The Next Generation of Glycan Sample Preparation …...H-Class Bio System configured with FLR and...

[ GLYCAN ANALYSIS SOLUTIONS ]

The Next Generation of Glycan Sample Preparation and Analysis

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What if... identifying WHERE glycans are present on a glycoprotein

becomes part of your routine workflow?

4

[ GLYCAN ANALYSIS SOLUTIONS ] [ GLYCAN ANALYSIS SOLUTIONS ]

RELEASED GLYCAN

ANALYSIS

MONOSACCHARIDE SIALIC ACID

COMPOSITION

Met

hod

com

plex

ity

Increasing information

MIDDLE UP/DOWN - SUBUNIT ANALYSIS

GLYCOPEPTIDE MAPPING

INTACT PROTEIN ANALYSIS

Bringing Together Complementary Techniques to Streamline Glycan AnalysisWaters offers complete, workflow-based approaches for analyzing glycoproteins at all structural levels.

¡ Glycoprotein profiling ¡ Released glycan characterization and monitoring

¡ Middle up/down - Subunit analysis ¡ Monosaccharide/Sialic acid composition

¡ Glycopeptide mapping

2


Intact Protein Analysis Workflow

INJECTION ON COLUMN FOR LC ANALYSISACQUITY UPLC® Glycoprotein BEH Amide, 300Å Column,

and MassPREP™ Desalting Cartridge

ANALYSIS ON LCWITH TUV OR FLR DETECTORACQUITY UPLC

Systems

GLYCOPROTE IN

Glycoprotein Performance Test Standard

Subunit Analysis Workflow

Glycopeptide Mapping Workflow

Monosaccharide/Sialic Acid Composition Workflow

INJECTION ON COLUMN FOR LC ANALYSIS

ACQUITY UPLC Glycoprotein BEH Amide, 300Å Column, and Glycoprotein Performance Test Standard


ACQUITY UPLC Glycoprotein BEH Amide, 300Å Column, and Glycoprotein Performance Test Standard

LABEL WITH A FLUOROPHORE

ANALYSIS ON LCWITH UV DETECTOR

ACQUITY UPLC Systems

ANALYSIS ON LC WITH TUV DETECTOR

ACQUITY UPLC Systems

Injection on Column for LC Analysis

ACQUITY UPLC BEH C18, 130Å, 1.7 μm,

2.1 x 100 mm Column

IdeS ENZYMATIC DIGESTION WITH OPTIONAL REDUCTION

REDUCTION, ALKYLATION, AND ENZYME DIGESTION(S)

Glycosylated and non-glycosylated peptide fragments

RELEASE OF MONOSACCHARIDES

Hydrolyze with acid

Intact mAb Mass Check Standard



GLYCOPROTE IN

GLYCOPROTE IN

GLYCOPROTE IN

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MASS CONFIRMATION WITH QTof MS

Xevo® G2-XS QTof MS, andSYNAPT® G2-Si HDMS

DATA PROCESSINGUNIFI,® and

MassLynx® Software

MASS CONFIRMATION USING QTof MS

Xevo G2-XS QTof MS, and SYNAPT G2-Si HDMS

PEPTIDE MAPPING ANALYSIS USING QTof MS

Xevo G2-XS QTof, and SYNAPT G2-Si HDMS

Analysis on LCwith FLR Detector

Alliance HPLC orACQUITY UPLC Systems

DATA PROCESSINGMassLynx Software

DATA PROCESSINGMassLynx, and UNIFI Software

DATA PROCESSINGEmpower® 3 Software

50

45

40

35

30

25

20

EU

15

10

5

0

10 15 20 25 30 min-5

1

2

3 4 5

6

1. N-acetylglucosamine (GlcNAc)2. N-acetylgalactosamine (GalNAc)3. Galactose (Gal)4. Mannose (Man)5. Glucose (Glc)6. Fucose (Fuc).

