proteins, potentially affecting immunogenicity, pharmacokinetics and pharmacodynamics.1 This makes the characterization of N-glycans an essential part of the biotherapeutic development process. N-glycans do not contain a chromophore or fluorophore suitable for online detection with standard liquid chromatography (LC) techniques, so they are commonly derivatized with fluorescent tags such as 2-AB by reductive amination chemistry (Figure 1) after enzymatic release with PNGase F. The most commonly used reducing agent used for this purpose is sodium cyanoborohydride.2 Traditional 2-AB labeling methods require multiple steps including drying released glycans prior to labeling and subsequent cleanup of excess dye label that can take hours if not days to complete.3 GlykoPrep with traditional reductive amination dyes such as 2-AB streamlined the sample
Figure 1. Labeling of enzymatically released N-glycans with 2-AB. The primary amine of the dye attacks the vcarbonyl carbon of the acyclic reducing sugar to form a partial Schiff’s base. The imine group of the Schiff’s base is chemically reduced to give the labeled glycan.
O
H2N
H2N
H2N
O
O
O
OHOH
Glycan Glycan
NHAcNHAc
OHOH
OH
OH
CH2O(R) CH2O(R)
CH2O(R)
HN
Glycan
NHAc
N
Schiff’s Base
Reductant
Labeled glycanCyclic/acyclic
2-AB reductive amination of N-glycans
2-AB
2-AB
+
preparation process allowing the process to be completed in four to five hours. Gly-X N-Glycan Prep with 2-AB Express is a simplified and rapid workflow, where in solution deglycosylation releases N-glycans in five minutes and
2-AB labeling occurs on a solid-state matrix, where excess dye is washed away with acetonitrile before eluting labeled samples with DI water. Samples are typically ready for UHPLC analysis in two hours (Figure 2).
Figure 2. N-glycan sample preparation workflows: Agilent GlykoPrep and Agilent AdvanceBio Gly-X with 2-AB Express.
Dry down30 to 60 min
• Solid phase• Vacuum driven
Cleanup15 to 30 min
Denature5 min
Deglycosylate15 to 60 min
Label60 min
Agilent GlykoPrep with 2-ABTotal time ~3.5 hours
On-matrix deglycosylationIn-solution labelingOn-matrix cleanup
Automation-ready format
Agilent AdvanceBio Gly-X with 2-AB Express
Total time ~2 hoursIn-solution deglycosylation
On-matrix labeling and cleanupAutomation-ready format
N-glycan sample prep workflows
Cleanup
10 to 15 min
Denature
3 min
Deglycosylate
5 min
Label
60 min
No dry down
sodium cyanoborohydride
or alternative developmental
reductant
3
Methods
Sample preparationN-Glycan samples from MabThera (lot no. H0102B03), Enbrel (lot no. R170724), and NIST monoclonal antibody reference material 8671 (NISTmAb) (lot no. 14HB-D-002) were prepared with Agilent’s Glyko-Prep Rapid N-Glycan Prep with 2-AB (GP96NG-AB) and Agilent AdvanceBio Gly-X N-Glycan Prep with 2-AB Express (GX96-2AB) (formerly Prozyme) kits, following standard recommended protocols using 50 µg of protein per preparation. All samples were prepared in triplicate.
HILIC-UHPLC analysisEluted samples were adjusted to a final volume of 100 µL prior to analysis (1 µL injection). Glycans were separated by hydrophilic interaction liquid chromatography (HILIC). HILIC-UHPLC separation was performed on an Amide 2.1 × 150 mm, 1.7 µm column using 25 to 38% 50 mM ammonium formate pH 4.4, between 2.5 to 50 minutes at a flow rate of 0.4 mL/min. Labeled glycans were monitored by fluorescence detection Ex/Em (nm) 360/428. Glycan assignments were made by comparison to existing Agilent data, mass determination by LC/MS,4,5 and for the NISTmAb, published data.6,7
Results and discussionHILIC-UHPLC analysis of 2-AB labeled N-glycans showed comparable results between the two different sample preparation methods GlykoPrep with 2-AB and, Gly-X with 2-AB Express
• MabThera: Like most monoclonal antibodies produced in CHO cells, the glycosylation pattern of MabThera consists of mostly neutral biantennary glycans where G0F, G1F[6]/G1F[3] and G2F predominate with low levels of siaylated and high mannose species (Figure 3).
Figure 3. Overlay of 2-AB UHPLC fluorescence profiles of N-glycans from MabThera prepared with Agilent GlykoPrep with 2-AB and Agilent AdvanceBio Gly-X with 2-AB Express labeling.
MabThera 2-AB
G0
G0F
G1[6]
G1F[6]
G1F[3] G2F
G0F-NMan5
A2FA1F
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Agilent AdvanceBio Gly-X with 2-AB Express
Agilent GlykoPrep 2-AB
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• Enbrel: In contrast to MabThera, the glycosylation profile of Enbrel contains much higher levels of sialylation as well as afucosyl glycans as seen with A1F, A2F, A1, and A2 (Figure 4).
