background technical overview case studies product information

©2015 Waters Corporation 2

Outline

Background

Technical Overview

Case Studies

Product Information


Rapid or

Traditional

LC or LCMS

Quantitative or Qualitative

Cost/ROI

Training Requirements

The Road to Glycan Analysis Without Compromise

RELEASED GLYCAN ANALYSIS

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Conventional Workflow

16 hrs

Conventional

3 hrs

0.1 hrs

(Pre-Labeling Clean-Up) (Drying) 1-3 hrs

(Drying) 2 hrs

>1 work day


Streamlined and Simplified Workflow

16 hrs

Conventional

3 hrs

0.1 hrs

(Pre-Labeling Clean-Up) (Drying) 1-3 hrs

(Drying) 2 hrs

>1 work day

<15 min

5 min

30 min

GlycoWorks RapiFluor-MS N-Glycan Kit

10 min


Sample Preparation Workflow

Step 1

Step 2

Step 3


Rapid Deglycosylation RapiGest SF Assisted

1% RapiGest SF Surfactant

2 min ≥90˚C

GlycoWorks Rapid Buffer

5 min 50˚C

GlycoWorks Rapid PNGase F

Enzymatic Deglycosylation

Customized Buffer specific for RapiFluor-MS™


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 min Heat Denaturation

5 min 50˚C

5 min 50˚C

No PNGase F (control)

Rapid Deglycosylation Completeness measured by Intact Mass Analysis


Rapid Deglycosylation Completeness measured by gel shift assays

(-) Negative Control (no enzyme) (+) Positive Control (SDS/DTT + PNGase F)

(R) Rapid Deglycosylation (RapiGest, Rapid PNGase F, 10 min)

Positive control: denaturation with DTT-SDS (incompatible with MS applications), followed by PNGase F treatment is a well established method for complete N-glycan removal. • Complete deglycosylation is achieved with the 2 step rapid method

across a wide range of samples. • Compatible with rapid labeling and MS analysis.



Step 1

Step 2

Step 3


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 Conventions Reductive Amination is Laborious


RapiFluor-MSTM Reagent Built 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

Rapid Fluorescence

Patent Pending

RapiFluor-MS


+

+

H2O

+ + CO2

rapiFluor-MS

Monoisotopic mass shift (from glycosylamine)312.1586 Da

Glycosylamine+C17H20N4O2

RapiFluor-MS Reagent Rapid Reaction Kinetics

NHS Carbamate Rapid Tagging Group

Tertiary Amine MS-Active Charge Tag

Reaction Time = Seconds Procedure Time:

Quinoline Fluorophore

Glycan

Glycan

Highly stable urea linkage

ΔMass

312 Da


Unparalleled FLR and MS Sensitivity

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*

RapiFluor-MS vs Instant: 2x ↑ in FLR and 1000x ↑ in MS RapiFluor-MS vs Traditional: 14x ↑ in FLR and 160x ↑ in MS RapiFluor-MS vs PCA: 14x ↑ in FLR and 3x ↑ in MS



Step 1

Step 2

Step 3


Labeled Glycan Labeled 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 when comparing neutrals to tetrasialyated upon SPE processing

FLR

hIgG and fetuin N-glycans

Robustness Quantitative Extraction

FLR


FA2 Rep #1

1.6 pmol Rep #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

Robustness High Yield and Minimal Bias

0E+0

2E+6

3 4 5 6 7 8 9 10 11 12 13


Case Study #1: 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


FA2BG2S2

How RapiFluor-MS™ Helps Enhanced Electrospray Ionization

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


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





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





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





500 2000 m/z

500 2000 m/z

500 2000 m/z

500 2000 m/z

2.9e4

1.2e5

3.5e6 2+

3+

2+ 2+

1+

FA2

2-AB RapiFluor-MS

Example courtesy of M.Lauber


Time (minutes)

5.50 10.00 15.00 20.00 25.00

1: TOFMSES+BPI

6.93e6

1: TOFMSES+BPI

6.93e6

Conventional 2-AB Glycan from 10 µg of Protein 1.5 Day Sample Prep

RapiFluor-MS Glycan from 0.1 µg Protein <1 hr Sample Prep 100x

Sensitivity

Example courtesy of Y. Yu

10 20 min

0.3 ugp/ul Refi ma

m/z800 1000 1200 1400 1600

%

0

100

RapiFluor-MS-NIST Refimab_yqy_11212014_01 443 (7.810) Sm (SG, 100x5.00); Cm (442:446)2.07e5

