from wcbp 2015: glycoworks rapifluor-ms for glycan profiling

©2015 Waters Corporation 1

The Road to Glycan Analysis

Without Compromise

WCBP 2015

Waters Technical Seminar

Jan 27, 2015 Washington, DC


Today’s Agenda

Overview and Introduction

RapiFluor-MS N-Glycan Labeling: A breakthrough technology for released glycan LC and MS analysis

Matthew A. Lauber (Consumables Business Unit, Waters)

RapiFluor-MS Technology & Glycan Characterization

Ying Qing Yu (Biopharmaceutical Sciences, Waters)

Impact of RapiFluor-MS Technology on Released Glycan Profile Monitoring

Sean M. McCarthy (Biopharmaceutical Sciences, Waters)

Scientific Panel – Questions & Discussion


Rapid Preparation

DeglycosylationLabeling

SPE

30 min

High Sensitivity

HILIC-Fluorescence-MSFluorophore

+MS Charge Tag

Fluorescence MS

Glycosylation of Biotherapeutics

N-glycolylneuraminic acid

Fucose

GlcNAc

Mannose

Galactose

N-acetylneuraminic acid

Immunogenic (αGal / N-glycolylneuraminic acid)

Low Half Life(high mannose)

Anti-Inflammatory(sialylation)

Effector Functions (ADCC/CDC)(fucosylation/galactosylation)

Overall profile sensitive to manufacturing conditions

N-glycosylation is a quality attribute of biotherapeutics

Glycosylation profiles are characterized and routinely monitored

Anal Chem 2013, 85 (2), 715-36.


Glycoprotein CharacterizationMultiple Strategies – Complementary Information


Customer voice brought us focus

Compatible with both MS and optical

detection

Just compatible with MS detection

Just compatible with Fluorescence

Provides rapid labeling versus many existing

offerings

Just compatible with UV detection

Reagents are less toxic versus many existing

offerings Lower cost per sample

Labeling reagent and protocol is automation

friendly.

1.7

1.8

1.9

2

2.1

2.2

2.3

0 20 40 60 80 100 120 140

Me

an

Mentions

More

Im

port

ant

Less I

mport

ant

Lower Interest Higher Interest


Rapid or

Traditional

LC or LCMS

Quan or Qual

Cost/ROI

TrainingRequirements

The Road to Glycan Analysis Without Compromise

RELEASED GLYCAN ANALYSIS

http://www.globaldemocracy.org/media/gd.org-pic-82.jpg

http://www.globaldemocracy.org/media/gd.org-pic-82.jpg


Rapid Preparation

DeglycosylationLabeling

SPE

30 min

High Sensitivity

HILIC-Fluorescence-MSFluorophore

+MS Charge Tag

Fluorescence MS

Released Glycan AnalysisHILIC Profiling

5 Hoursto

2 Days

0.0E+0

3.3E+6

3 4 5 6 7 8 9 10

FLR

Conventional2-AB

5.3e2

m/z500 1000 1500 2000

%

0

100

m/z500 1000 1500 2000

%

0

100

m/z500 1000 1500 2000

%

0

100

m/z500 1000 1500 2000

%

0

100

RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6

2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4

2AB_hIgG_halfload_20Nov14_10 495 (8.689)529

RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5

500 2000 m/z

2+

MS


N-Glycan(Glycosylamine)

N-Glycan( Acyclic Free Reducing End)

Mild Acid

Hydrolysis

PNGase F

+

ProteinN-Glycan

Released N-Glycan

(Glycosylamine)

Protein

+

HOAc

DMSO

N-Glycan(Free Reducing End) 2-AB

N-Glycan(2-AB Labeled)

Reduction

NaBH3CN

SPE

Reductive Amination (conventional)

• Anhydrous sample• Numerous chemical

conversions• Laborious• Heterogenous reaction

products

Rapid Tagging

Glycosylamine labeling circumvents these issues

Out with ConventionsReductive Amination is Laborious


RapiFluor-MSTM ReagentBuilt Upon Our Expertise

From Waters’ expertise in rapid, fluorescence labeling of amino acids

Enhanced chemical properties for glycan analysis:• Rapid Tagging• Efficient Fluorescence• Enhanced Ionization Efficiency

AccQ·Fluor™

RapidFluorescence

Patent Pending

RapiFluor-MS


+

+

H2O

+ + CO2

rapiFluor-MS

Monoisotopic mass shift (from glycosylamine)312.1586 Da

Glycosylamine+C17H20N4O2

RapiFluor-MS ReagentRapid Reaction Kinetics

NHS Carbamate Rapid Tagging Group

Tertiary AmineMS-Active Charge Tag

Reaction Time = Seconds

Procedure Time:

Quinoline Fluorophore

Glycan

Glycan

Highly stable urea linkage

ΔMass

312 Da


RapiFluor-MS Reagent Sensitivity Comparison – Instant AB

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

0.0E+0

1.7E+6

4.5 5 5.5 6 6.5 7 7.5

FLR

MS(BPI)

FLR

MS(BPI)

FA2

FA2

FA2

FA2

RapiFluor-MS™ Labeled N-Glycans

Instant AB™ Labeled N-Glycans

min

min

RapiFluor-MSTM Labeled N-Glycans

Instant ABTM Labeled N-Glycans

Instant AB is a trademark of Prozyme Inc.

