©2015 Waters Corporation 1
The Road to Glycan Analysis
Without Compromise
WCBP 2015
Waters Technical Seminar
Jan 27, 2015 Washington, DC

©2015 Waters Corporation 2
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

©2015 Waters Corporation 3
Rapid Preparation
Deglycosylation
Labeling
SPE
30 min
High Sensitivity
HILIC-Fluorescence-MS
Fluorophore
+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.

©2015 Waters Corporation 4
Glycoprotein Characterization
Multiple Strategies – Complementary Information

©2015 Waters Corporation 5
Customer voice brought us focus
Compatible with both
MS and optical
detection
Just compatible with
MS detection
Just compatible with
Fluorescence
Providesrapid labeling
versusmany existing
offerings
Just compatible with
UV detection
Reagents are less toxic
versusmany existing
offerings Lower cost per sample
Labelingreagent 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
Mean
Mentions
MoreImportantLessImportant
Lower Interest Higher Interest

©2015 Waters Corporation 6
Rapid
or
Traditional
LC or LCMS
Quan or Qual
Cost/ROI
Training
Requirements
The Road to Glycan Analysis Without Compromise
RELEASED GLYCAN ANALYSIS

©2015 Waters Corporation 7
RapiFluor-MS N-Glycan Labeling:
A breakthrough technology for
released glycan LC and MS analysis

©2015 Waters Corporation 8
Rapid Preparation
Deglycosylation
Labeling
SPE
30 min
High Sensitivity
HILIC-Fluorescence-MS
Fluorophore
+MS Charge Tag
Fluorescence MS
Released Glycan Analysis
HILIC Profiling
5 Hours
to
2 Days
0.0E+0
FLR
Conventional
2-AB
5.3e2
m/z
500 1000 1500 2000
%
0
100
m/z
500 1000 1500 2000
%
0
100
m/z
500 1000 1500 2000
%
0
100
m/z
500 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

©2015 Waters Corporation 9
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

©2015 Waters Corporation 10
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

©2015 Waters Corporation 11
+
+
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

©2015 Waters Corporation 12
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

©2015 Waters Corporation 13
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
0.3
0
100
200
300
400
Compound 4 Compound 1
ReponseFactors
(FA2PeakAreaperSampleofN-Glycans
from1µgofAnti-CitrininIgG/1000)
FA2
FA2
FA2
FA2
RapiFluor-MS
Labeled
Instant AB
Labeled
FLR
MS
(BPI)
ResponseFactors
RapiFluor-MS Labeled N-Glycans
Instant AB Labeled N-Glycans
min
min
~2x
nearly
1000x
300x
Zoom

©2015 Waters Corporation 14
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
RelativePerformance(%)
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

©2015 Waters Corporation 15
10 min 5 min 10 min
30 min
Patent Pending
Simplified Workflow
Total
Sample Prep Time
Conventional
5 Hours
to
2 Days
GlycoWorks™
RapiFluor-MS™ N-Glycan Kit
Direct Analysis
(Organic Solvent Dilution)

©2015 Waters Corporation 16
Rapid Deglycosylation
RapiGest™ SF Assisted
1% RapiGest SF
Surfactant
2 min
≥80˚C
GlycoWorks
Rapid Buffer
5 min
50˚C
GlycoWorks
Rapid PNGase F
Enzymatic
Deglycosylation

©2015 Waters Corporation 17
140000 145000 150000 1550
%
0
100
VicamIgG_PNGaseFnoDTT_7p5ug_100414 638 (10.803) M1 [Ev-612128,
145345
mass
140000 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
3.49e3146786
146952
mass
140000 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
RapiGest™ SF Assisted

©2015 Waters Corporation 18
Labeled Glycan
Labeled Deglycosylated Protein
Byproducts
Collect
Labeled Glycan
Reaction Byproducts
Robust HILIC SPE

©2015 Waters Corporation 19
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
RelativeAbundance(%)
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
Robustness
Quantitative Extraction
FLR

©2015 Waters Corporation 22
FA2
Rep #1
1.6 pmol
Rep #2
1.7 pmol
100% Theoretical Yield = 2.3 pmol
1.5x107 pg IgG 1 pmol
150,000 pg
2 pmol glycan
1 pmol IgG
0.45 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

©2015 Waters Corporation 23
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

©2015 Waters Corporation 24
GlycoWorks™ RapiFluor-MS™ Kit
Smart Workflow with No Compromise
Available
February 2015

©2015 Waters Corporation 25
Poster
WCBP 2015 Poster P-216-W
Rapid Preparation
of N-Glycans Using a Novel
Fluorescence and MS Active
Labeling Reagent
Download pdf

©2015 Waters Corporation 26
RapiFluor-MS:
Enhanced Workflows
for Glycan Characterization

©2015 Waters Corporation 27
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
 GlycoWorks™ RapiFluor-MS™ N-Glycan Kit
 ACQUITY UPLC Glycan BEH Amide Column

©2015 Waters Corporation 28
UNIFI® Glycan Workflows
Workflows support conventional glycan labels
and new RapiFluor-MS™ label technology
HILIC FLR
GU +Accurate Mass
HILIC FLR
GU + DDA MS/MS
Automated
Data
Acquisition
Processing
Review and
Reporting