Results

Results

0.0

0.5

1.0

0 5 10 15 20 25 30 35 40 45 min

A 214

Glycoprotein BEH Amide 300Å 1.7 µm

Non-glycosylatedPeptides

Glycopeptides

Lys-C

Results

0.00

0.04

0.08

0.12

0.16

6 7 8 9 10 11 12 13 min

A21

4

Fc/2 Glycoforms

Fd’

LC Results

EU

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

5 10 15 20 min

1

2

3

4

5

6

Glycoprotein Performance Test Standard

Peak Species Rs

1 RNase A - 2 RNase B (+Man 5) 21.2 3 RNase B (+Man 6) 3.5 4 RNase B (+Man 7) 2.7 5 RNase B (+Man 8) 2.6 6 RNase B (+Man 9) 3.1

RapiFluor-MS labeled N-glycans

monitored using an ACQUITY UPLC

H-Class Bio System configured with FLR

and ACQUITY QDa mass detection for

confirmation of known structures and

mass data for unassigned peaks.

A2

– 81

5.1

FA2

– 88

8.2

FA2B

– 9

89.6

A2G

1a –

896

.2A

2G1b

– 8

96.0

FA2G

1a –

969

.3

FA2B

G1a

– 1

070.

7FA

2G1b

– 9

69.2

0

240

480

min10 15 20 25 30

Inte

nsity

0

1.6x106

3.2x106

min10 15 20 25 30

EU

FA2B

G1b

– 1

070.

6A

2G2

– 97

7.1

FA2G

2 –

1050

.3FA

2BG

2 –

1151

.8FA

2G1S

1 –

1114

.6

UN

AS

SIG

NE

D –

811

.1U

NA

SS

IGN

ED

– 1

122.

4

FA2G

2S1

– 11

96.0

FA2B

G2S

1 –

865.

3

FA2G

2S2

– 89

4.7

FA2B

G2S

2 –

962.

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ROUTINE MONITORING

Explore the possibilities

Due to the increased ionization efficiency and higher proton affinity of RapiFluor-MS, mass detection

of glycans using the ACQUITY QDa Detector is finally possible.

¡ Provides unrivaled sensitivity for FLR and MS detection.

¡ Offers greater confidence with mass confirmation of FLR peaks.

¡ Reduce chromatography analysis times with targeted MS glycan profiling for rapid process development.

¡ Can be easily integrated with existing GMP-compliant-ready UPLC workflows with Empower 3 Software.

Glycan Monitoring with ACQUITY UPLC H-Class Bio System with FLR and ACQUITY QDa Detection

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Introducing the ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Column

You need to know WHAT your glycan structures are, WHY not also know WHERE they are…

With Waters, now you can combine approaches for comprehensive characterization. The ACQUITY UPLC® Glycoprotein

BEH Amide 300Å Column’s wide pore stationary phase and optimized amide bonding, provides unprecedented, MS

compatible HILIC-based separations for the analysis of intact glycoproteins, glycoprotein fragments, and glycopeptides.

This new, patent pending column technology allows access to information about:

¡ Site-specific glycan structures

¡ Glycan occupancy

One column that brings separation opportunities never seen before…

HILIC INTACT PROTEIN

0-LINKS N-LINKS

HILIC PROTEIN SITE OCCUPANCY

HILIC PROTEIN SUBUNIT

HILIC GLYCOPEPTIDE MAPPING

HILIC Separations of Glycosylated Protein Fragments.

0.00

0.04

0.08

0.12

0.16

6 7 8 9 10 11 12 13 min

A21

4

Fc/2 Glycoforms

Fd’

LC

Glycopeptide Mapping of a Lys-C Digested mAb.

0.01

0.04

0.07

25 26 27 28 29 30 31 32 33 34 35 36 37 38 min

A21

4

A1

M5

FA2G1

FA2G2FA1G1

A2G1

A2FA1

FA2-17.03 Da

FA2G1-17.03 Da

FA2G1’

FA2

HC:K16TKPREEQYNSTYRVVSVLTVLHQDWLNGK

Glycoforms

Intact Protein HILIC of Ribonuclease Glycoforms. Resolving High Mannose N-Glycosylation.