300X Zoom


RapiFluor-MS Reagent Sensitivity Comparison – Instant AB

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

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

Reponse F

acto

rs(F

A2 P

eak A

rea p

er

Sam

ple

of

N-G

lycans

from

1 µ

g o

f Anti-C

itrinin

Ig

G /

1000)

FA2

FA2

FA2

FA2

RapiFluor-MSLabeled

Instant ABLabeled

FLR

MS(BPI)

Response F

acto

rs

RapiFluor-MS Labeled N-Glycans

Instant AB Labeled N-Glycans

min

min

~2x

nearly1000x

300x Zoom


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*

(*) Comparative result extrapolated from a published comparison of N-glycans, wherein it was found that procainamide provided comparable fluorescence and up to 50 fold greater ESI-MS sensitivity when compared to 2-AB(Klapoetke et al. 2010).

RapiFluor-MS Reagent Sensitivity Comparison


10 min 5 min 10 min

30 min

Patent Pending

Simplified Workflow

Total Sample Prep Time

Conventional5 Hours

to 2 Days

GlycoWorks™ RapiFluor-MS™ N-Glycan Kit

Direct Analysis(Organic Solvent Dilution)


Rapid DeglycosylationRapiGest™ SF Assisted

1% RapiGest SFSurfactant

2 min≥80˚C

GlycoWorks Rapid Buffer

5 min50˚C

GlycoWorksRapid PNGase F

EnzymaticDeglycosylation


mass140000 145000 150000 155000 160000

%

0

100

VicamIgG_PNGaseFnoDTT_7p5ug_100414 638 (10.803) M1 [Ev-612128,It15] (Gs,1.500,1994:5000,1.00,L30,R30); Cm (633:642)1.71e4145345

mass140000 145000 150000 155000 160000

%

0

100

VicamIgG_PNGaseFnoDTT_7p5ug_100414 654 (11.074) M1 [Ev-629947,It13] (Gs,1.500,1955:4983,1.00,L30,R30); Cm (650:658)3.49e3146786

146952

mass140000 145000 150000 155000 160000

%

0

100

VicamIgG_PNGaseFnoDTT_7p5ug_100414 671 (11.361) M1 [Ev-562807,It10] (Gs,1.500,1993:5000,1.00,L30,R30); Cm (666:679)352148401

148240

148560148.4 kDa

145.3 kDa

146.8 kDa

2 Step

2 minHeat Denaturation

5 min50˚C

5 min50˚C

No PNGase F(control)

Rapid DeglycosylationRapiGest™ SF Assisted


Labeled GlycanLabeled Deglycosylated Protein

Byproducts

Colle

ct

Labeled Glycan

Reaction Byproducts

Robust HILIC SPE


0.0E+0

1.2E+6

10 15 20 25 30 35

Positive Control

0.0E+0

1.2E+6

10 15 20 25 30 35

2x SPE

FA2

FA2G2S1

A3S1G3S3

1x SPE

0

5

10

15

20

25

30

Rela

tive A

bundance (

%)

Positive

Control

SPE

Processed

A3G3S3

1x SPE

2x SPE

min

5.7%6.1%

GlycoWorks HILIC SPE of RapiFluor-MS N-glycans is quantitative No significant deviation in the glycan profile upon SPE processing