• NISTmAb: Similar to MabThera, the NISTmAb contains mostly G0F, G1F[6]/[3], and G2F. However, the NISTmAb also contains glycans with single and double galactose-α(1,3)-galactose epitopes on neutral species (Figure 5) as in published data.6
Enbrel 2-AB
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G0
G0F
Man5
G1[6]
G1F[6]
G1F[3]
G2
G2F
A1
A2
A2F
G0F-N
G1FS1
A1F
EU
Minutes8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Agilent AdvanceBio Gly-X Express
Agilent GlykoPrep 2-AB
Figure 4. Overlay of Agilent 2-AB UHPLC (formerly ProZyme) fluorescence profiles of N-glycans from Enbrel prepared with Agilent AdvanceBio Gly-X with 2-AB Epxress labeling and Agilent GlykoPrep with 2-AB.
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64 NISTmAb 2-AB
G0F
G1[6]
G1F[6]
G1F[3]G2F
G0F-NMan5
G1FS1-NG2F +1αGal
G2F + 2αGal
G1F-N +1αGal
Agilent AdvanceBio Gly-X Express
Agilent GlykoPrep 2-AB
Minutes8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
EU
Figure 5. Overlay of 2-AB UHPLC fluorescence profiles of N-glycans from NISTmAb prepared with Agilent AdvanceBio Gly-X N-Glycan Prep with 2-AB Express and Agilent GlykoPrep Rapid N-Glycan Prep with 2-AB.
0
5
10
15
20
25
GlykoPrepwith 2-AB
GlykoPrepwith 2-AB
Gly-Xwith 2-ABExpress
GlykoPrepwith 2-AB
Gly-Xwith 2-ABExpress
MabThera Enbrel NISTmAbTo
tal
FLR
Gly-Xwith 2-ABExpress
×106
Figure 6. Comparison of 2-AB total fluorescence response. Glycans from equivalent amounts of glycoprotein (MabThera, Enbrel, and the NISTmAb) prepared were prepared with Agilent AdvanceBio Gly-X N-Glycan Prep with 2-AB Express and Agilent GlykoPrep Rapid N-Glycan Prep with 2-AB. All eluted samples were adjusted to 100 µL final volume prior to UHPLC – FLR analysis (1 µL injection).
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• Higher total fluorescence signal was observed for Gly-X sample preparation versus GlykoPrep for molecules MabThera and Enbrel. NISTmAb total fluorescence was comparable for both Gly-X and GlykoPrep.
• The two sample preparation methods provided similar reported relative % areas for the three molecules tested (Figure 7).
Figure 7. Comparison of relative % areas for N-glycans prepared with Agilent GlykoPrep with 2-AB and Agilent AdvanceBio Gly-X with 2-AB Express. (A) MabThera, (B) Enbrel, (C) NISTmAb. Glycans >0.5% relative area reported for MabThera/Enbrel and >1.0% for NISTmAb.
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G0F-N G0 G0F Man5 G1[6] G1F[6] G1F[3] G2F A1F A2F
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ve %
are
a
A MabThera
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G0 G0F Man[5] G1[6] G1F[6] G1F[3] G2 G2F G1FS1 A1 A1F A2 A2F
B Enbrel
Re
lati
ve %
Are
a
G0F Man5 G1[6] G1F[6] G1F[3] G1F-N +1αGal
G2F G2F + 1αGal
G2F +2αGal
C NISTmAb
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G0F-N
Agilent AdvanceBio Gly-X with 2-AB Express
Agilent GlykoPrep with 2-AB
Agilent AdvanceBio Gly-X with 2-AB Express
Agilent GlykoPrep with 2-AB
Agilent AdvanceBio Gly-X with 2-AB Express
Agilent GlykoPrep with 2-AB
www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019 Printed in the USA, July 31, 2019 5994-0945EN TN 4012 Rev B
Conclusion• Agilent GlykoPrep Rapid N-Glycan
Prep with 2-AB labeling of released N-glycans offers reproducible and reliable results that can be directly compared to historic data.
• Agilent AdvanceBio Gly-X N-Glycan Prep with 2-AB Express offers a rapid workflow with traditional 2-AB labeling without the need for lengthy dry down steps, shortening the time to result.
• Preparation of 2-AB labeled N-glycans from MabThera, Enbrel and NISTmAb with GlykoPrep and Gly-X results in comparable data in terms of total fluorescence signal and reported relative percent areas.
• The NISTmAb N-glycan profile is consistent with published data.
References1. Liu, L. Antibody glycosylation and its
impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins. J. Pharm. Sci. 2015, 104(6), 1866–1884.
2. Ruhaak, R.; et al. Glycan labeling strategies and their use in identification and quantification. Anal Bioanal Chem. 2010, 397(8), 3457–3481.
3. Aich, U.; et al. State-of-the-art technologies for rapid and high-throughput sample preparation and analysis of N-glycans from antibodies. Electrophoresis 2016, 37(11), 1468-88.
4. Kimzey; et al. Development of an instant glycan labeling dye for high throughput analysis by mass spectrometry. Agilent poster, ASMS 2015.
5. Yan; et al. Comparison of common fluorescent labels for liquid chromatography analysis of released N-linked glycans. Agilent poster, ASMS 2017.
6. De Leoz, M.L.A.; et al. NISTIR 8186: NIST Interlaboratory Study on the Glycosylation of NISTmAb, a Monoclonal Antibody Reference Material, June 2015 to February 2016. 2017, http://nvlpubs.nist.gov/nistpubs/ir/2017/NIST.IR.8186.pdf
7. Hilliard, M.; et al. Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis. MAbs 2017, Sep 12:1-11. doi: 10.1080/19420862.2017.1377381. [Epub ahead of print].