0

0.02

0.04

0.06

6 8 10 12 14 16 18 20 min

A21

4

+Man5

+Man6

+Man7

+Man8

+Man9

RNase Baglycosylated

HILIC BEH Amide300Å

0E+0

Agl

ycos

ylat

ed

PNGase F * Truncated

C-terminus

7 15 min 8 9 10 11 12 13 14

O-linkedGlycan

Intri

nsic

Flu

ores

cenc

e D

etec

tion

* *

*

O-Linked Glycan

N-Linked Glycans

Rapid Deglycosylation

O-Linked Glycan

HILIC of N-Deglycosylated Intact Epoetin Alpha (EPO) to Characterize the Occupancy and Heterogeneity of O-glycosylation.

Intri

nsic

Flu

ores

cenc

e D

etec

tion

Bevacizumab

1 N-Glycan Variant 4.9%

Assaying Glycan Occupancy on Intact mAbs.

5 10 min

What if... released N-glycan analysis could be

both streamlined and highly sensitive?

Finally, a smart workflow with no compromises.

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Introducing the GlycoWorks RapiFluor-MS N-Glycan Kit

Waters, the leader in innovations for biotherapeutic analysis, has reimagined the workflow for released N-glycan

analysis. Our new reagent, RapiFluor-MS,™ features the ionization capability necessary to maximize the information

generated by MS detection. Our new workflow speeds up deglycosylation and streamlines labeling.

It’s the smart workflow that biopharmaceutical laboratories have been waiting for—you no longer have to choose

between a “quick” labeling tool and sensitivity for mass spectrometry; no more labor-intensive and complicated

processes that leave room for error.

With Waters, there are no compromises between speed and sensitivity in sample preparation—only gains in

confidence in your analytical results.

FLR QUANTIFICATIONMS PERFORMANCESPEED/THROUGHPUT

Join Waters in thinking outside the box for released glycan analysis.

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DATA PROCESSING

UNIFI,, MassLynx, and Empower Software

GLYCOPROTEIN

RELEASED N-GLYCAN

ANALYSIS ON LCWITH FLR DETECTOR

Alliance HPLC orACQUITY UPLC Systems

CHARACTERIZATION OR MASS CONFIRMATION

ACQUITY® QDa® Detector Xevo G2-XS QTof MSSYNAPT G2-Si HDMS

0.0E+0

1.2E+6

10 12 14 16 18 20 22 24 26 28 30 min

0.0E+0

1.2E+6

10 12 14 16 18 20 22 24 26 28 30 min

MS

FLR

Results

RELEASED N-GLYCANS BY

DEGLYCOSYLATION

GlycoWorks™ Rapid PNGase F and Buffer, and RapiGest™ SF Surfactant

LABELED GLYCANS

GlycoWorks RapiFluor-MS

Labeling

PURIFIED GLYCANS

GlycoWorks HILIC µElution Plate or Cartridges


Glycan BEH Amide, 130Å Column, Glycan Performance Test Standard,

and Dextran Calibration Ladder

GLYCOPROTE IN


How does it work?

Released N-glycan analysis is an information-rich technique, but it has

involved complicated sample preparation procedures. Once glycans

are released by PNGase F, labeling is required for effective separation

and detection. Using Waters’ new protocol, you now have a versatile

workflow for:

¡ Glycan identification ¡ Glycan structure ¡ Glycan monitoring

See the science behind our NEW GlycoWorks RapiFluor-MS Reagent, which enables new capabilities for glycan analysis, monitoring and characterization. Watch our video at waters.com/SeeTheScience

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RELEASED N-GLYCANS

GlycoWorks Rapid PNGase F and Buffer, and RapiGest

SF Surfactant

LABELED GLYCANS

GlycoWorks RapiFluor-MS Labeling

GLYCOPROTEIN


PURIFIED GLYCANS

GlycoWorks HILIC µElution Plate or Cartridges

What if released N-glycan sample preparation was fast and simplified?

¡ Easily and quickly prepare labeled released N-glycan samples.

¡ Minimize error and sample loss.

¡ Easily train analysts and transfer methods throughout your organization.

3 STEPS, 30 MINUTES

< 15 minutes

10 minutes

5 minutes

STEP 1 STEP 2 STEP 3

Watch a short 2 minute RapiFluor-MS N-glycan Kit demonstration video.

www.waters.com/RapiFluorMS


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What if you could obtain unrivaled FLR and MS sensitivity?

