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Introducing Introducing GlycoWorksGlycoWorks RapiFluorRapiFluor--MS: MS: Label Label NN--GlycansGlycans Simply and Rapidly Without Simply and Rapidly Without
Sacrificing SensitivitySacrificing Sensitivity
©2015 Waters Corporation 1
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� Recorded version of today’s presentation
©2015 Waters Corporation 2
� PDF Copies of today’s slides
� Discount Offers on GlycoWorks RapiFluor MS Products
� Product specific information
� Reference materials
Today’s Presenters
� Matthew Lauber, Ph.D - Senior Research Chemist, Waters Corporation
� Matthew Lauber is a Senior Applications Chemist in the Chemistry Applied Technology group. He has an academic background in protein chemistry and proteomic methods for the structural characterization of biomolecular complexes. Most recently Matt has worked extensively on chemistry products for bioseparations, with a specific focus on the new
©2015 Waters Corporation 3
bioseparations, with a specific focus on the new GlycoWorks – RapiFluor MS kit we will discuss today.
� Paula Magnelli, Ph.D – New England Biolabs
� Dr. Paula Magnelli is a Scientist at NEB GlycobiologyGroup. Paula is responsible for developing and characterizing novel glycosidases, creating methods for glycomics and glycoproteomics research with an emphasis in therapeutic glycoproteins workflows and glycansequencing.
©2015 Waters Corporation 4
Introducing GlycoWorks RapiFluorIntroducing GlycoWorks RapiFluor--MS: MS: Label NLabel N--Glycans Simply and Rapidly Glycans Simply and Rapidly
Without Sacrificing SensitivityWithout Sacrificing Sensitivity
Matthew A. Lauber, Ph.D.Paula Magnelli, Ph.D.
Glycosylation of Biotherapeutics
©2015 Waters Corporation 5
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
� N-glycan profiles are frequently characterized in detail and routinely monitored
Anal Chem 2013, 85 (2), 715-36.
Glycoprotein CharacterizationMultiple Strategies – Complementary Information
©2015 Waters Corporation 6
Released Glycan AnalysisHILIC Profiling
Conventional
©2015 Waters Corporation 7
5 Hoursto
2 Days
FLR
Conventional2-AB 5.3e2
500 2000 m/z
2+
Released Glycan AnalysisRecent Developments
Procainamide
Klapoetke, S.; Zhang, J.; Becht, S.; Gu, X.; Ding, X., J Pharm Biomed Anal 2010, 53 (3), 315-24.
Rapid Tagging for MS
Rapid
©2015 Waters Corporation 8
Rapid Tagging for MS
Rapid Tagging for FLR
FLR
MSCook, K. S.; Bullock, K.; Sullivan, T., Biologicals 2012, 40 (2), 109-17.
Gong, B.; Hoyt, E.; Lynaugh, H.; Burnina, I.; Moore, R.; Thompson, A.; Li, H., Anal BioanalChem 2013, 405 (17), 5825-31.
Part I
Development of the GlycoWorks
©2015 Waters Corporation 9
Development of the GlycoWorks RapiFluor-MS N-Glycan Kit
Mild AcidHydrolysis
PNGase F
+
ProteinN-Glycan
Released N-Glycan
(Glycosylamine)
Protein
Reductive Amination (conventional)
• Relies on numerous chemical conversions
• Laborious• Time consuming • Often produces
Rapid Tagging
Glycosylamine labeling with rapid tagging reactive groups circumvents these issues
Glycan Labeling Techniques
©2015 Waters Corporation 10
N-Glycan(Glycosylamine)
N-Glycan( Acyclic Free Reducing End)
+
HOAcDMSO
N-Glycan(Free Reducing End) 2-AB
N-Glycan(2-AB Labeled)
Reduction
NaBH3CN
SPE
• Often produces heterogenous reaction products
these issues