©2015 Waters Corporation 29
RapiFluor-MS™ Labeling for Characterization
Greater than 100x MS response over 2AB labeling
BPI MS
Sample: NIST RM 8670 mAb lot #3F1b

©2015 Waters Corporation 30
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

©2015 Waters Corporation 31
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

©2015 Waters Corporation 32
UNIFI® Scientific Library for
Confident Glycan Assignments

©2015 Waters Corporation 33
Powerful UNIFI® Reporting Architecture
Simplifies Communication of Results
Example

©2015 Waters Corporation 34
UNIFI® Glycan DDA Workflow

©2015 Waters Corporation 35
Optional UNIFI® Export to SimGlycan
for MS/MS Database Search
A2G2S1

©2015 Waters Corporation 36
Enhanced MS/MS with RapiFluor-MS™ Labeling
FLR
BPI MS
MSMS
MS performance extends to fragmentation data

©2015 Waters Corporation 37
Enhanced MSMS with RapiFluor-MS Labeling
MS performance extends to MS/MS fragmentation data

©2015 Waters Corporation 38
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

©2015 Waters Corporation 39
RapiFluor-MS:
Enhanced Workflows
for Glycan Monitoring

©2015 Waters Corporation 40
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 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
Fluorescence(relative)
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 HPLC
RapiFluor-MS™ Labeled Glycans

©2015 Waters Corporation 41
Glycan Monitoring
 ACQUITY UPLC® H-Class Bio System
 ACQUITY UPLC Column Manager
 ACQUITY UPLC FLR Detector
 ACQUITY® QDa® Mass Detector
 Empower® or MassLynx® Informatics
 GlycoWorks™ RapiFluor-MS™ N-Glycan Kit
 ACQUITY UPLC Glycan BEH Amide Column

©2015 Waters Corporation 42
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®

©2015 Waters Corporation 43
Automated Start Up Provides
Robust, 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

©2015 Waters Corporation 44
Routine N-Glycan Detection with
Comparable 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

©2015 Waters Corporation 45
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
Intensity
0.0
1.6x106
3.2x10 6
Minutes
10.00 15.00 20.00 25.00 30.00
896.0
Intensity
0.0
12000.0
24000.0
m/z
600 1200
A2G1b
0.5% RPA
888.2
Intensity
0
300000
600000
m/z
600 1200
FA2
20.5% RPA
EU

©2015 Waters Corporation 46
Intuitive GMP compliant Reporting
Empower® integration enables annotation of peaks with names and m/z
A2-815.1
FA2-888.2
FA2B-989.6
A2G1a-896.2
A2G1b-896.0
FA2G1a-969.3
FA2G1b-969.2
FA2BG1a-1070.7
FA2BG1b-1070.6
A2G2-977.1
FA2G2-1050.3
FA2BG2-1151.8
FA2G1S1-1114.6
FA2G2S1-1196.0
FA2BG2S1-865.3
FA2G2S2-894.7
FA2BG2S2-962.2
0.00
240.00
480.00
Minutes
10.00 15.00 20.00 25.00 30.00
EU

©2015 Waters Corporation 47
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

©2015 Waters Corporation 48
Rapid Screening Process
for Development Samples
EU
0
20
40 FLR
Intensity
0
1x106
2x106
Minutes
1 2 3 4 5 6
SIR Overlay
Trastuzumab N-Glycan Analysis
RapiFluor-MS™ labeled glycans: 10 minute method
Intensity
0
50000
Intensity
0
200000
Intensity
0
1x10
6
2x10
6
Intensity
0
50000
100000
Intensity
0
1x10
6
Intensity
0
200000
400000
Intensity
0
20000
Minutes
1 2 3 4 5 6
A2G(4)1
895.9 m/z
F(6)A2
887.9 m/z
F(6)A2G(4)1
968.9 m/z
F(6)A2G(4)2
1049.9 m/z
A2
814.8 m/z
F(6)A2G(4)2S1
1195.5 m/z
M5
774.1 m/z

©2015 Waters Corporation 49
Monitoring Glycan Ratios
Keeping Tabs on Mannose 5
Mannose 5 spiked in
Low Medium High
Inj 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
Intensity
0
60000
120000
180000
240000
Minutes
2.00 2.50 3.00 3.50 4.00
Intensity
0.0
35000.0
70000.0
105000.0
140000.0
Minutes
2.00 2.50 3.00 3.50 4.00
F(6)A2G(4)1
SIR: 968.9 m/z
M5
SIR: 774.1 m/z
Man5: F(6)A2G(4)1 Ratio

©2015 Waters Corporation 50
Released N-Glycan UPLC Analysis Workflows
SAMPLE PREP SEPARATION
DETECTION &
INFORMATICS
GlycoWorks™ Kits
RapiFluor-MS™
N-Glycan Kit
ACQUITY® FLR/QDa
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

©2015 Waters Corporation 51
Poster
WCBP 2015 Poster P-206-W
Routine Monitoring of
N-Glycans Using a Novel
Labeling Reagent with
Fluorescence and Mass
Detection
Download pdf

©2015 Waters Corporation 52
RapiFluor-MS N-Glycan Labeling:
A breakthrough technology for
released glycan LC and MS analysis