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FLUORESCENT AND MS-ACTIVE LABELING REAGENT FOR THE ANALYSIS OF N-GLYCANS

Darryl W. Brousmiche, Matthew A. Lauber, Qi Wang, Stephan M. Koza, Ying-Qing Yu

Waters Corporation, 34 Maple Street, Milford, MA, USA 01757

Traditional sample preparation techniques for the analysis of N-glycans have either been time-consuming and labor intensive, or require compromises in sensitivity.1 These restrictions result in

throughput issues, or limit the level of detail that can be obtained regarding the glycosylation of a sample. In the case of one of the most frequently used labeling compounds, 2-aminobenzamide

(2-AB), the resulting glycans can be readily detected by fluorescence but are difficult to detect by electrospray ionization mass spectrometry (ESI-MS). Variations of conventional approaches for

N-glycan sample preparation have been explored, but have not presented solutions that combine the desired attributes of simplicity, high MS sensitivity, and high throughput. For example, rapid

tagging procedures that yield labeled glycans in minutes have been developed2, however, they did not provide the enhanced ionization efficiencies needed in modern N-glycan MS analyses.

As part of our R&D work into improving biopharmaceutical workflows, we have developed a novel labeling reagent, RapiFluor-MS®, which reacts rapidly with released N-glycans and provides

unrivalled sensitivity for their detection through a highly fluorescent chromophore and a tertiary amine for enhancing MS detection.3,4

By incorporating this reagent into an optimized sample

preparation workflow for deglycosylation and HILIC-based sample cleanup, we have been able to achieve highly quantitative recovery of the tagged glycans, that are ready for analysis, in under an

hour. This poster will provide a detailed overview of this reagent and workflow, along with comparisons to other commercially available tagging solutions.

Sample Preparation

To compare response factors for Instant AB, and RapiFluor-MS tagged glycans, labeling was performed with equivalent molar excesses on a common protocol. Crude reaction mixtures were directly analyzed by HILIC-FLR-MS in order to avoid potential biases from SPE clean-up procedures.

For the 2-AB vs. RapiFluor-MS labeled glycans comparisons, equivalent quantities of labeled N-glycans from pooled human IgG were utilized. Column loads were calibrated using external quantitative standards.

To compare MS response factors of Instant PC and RapiFluor-MS tagged N-glycans as part of the GlykoPrep, Gly-X

, GlycoWorks kit workflows, samples

were prepared using the manufacturer’s suggested protocols and analyzed using a Xevo

® G2-XS QToF.

LC Method Conditions

LC system: ACQUITY UPLC H-Class Bio System Sample Temp.: 5 °C ColumnTemp.: 60 °C Flow Rate: 0.4 mL/min Fluorescence Detection: Ex 265 / Em 425 nm (RapiFluor-MS) Ex 278 / Em 344 nm (Instant AB) Ex 330 / Em 420 nm (2-AB) Ex 285 / Em 345 nm (Instant PC) 5 Hz scan rate; Gain + 1 Column: ACQUITY UPLC Glycan BEH Amide 130 Å 1.7 µm, 2.1 x 50 mm Gradient: Mobile Phase A: 50 mM NH4HCO2, pH 4.4 (LC-MS grade)

Mobile Phase B: ACN (LC-MS grade)

Time (min) Flow Rate (ml/min) %A %B Curve 0.0 0.4 25 75 6 11.7 0.4 46 54 6 12.2 0.2 100 0 6 13.2 0.2 100 0 6 14.4 0.2 25 75 6 15.9 0.4 25 75 6 18.3 0.4 25 75 6

MS Conditions

MS system: Synapt® G2-S HDMS Xevo G2-XS QToF

Analyzer mode: ESI+, TOF MS ESI+, TOF MS Capillary voltage: 3.0 kV 2.2 kV Cone voltage: 80 V 75 V Source temp.: 120 °C 120 °C Desolvation temp.: 350 °C 500 °C Desolvation gas flow: 800 L/Hr 600 L/Hr Acquisition: 500–2500 m/z, 1 Hz 700–2000 m/z, 0.5 Hz Data management: MassLynx MassLynx

1. Mechref, Y. et al, Quantitative glycomics strategies. Mol Cell Proteomics 2013, 12 (4), 874-84. 2. Cook, K. S. et al, Development and qualification of an antibody rapid deglycosylation method.

Biologicals 2012, 40 (2), 109-17. 3. Lauber, M.A. et al, Rapid preparation of released N-Glycans for HILIC analysis using a

labeling reagent that facilitates sensitive fluorescence and ESI-MS detection. Anal Chem, 2015, 87, 5401-09

4. US Patent Applications 20140179011 and 20140350263 5. Klapoetke, S. et al, The evaluation of a novel approach for the profiling and identification of N-

linked glycan with a procainamide tag by HPLC with fluorescent and mass spectrometric detection. J Pharm Biomed Anal 2010, 53 (3), 315-24.

6. For more experimental details and additional results, see Waters Application Note “Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent ”

RESULTS AND DISCUSSION

Synthesized based on rational design considerations. Affords rapid labeling kinetics, high fluorescence quantum

yields, and significant MS detectability.

Ambient, aqueous reaction conditions.

5 minute reaction time. Yields highly stable urea linkage.

RapiFluor-MS - A New N-Glycan Labeling Reagent

Reaction Pathways for Derivitization

of an N-Glycan

CONCLUSIONS

REFERENCES

METHODS

Relative Response Factors

High-Sensitivity Fluorescence and MS Detection

Tagged equivalent quantities of released N-glycans

(0.4 µg of a murine intact mAb) with RapiFluor-MS and an

alternative rapid labeling reagent - Instant AB™.