800 1600 m/z

1130.921, 2+

Characterization

High Resolution LC-MS(/MS)

Direct Sensitivity Comparison using UPLC/FLR/Xevo G2-XS

FA2G2Ga1


50 µg of a Dextran Ladder

Time5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

EU

x 1

0e

4

50000.004

100000.008

150000.016

200000.016

250000.016

300000.031

*It is recommended to use of cubic spline fitting

Time4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

%

0

GU Monoisotopic

Mass (Da)

m/z

(monoisotopic)

[M+H]+

m/z

(monoisotopic)

[M+2H]2+

2 699.333 700.340 350.674

3 861.391 862.398 431.703

4 1023.449 1024.456 512.732

5 1185.507 1186.514 593.761

6 1347.565 1348.572 674.790

7 1509.623 1510.630 755.819

8 1671.681 1672.688 836.848

9 1833.739 1834.746 917.877

10 1995.797 1996.804 998.906

11 2157.855 2158.862 1079.935

12 2319.913 2320.920 1160.964

13 2481.971 2482.978 1241.993

14 2644.029 2645.036 1323.022

15 2806.087 2807.094 1404.051

16 2968.145 2969.152 1485.080

17 3130.203 3131.210 1566.109

18 3292.261 3293.268 1647.138

3

8 13

9 10 11 12

7 6

5 4

Patent Pending

GlycoWorks RapiFluor-MS Standards RapiFluor-MS™ Dextran Calibration Ladder

FLR

MS (BPI)


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

RFMS labeled Dextran and System Performance Standard (hIgG) are now available to support this GU workflow


UNIFI Scientific Library for Confident Glycan Assignments


Enhanced MSMS with RapiFluor-MS Labeling RFMS Label MS performance extends to MS/MS fragmentation data


Case Study #2: 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

Simple sample preparation with simple mass detection


FA2BG2S2

How RapiFluor-MS™ Helps Higher Charge State for QDa Compatibility

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





m/z500 1000 1500 2000

%

0

100

m/z500 1000 1500 2000

%

0

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m/z500 1000 1500 2000

%

0

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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

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

m/z500 1000 1500 2000

%

0

100





500 2000 m/z

500 2000 m/z

500 2000 m/z

500 2000 m/z

2.9e4

1.2e5

3.5e6 2+

3+

2+ 2+

1+

FA2

2-AB RapiFluor-MS

Example courtesy of M.Lauber


Routine N-Glycan Detection with Equivalent FLR and MS response

Minutes 5 10 15 20 25 30 35 40

Minutes

5 10 15 20 25 30 35 40

Detection across a broad range of glycoforms:

IgG Simple bi-antennery structures

RNase B High mannose structures

Fetuin Large, complex structures

MS

FLR


IgG Glycan Profile and Structure Confirmation Using the 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

1.6x10 6

3.2x10 6

Minutes 10.00 15.00 20.00 25.00 30.00

896.0

Inte

nsity

0.0

12000.0

24000.0

m/z 600 1200

A2G1b 0.5% RPA

888.2

Inte

nsity

0

300000

600000

m/z 600 1200

FA2 20.5% 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

Minutes 2 3 4 5

10 minute

Method

EU

0

10

20

Minutes 5 10 15 20

55 minute

Method


Released N-Glycan UPLC Analysis Workflows

SAMPLE PREP SEPARATION DETECTION & INFORMATICS

GlycoWorks Kits RapiFluor-MS N-Glycan Kit ACQUITY FLR/QDa

Detectors and Empower 3

Software

FLR/Xevo G2-XS QTof MS and 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 Quantification GU Retention

MS Confirmation

FLR Quantification GU Retention

Accurate Mass Confirmation MS/MS Fragmentation

FLR

TIC

MS/MS


30.00 10.00 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 HPLC XBridge 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 UPLC ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7µm 2.1 X 150 mm Flow 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

Case Study #3: Transferability between UPLC and HPLC


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


RapiFluor-MS

Glycoprotein

Analysis-Ready N-glycans

30 min


GlycoWorks RapiFluor-MS N-Glycan Kit: Complete Sample Preparation Kit


Questions

background technical overview case studies product information

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