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
RapiFluor-MS
AccQ·Fluor
RapidFluorescence
©2015 Waters Corporation 11
Patent PendingExclusive Licensing
RapiFluor-MS
Mechanism for Rapid Tagging of Glycosylamines with RapiFluor-MS
RapiFluor-MS ReagentRapid Labeling Kinetics
+
H O
rapiFluor-MS
∆mass
NHS Carbamate Rapid Tagging Group
Quinoline Fluorophore
Tertiary AmineMS-Active Charge Tag
©2015 Waters Corporation 12
+
H2O
+ + CO2
Monoisotopic mass shift (from glycosylamine)312.1586 Da
Glycosylamine+C17H20N4O2
∆time = seconds
∆mass (glycosylamine) = 312.16 Da(reducing end) = 311.18 Da
Quinoline Fluorophore
Revolutionizing N-Glycan Sample PrepSensitivity Comparison – Instant ABTM
0.0E+0
2.6E+6
4.5 5 5.5 6 6.5 71.7E+6
FLR
MS(BPI)
342.8
233.7
300
400FA2
FA2
FLR
MS(BPI)
Res
ponse
Fac
tors
(FA2 P
eak
Are
a per
Sam
ple
of
N-G
lyca
ns
µg o
f In
tact
mAb M
ass
Chec
k Std
/ 1000)
RapiFluor-MS Labeled N-Glycans
©2015 Waters Corporation 13
0.0E+0
2.6E+6
0.0E+0
1.7E+6
4.5 5 5.5 6 6.5 7
300x zoom
0.0E+0
4.5 5 5.5 6 6.5 7
FLR
MS(BPI)
179.9
0.30
100
200
FA2
FA2
RapiFluor-MSLabeled
Instant ABLabeled
Res
ponse
Fac
tors
(FA2 P
eak
Are
a per
Sam
ple
of
Nfr
om 1
µg o
f In
tact
mAb M
ass
Chec
k Std
/ 1000)
Instant AB Labeled N-Glycans
min
min
Revolutionizing N-Glycan Sample PrepQuantitative Analysis to Compare to Convention
DifferentMechanisms
H O
rapiFluor-MSReductive Amination
2-ABRapid TaggingRapiFluor-MS
3.3E+6
RapiFluor-MS Labeled Human IgG N-Glycans
FA2 (2.2 pmol)(Area = 229.6 x 103)
FLR
y = 113033x - 11366 Quantity (pmol)
hIgG FA2 232105 227079 229592 2.2
©2015 Waters Corporation 14
0.0E+0
4.0E+6
3 4 5 6 7 8 9 10
0.0E+0
3 4 5 6 7 8 9 10
FA2(Area = 275.5 x 103)
FLR
MS(BPI)
RapiFluor-MS Label(pmol)
y = 113033x - 11366R² = 1
0E+0
2E+5
4E+5
6E+5
8E+5
1E+6
0 5 10
Peak A
rea
(Flu
ore
scence
-Ex 2
65 n
m /
Em
425 n
m)
3.0E+5
FA2 (2.6 pmol)(Area = 19.3 x 103)
FLR
y = 7721.8x-834.17 Quantity (pmol)
hIgG FA2 19665 18955 19310 2.6
Revolutionizing N-Glycan Sample PrepQuantitative Analysis to Compare to Convention
DifferentMechanisms
H O
rapiFluor-MSReductive Amination
2-ABRapid TaggingRapiFluor-MS
2-ABLabeled Human IgG N-Glycans
©2015 Waters Corporation 15
0.0E+0
3.0E+4
3 4 5 6 7 8 9 10
0.0E+0
3 4 5 6 7 8 9 10
FA2(Area = 2.0 x 103)
FLR
MS(BPI)
2-AB Label(pmol)
y = 7721.8x - 834.17R² = 1
0E+0
1E+4
2E+4
3E+4
4E+4
5E+4
6E+4
7E+4
0 5 10
Peak A
rea
(Flu
ore
scence
-Ex 3
30 n
m /
Em
420 n
m)
Revolutionizing N-Glycan Sample PrepSensitivity Comparison – 2-AB
4.0E+6
0.0E+0
3.3E+6
3 4 5 6 7 8 9
FA2(2.2 pmol)
FA2
105.8
126.9
100
150
FLR
MS(BPI)
RapiFluor-MS Labeled N-Glycans from Pooled Human IgG
FLR
MS(BPI)
Res
ponse
Fac
tors
of L
abel
ed F
A2 G
lyca
n/
1000)
©2015 Waters Corporation 16
0.0E+0
4.0E+6
3 4 5 6 7 8 9
4 5 6 7 8 9
0.0E+0
3 4 5 6 7 8 9
100x zoom
0.0E+0
3.3E+6
3 4 5 6 7 8 9
FA2 (2.6 pmol)
FA2 7.4
0.8
0
50
RapiFluor-MSLabeled
FLR
MS(BPI)
2-ABLabeled
Res
ponse
Fac
tors
(Pea
k Are
a per
pm
olof
Lab
eled
FA2 G
lyca
n
2-AB Labeled N-Glycans from Pooled Human IgG
50
60
70
80
90
100
50
60
70
80
90
100
Fluorescence MS (BPI)
Instant AB Labeled
RapiFluor-MS LabeledRel
ativ
e Pe
rform
ance
(%
)
52.5
Revolutionizing N-Glycan Sample PrepSensitivity Comparison
©2015 Waters Corporation 17
0
10
20
30
40
50
0
10
20
30
40
50
2-AB Labeled
Rel
ativ
e Pe
rform
ance
(%
)
7.0
0.1 0.6
Procainamide Labeled
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).