Utilized chromatographic peak areas for the fucosylated,

biantennary FA2 glycan to compare fluorescence and MS

response factors.

RapiFluor-MS tagged glycans produce 2x higher

fluorescence signal and 780x higher MS signal.

RapiFluor-MS was also compared to conventional

reductive amination tags - 2-AB and procainamide.

Response factors were normalized to the fluorescence

and MS signals of the RapiFluor-MS labeled N-glycans.

Significantly higher responses were observed for the

RapiFluor-MS labeled glycans.

Optimized SPE Workflow - GlycoWorks®

Final clean-up prior to analysis is achieved via a HILIC

mechanism, using an aminopropyl silica SPE sorbent,

which selectively extracts the labeled glycans from

reaction by-products.

Significant chromatographic improvements are observed

for Rapi-Fluor-MS labeled glycans (pooled human IgG/

bovine fetuin sample, 0.4 µg glycoprotein) following this

SPE protocol. (A) - No SPE; (B) - After SPE.

SPE results in no significant compromise in the accuracy

of the relative abundances determined for a wide range

of N-glycans. They remain essentially constant for one

vs. two passes through the HILIC SPE (C).

0

5

10

15

20

25

30

Rela

tive A

bundance (

%)

Positive

Control

SPE

Processed

0.0E+0

1.9E+6

0 5 10 15

0.0E+0

2.3E+6

0 5 10 15

After SPE

(1x SPE)

FA2 FA2G2S1

A3G3S3

A3S1G3S3

C

B

A No SPE

(Crude Reaction Mixture)1x SPE

2x SPE

min

Conventional N-glycan sample preparation is time

consuming due to lengthy deglycosylation labeling and

sample preparation steps (up to 16 hours).

A three-step optimized workflow was developed, going

from glycoprotein to injection, in 30 minutes with an

overall yield of 73%.

Complete deglycosylation is achieved in ~ 10 min using

RapiGest™ surfactant, a novel, rapid PNGase F and a

custom buffer.

Comparative MS Sensitivity

Unprecedented MS and fluorescence sensitivity have

been achieved for labeled glycans using RapiFluor-MS.

Preparation of labeled N-glycans - from glycoprotein to

analysis ready sample - can be done in under an hour.

Accurate profiling, based on robust SPE, is achieved for

neutral to tetrasialylated N-glycans.

A simple, efficient, streamlined protocol is provided with

the GlycoWorks RapiFluor-MS N-Glycan Kit.

(*) Comparative result extrapolated from a published comparison of N-glycans.5

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Fluorescence MS (BPI)

Instant AB Labeled

RapiFluor-MS Labeled

2-AB Labeled

Rela

tive P

erf

orm

ance (%

)

52.5

7.0

0.1 0.6

Procainamide Labeled

0

10

20

30

40

50

60

70

80

90

100

30.0*

7.0*

0.0E+0

2.6E+6

4.5 5.0 5.5 6.0 6.5 7.0 7.5

0.0E+0

2.6E+6

4.5 5 5.5 6 6.5 7 7.5

0.0E+0

1.7E+6

4.5 5 5.5 6 6.5 7 7.5

0.0E+0

3.0E+3

4.5 5 5.5 6 6.5 7 7.5

300x zoom

0.0E+0

1.7E+6

4.5 5 5.5 6 6.5 7 7.5

FLR

MS(BPI)

FLR

MS(BPI)

342.8

179.9

233.7

0.30

100

200

300

400

Compound 4 Compound 1

Repo

nse

Fact

ors

(FA2

Pea

k Ar

ea p

er S

ampl

e of

N-G

lyca

ns

from

1 µ

g of

Ant

i-Citr

inin

IgG

/ 1

000)

A FA2

FA2

FA2

FA2

RapiFluor-MS

Labeled

Instant AB

Labeled

B

C FLR

MS(BPI)

Resp

onse

Fac

tors

(FA2

Pea

k Ar

ea p

er S

ampl

e of

N-G

lyca

ns

from

1 µ

g of

Inta

ct m

AbM

ass

Chec

k St

anda

rd /

100

0)

RapiFluor-MS Labeled N-Glycans

from Intact mAb Mass Check Standard (0.4μg)

Instant ABTM Labeled N-Glycans

from Intact mAb Mass Check Standard (0.4μg)

min

min

2.6 E+6

0

0

4.5

1.7 E+6

5 5.5 6.5 6 7 min

4.5 5 5.5 6.5 6 7 min

300 x Zoom

FLR FA2 FA2G1 FA2G2

MS (BPI)

FLR

0

2.6 E+6

0

1.7 E+6

MS (BPI)

Relative responses of commercially available MS active

N-glycan tagging agents were measured using the

manufacturer’s kit and recommended protocols with a

murine intact mAb.

Almost 2x higher MS signal was observed for the

GlycoWorks® RapiFluor-MS kit and protocol.

GlycoWorks RapiFluor-MS

GlykoPrep® Instant PC™

Gly-X™ Instant PC™

400

200

0

400

233 247

Instant AB, Instant PC, Gly-X and GlykoPrep are trademarks of Prozyme, Inc. GlycoWorks, RapiFluor-MS, Xevo and Synapt are trademarks of Waters Corporation, registered in the U.S. and other countries. RapiGest is a trademark of Waters Corporation.

INTRODUCTION

HPLC 2017: Poster APP8-P08-Tu Contact: Darryl_Brousmiche@Waters.com