¡ Improve product quality understanding with in-depth characterization of previously unidentified peaks with fluorescence quantification obtained with equivalent mass spectral response.

¡ Accelerate decision making.

¡ More easily identify low abundance N-linked glycans.

UNRIVALED SENSITIVITY

14x fluorescence and 160x mass spectral response when compared to traditional labels (2-AB and procainamide). 2x fluorescence and 1,000x mass spectral response when compared to instant labels.

*Based on published theoretical values.

0

10

20

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60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Pe

rform

ance

(%)

52.5

7.00.1 0.6

30.0*

7.0*

Fluorescence MS (BPI)

2X

1000X

3X

160X14X

Instant AB Labeled

RapiFluor-MS Labeled

2-AB Labeled

Procainamide Labeled

NH

O

NH

NNH

O

N

Glycan

NH

O

NHGlycan

O

NH2

NHGlycan

NH2O

NHGlycan

O

NHN

Proc

ainam

ide*

RapiF

luor-M

S

2-AB

Insta

nt A

B

RapiF

luor-M

S

2-AB

Insta

nt A

B

Proc

ainam

ide*

See how you can bring extraordinary possibilities to your everyday analysis.

www.waters.com/XevoG2XS


0.0E+0

1.2E+6

10 12 14 16 18 20 22 24 26 28 30 min

0.0E+0

1.2E+6

10 12 14 16 18 20 22 24 26 28 30 min

MS

FLR

The increased ionization efficiency of RapiFluor-MS allows for quantitative fluorescence paired with mass detection with the ACQUITY QDa Detector.

What if mass data was easily and always available for routine glycan analysis?

¡ Increase confidence in glycan profile monitoring with routine mass detection.

¡ Obtain information about manufacturing changes with quick sample turnaround.

¡ Resolve method development and transfer issues quickly.

CONFIDENCE

Detailing the N-glycan Profile of a mAb

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Watch a short 1 minute video on how you can bring the power of mass detection to your lab.

www.waters.com/QDa


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Released N-Glycan Sample Preparation Complete KitsDescription Part No.

GlycoWorks RapiFluor-MS N-Glycan Starter Kit—96 Sample

Kit contains: GlycoWorks Deglycosylation Module, GlycoWorks Labeling Module, GlycoWorks Clean-up Module, GlycoWorks Sample Collection Module, ACQUITY UPLC Glycan BEH Amide, 1.7 μm, 2.1 x 150 Column, Mobile Phase Concentrate: Ammonium Formate

176003635

GlycoWorks RapiFluor-MS N-Glycan Kit—96 Sample

Kit contains: GlycoWorks Deglycosylation Module, GlycoWorks Labeling Module, GlycoWorks Clean-up Module, GlycoWorks Sample Collection Module

176003606

GlycoWorks RapiFluor-MS N-Glycan Starter Kit—24 sample

Kit contains: GlycoWorks Deglycosylation Module (24 sample), GlycoWorks Labeling Module (24 sample), GlycoWorks Clean-up Module, GlycoWorks Sample Collection Module, ACQUITY UPLC Glycan BEH Amide, 1.7 μm, 2.1 x 150 mm Column, Ammonium Formate Solution – Glycan Analysis

176003712

GlycoWorks RapiFluor-MS N-Glycan Kit—24 sample

Kit contains: GlycoWorks Deglycosylation Module (24 sample), GlycoWorks Labeling Module (24 sample), GlycoWorks Clean-up Module, GlycoWorks Sample Collection Module

176003713

GlycoWorks RapiFluor-MS N-Glycan Refill Kit—24 sample

Refill Kit contains one of each: GlycoWorks Deglycosylation Module (25 sample) and the GlycoWorks Labeling Module (24 sample)176003714

Ordering Information

Released N-Glycan Standards and AccessoriesDescription Part No. Description Part No.