FLR

hIgG and fetuin N-glycans

RobustnessQuantitative Extraction

FLR


FA2Rep #1

1.6 pmolRep #2

1.7 pmol

100% Theoretical Yield = 2.3 pmol

1.5x107 pg IgG1 pmol

150,000 pg

2 pmol glycan

1 pmol IgG0.45 pmol FA2

1 pmol total

glycan pool

10 μL injection

400 μL

sample

prepared

X X X X = 2.3 pmol

Step Yield Testing to confirm minimal bias

Deglycosylation Complete Intact mass analysis

Gel shift assays Subunit LC-MS

Labeling >95% Released glycan profile vs subunit derived glycan information

SPE ~74% Recovery measurements

Glycan profile before vs after SPE

Entire Workflow(experimentally

determined)~73% Yield

RobustnessHigh Yield and Minimal Bias

0E+0

2E+6

3 4 5 6 7 8 9 10 11 12 13


Summary GlycoWorks™ RapiFluor-MS™ N-Glycan Kit

Simple, streamlined protocol

Fast and complete deglycosylation

Rapid and efficient labeling

Unbiased and robust SPE for neutral to

tetrasialylated N-glycans

Unprecedented FLR and MS sensitivity

GlycoWorks RapiFluor-MS N-Glycan Kit

RapiFluor-MS

Glycoprotein

Analysis-Ready N-glycans

30 min


GlycoWorks™ RapiFluor-MS™ KitSmart Workflow with No Compromise

Available February 2015


Poster

WCBP 2015 Poster P-216-W

Rapid Preparation of N-Glycans Using a Novel Fluorescence and MS Active

Labeling Reagent

Download pdf

http://www.waters.com/waters/library.htm?lid=134829545


RapiFluor-MS:Enhanced Workflows for Glycan Characterization


Glycan Characterization

ACQUITY UPLC® H-Class Bio System

ACQUITY UPLC Column Manager

ACQUITY UPLC FLR Detector

Xevo® G2-XS QTof MS

UNIFI® Glycan Application Solution

or MassLynx® Informatics


ACQUITY UPLC Glycan BEH Amide Column


UNIFI® Glycan Workflows

Workflows support conventional glycan labels and new RapiFluor-MS™ label technology

HILIC FLRGU +Accurate Mass

HILIC FLRGU + DDA MS/MS

Automated Data

Acquisition

Processing

Review andReporting


RapiFluor-MS™ Labeling for Characterization

Greater than 100x MS response over 2AB labeling

BPI MS

Sample: NIST RM 8670 mAb lot #3F1b


HILIC FLR GU + Accurate Mass

RapiFluor-Dextran Ladder Retention Time Calibration Curve

Assign GU values to N-glycans

BPI MS for accurate mass confirmation

GU

Method Robustness and Transferability, Confident Assignments

RapiFluor-MS Labeled Dextran and System Performance Standard (hIgG) are now available to support this GU workflow


UNIFI ® Scientific Library for Automated GU or GU+Mass Glycan Confirmation

Experimental GU value

FLR label

mass

Waters Glycan GU Library:

• Experimentally derived GU Retention (>10 injections/protein)

• Data from proteins representing spectrum of glycan diversity

• All entries confirmed with exoglycosidase digestion


UNIFI® Scientific Library for Confident Glycan Assignments


Powerful UNIFI® Reporting Architecture Simplifies Communication of Results

Example


UNIFI® Glycan DDA Workflow


Optional UNIFI® Export to SimGlycanfor MS/MS Database Search

A2G2S1


Enhanced MS/MS with RapiFluor-MS™ Labeling

FLR

BPI MS

MSMS

MS performance extends to fragmentation data


Enhanced MSMS with RapiFluor-MS Labeling

MS performance extends to MS/MS fragmentation data


Poster

WCBP 2015 Poster P-115-T

Developing a Scientific Library for UPLC/FLR/MS

Analysis of Released N-glycans Labeled with a Novel Labeling Reagent

Download pdf



RapiFluor-MS:Enhanced Workflows for Glycan Monitoring


30.0010.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

24.00 28.00 32.00 36.00 40.00 44.00 48.00 52.00 56.00 60.00 64.00

Alliance HPLCXBridge Glycan BEH Amide, 130Å, 2.5µm 3.0 X 225 mm total length (150 mm + 75 mm)Flow Rate: 0.56 mL/minute

H-Class BIO UPLCACQUITY UPLC Glycan BEH Amide, 130Å, 1.7µm2.1 X 150 mmFlow Rate: 0.40 mL/minute

Total Run Time = 55 minutes

Total Run Time = 121.3 minutes

Flu

ore

scence (

rela

tive)

Time (minutes)

Comparable sensitivity and resolution – 3x sample load / 2x increase in time

Transfer between labs with different LC equipment capabilities

Transferability between UPLC® and HPLCRapiFluor-MS™ Labeled Glycans


Glycan Monitoring

ACQUITY UPLC® H-Class Bio System

ACQUITY UPLC Column Manager

ACQUITY UPLC FLR Detector

ACQUITY® QDa® Mass Detector

Empower® or MassLynx® Informatics


ACQUITY UPLC Glycan BEH Amide Column


ACQUITY® QDa® Mass Detector A breakthrough product with mass appeal

Revolutionary innovative design

focused on ease of use for analysts

Empowering analytical chemists

everywhere with orthogonal mass

detection – added information with

every sample

Compact, robust and affordable:

Built for constant use with a wide

variety of chromatographic conditions

Seamlessly integrates with

Empower based HPLC & UPLC®


Automated Start Up ProvidesRobust, Reproducible Performance

Automated resolution and calibration occurs with each start-up

ensuring mass information is accurate and precise

ESI interface optimized for UPLC® performance to ensure

chromatographic resolution, sensitivity and throughput is preserved

Disposable sample aperature and capillary for easy maintenance

Graphic QDa®

monitor display enables easy viewing and adjustment of system parameters


Routine N-Glycan Detection with Comparable FLR and MS response

Minutes5 10 15 20 25 30 35 40

Minutes

5 10 15 20 25 30 35 40

Detection across a broad range of glycoforms:

IgGSimple bi-antennerystructures

RNase BHigh mannose structures

FetuinLarge, complex structures


IgG Glycan Profile and Structure Confirmation Using ACQUITY® QDa®

Key take away: Large dynamic range – can clearly see most abundant and least abundant glycoforms.

0.00

240.00

480.00

In

ten

sit

y

0.0

1.6x106

3.2x106

Minutes10.00 15.00 20.00 25.00 30.00

896.0

Inte

nsity

0.0

12000.0

24000.0

m/z600 1200

A2G1b0.5% RPA

888.2

Inte

nsity

0

300000

600000

m/z600 1200

FA220.5% RPA

EU


Intuitive GMP compliant Reporting

Empower® integration enables annotation of peaks with names and m/z

A2 -

815.1

FA2 -

888.2

FA2B -

989.6

A2G

1a -

896.2

A2G

1b -

896.0

FA2G

1a -

969.3

FA2G

1b -

969.2

FA2BG

1a -

1070.7

FA2BG

1b -

1070.6

A2G

2 -

977.1

FA2G

2 -

1050.3

FA2BG

2 -

1151.8

FA2G

1S1 -

1114.6

FA2G

2S1 -

1196.0

FA2BG

2S1 -

865.3

FA2G

2S2 -

894.7

FA2BG

2S2 -

962.2

0.00

240.00

480.00

Minutes

10.00 15.00 20.00 25.00 30.00

EU


Developing a Rapid Method for Glycan Analysis

EU

0

10

20

30

40

Minutes2 3 4 5

10 minute

Method

EU

0

10

20

Minutes5 10 15 20

55 minute

Method


Rapid Screening Process for Development Samples

EU

0

20

40 FLR

Inte

nsity

0

1x106

2x106

Minutes1 2 3 4 5 6

SIR Overlay

Trastuzumab N-Glycan Analysis

RapiFluor-MS™ labeled glycans: 10 minute method

Inte

nsity

0

50000

Inte

nsity

0

200000

Inte

nsity

0

1x106

2x106

Inte

nsity

0

50000

100000

Inte

nsity

0

1x106

Inte

nsity

0

200000

400000

Inte

nsity

0

20000

Minutes

1 2 3 4 5 6

A2G(4)1895.9 m/z

F(6)A2887.9 m/z

F(6)A2G(4)1968.9 m/z

F(6)A2G(4)21049.9 m/z

A2814.8 m/z

F(6)A2G(4)2S11195.5 m/z

M5774.1 m/z


Monitoring Glycan RatiosKeeping Tabs on Mannose 5

Mannose 5 spiked in

Low Medium HighInj 1 0.61 0.96 1.20

Inj 2 0.57 0.90 1.11

Inj 3 0.55 0.86 1.18

Mean 0.58 0.91 1.16

StDev 0.03 0.05 0.04

% RSD 5.44 5.47 3.85

Inte

nsity

0

60000

120000

180000

240000

Minutes2.00 2.50 3.00 3.50 4.00

Inte

nsity

0.0

35000.0

70000.0

105000.0

140000.0

Minutes2.00 2.50 3.00 3.50 4.00

F(6)A2G(4)1SIR: 968.9 m/z

M5SIR: 774.1 m/z

Man5: F(6)A2G(4)1 Ratio


Released N-Glycan UPLC Analysis Workflows

SAMPLE PREP SEPARATIONDETECTION & INFORMATICS

GlycoWorks™ KitsRapiFluor-MS™ N-Glycan Kit

ACQUITY® FLR/QDaand Empower® 3

Software

FLR/Xevo® G2-XS QTof MSand UNIFI® Scientific Information System

ACQUITY UPLC®

Glycan BEH Amide Column

Deglycosylation, Labeling and Clean-up in 30 min

Unmatched sensitivity for FLR and MS detection

FLR QuantificationGU Retention

MS Confirmation

FLR QuantificationGU RetentionAccurate Mass Confirmation

MS/MS Fragmentation

FLR

TIC

MS/MS


Poster

WCBP 2015 Poster P-206-W

Routine Monitoring of N-Glycans Using a Novel Labeling Reagent with Fluorescence and Mass

Detection

Download pdf