Vacuum Manifold Shims*, 3/set 186007986 96-Well Plate Extraction Manifold 186001831

Positive Pressure Manifold Spacer for the GlycoWorks RapiFluor-MS N-Glycan Kit*, 1/pack

186007987 Vacuum Pump 220 v/240 v 50 Hz 725000604

RapiFluor-MS Dextran Calibration Ladder, 50 μg/vial 186007982 Positive Pressure Manifold 186006961

RapiFluor-MS Glycan Performance Test Standard, 400 pmol total/vial

186007983Modular Heat Block for 1 mL Tubes, 1 mL tubes/96 wells

186007985

RapiGest SF, 10 mg vial 186002123 GlycoWorks Rapid Buffer, 5 mL 186008100

Ammonium Formate Solution – Glycan Analysis, 5000 mM

186007081 RapiGest SF, 3 mg vial 186008090

Intact mAb Mass Check Standard** 186006552ACQUITY UPLC Glycan BEH Amide, 1.7 μm, 2.1 x 150 mm Column

186004742

*Essential for kit use **Control Standard included in kit

Glycoprotein Columns and StandardDescription Pore Size Particle Size Dimensions Part No.

ACQUITY UPLC Glycoprotein BEH Amide and Std. 300Å 1.7 μm 2.1 x 5 mm Guard 3/pk with Std. 176003699

ACQUITY UPLC Glycoprotein BEH Amide and Std. 300Å 1.7 μm 2.1 x 50 mm with Std. 176003700



ACQUITY UPLC Glycoprotein BEH Amide and Std. 300Å 1.7 μm 2.1 x 100 MVK, 3/pk with Std. 176003703

Glycoprotein Performance Test Standard 186008010

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5.5 10.0 15.0 20.0 25.0 min

Glycan from 10 µg of proteinConventional 2-AB

1.5 day sample preparation

RapiFluor-MSGlycan from 0.1 µg protein<1 hour sample preparation

1: TOF MS ES+BPI

6.93e6

1: TOF MS ES+BPI

6.93e6

800 1600 m/z

FA2G2Ga11130.9423, 2+

O NH

ON

O

NHN

N

O

O

RapidFluorescence

MS

NHS Carbamate RapidTagging Group Quinolinyl

Fluorophore

Tertiary AmineCharge Tag

The next generation of glycan labeling

Built upon Waters’ expertise in rapid fluorescence labeling

of amino acids, RapiFluor-MS is designed with enhanced

chemical properties for superior glycan analysis. Within a

5-minute reaction, RapiFluor-MS modifies glycosylamines

generated by enzymatic release from a glycoprotein.

¡ Rapid tagging

¡ Efficient fluorescence

¡ Unrivaled mass spectral response


Assign low-level glycan components with more ease and confidence

Achieve unparalleled levels of MS and MS/MS sensitivity structural assignments by

combining RapiFluor-MS with the best-in-class Xevo G2-XS QTof or SYNAPT G2-Si

HDMS Mass Spectrometers.

Glycan Characterization by UPLC® FLR with Xevo G2-XS QTof Mass Spectrometer

Comparison of LC-MS chromatogram of 2AB-labeled N-glycans versus RapiFluor-MS labeled N-glycans from the same mAb. 100 fold greater ESI+ MS response was observed for RapiFluor-MS labeled sample. An MS spectrum (inset) shows the summed spectrum for a low abundance glycan within the sample.

CHARACTERIZATION

RapiFluor-MS Reagent

GlycoWorks RapiFluor-MS

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 508 478 2000 F: 508 872 1990 www.waters.com

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Waters, The Science of What’s Possible, ACQUITY UPLC, UPLC, Alliance, ACQUITY, QDa, UNIFI, SYNAPT, Empower, Xevo, MassPREP, and MassLynx are registered trademarks of Waters Corporation. RapiFluor-MS, GlycoWorks, and RapiGest are trademarks of Waters Corporation.

©2015 Waters Corporation. Printed in the U.S.A.July 2015 720005271EN KP-SIG

www.waters.com/glycans

The Next Generation of Glycan Sample Preparation …...H-Class Bio System configured with FLR and...

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Transcript of The Next Generation of Glycan Sample Preparation …...H-Class Bio System configured with FLR and...