Simplified Workflow
Conventional5 Hours
to 2 Days
Direct Analysis
©2015 Waters Corporation 18
10 min 5 min 10 min
30 min
Patent Pending
Total Sample Prep Time
GlycoWorks RapiFluor-MS N-Glycan Kit
Direct Analysis(Organic Solvent Dilution)
Powered by New England Biolabs®
:: Global leader in the production and supply of reagents for the life sciences
:: Innovator of technologies from basic molecular biology through to next generation
sequencing and high throughput genomics
:: Provider of best-in-class products with unparalleled technical support
:: Manufacturer of reagents for glycan analysis for over 20 years
©2015 Waters Corporation 19
2014
Rapid PNGase F
released
2007
K. lactis
glycoengineered
strains
1994
Chitin
research
begins
1974
NEB founded
1980 2000 2010
1992
PNGase F, Endo H &
first exos released
1992
Maltose Binding
Protein Expression
Kit Released
2014
Human GPI
glycoproteome
published
1990
1988
Dwek coins term
“glycobiology”
2005
Cellulase
research
begins
1990
NEB glycobiology
program formed
2011
Heparinases
released
2008
Novel
O-glycosidase
released
2000
α-N-Acetyl-
Galactosaminidase
released
2012
Endo S
released
Rapid DeglycosylationRapiGest SF and Glycoworks Rapid PNGase F
RapiGest SFSurfactant
GlycoWorks Rapid PNGase F
GlycoWorks Enzymatic
©2015 Waters Corporation 20
2 min80˚C
GlycoWorks Rapid Buffer
5 min50˚C
EnzymaticDeglycosylation
Revolutionizing N-Glycan Sample PrepRapid Deglycosylation
©2015 Waters Corporation 21
(-) 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.
�Rapid deglycosylation: complete unbiased N-glycan removal in 10 minutes, compatible with rapid labeling and mass spectrometry analysis.
Revolutionizing N-Glycan Sample PrepSpeed: Fast Deglycosylation
©2015 Waters Corporation 22
(-) Negative Control (no enzyme)
(R) Rapid Deglycosylation (RapiGest, Rapid PNGase F, 10 min)
(RT) Rapid Deglycosylation with 4mM TCEP
(+) Positive Control (SDS/DTT + PNGase F)
Revolutionizing N-Glycan Sample PrepSpeed: Fast Deglycosylation
©2015 Waters Corporation 23
5 min50˚C
+Rapid PNGase F
2 min80˚C
Glycoprotein +
Rapid Buffer+
Rapigest SF
Labeled GlycanLabeled Deglycosylated ProteinReaction Byproducts
Revolutionizing N-Glycan Sample PrepRobustness: Quantitative Extraction
©2015 Waters Corporation 24
Byproducts
Collect
Labeled Glycan
Revolutionizing N-Glycan Sample PrepRobustness: Quantitative Extraction
Mixture of hIgG and bovine fetuin N-glycans
2.3E+6 No SPE Crude Reaction Mixture
FA2FA2G2S1
A3S1G3S3
A3G3S3
FLR
RapiFluor-MS FA2
©2015 Waters Corporation 25
0.0E+0
1.9E+6
0 5 10 15
0.0E+0
0 5 10 15
After SPE (1x SPE)
FA2
SPE Recovery
~74%
0E+0
1E+8
2E+8
3E+8
4E+8
5E+8
6E+8
0 30 60 90 120 150 180
RapiFluor-MS FA2 Fluorescence Peak Area
SPE Elution Volume (µL)
Specified elution volume
0.0E+0
1.2E+6
FA2
FA2G2S1
A3S1G3S3
1x SPE
15
20
25
30
Rela
tive
Abundance
(%
)
Positive Control
SPE Processed
A3G3S3
1x SPE
2x SPE
Revolutionizing N-Glycan Sample PrepRobustness: Quantitative Extraction
FLR
©2015 Waters Corporation 26
10 15 20 25 30 35
Positive Control
0.0E+0
1.2E+6
10 15 20 25 30 35
2x SPE
0
5
10
Rela
tive
Abundance
(%
)
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
Revolutionizing N-Glycan Sample PrepRobustness: Optimized Labeling
� Labeling optimized for high yield and minimization of artifacts
Glycoprotein Concentration0.36 mg/mL
RapiFluor-MSConcentration 1.6E+5
G0F
Fluore
scen
ce P
eak
Are
aFA
2 F
luore
scen
ce P
eak
Are
a
Maximized yield
18 mM
36 mM
©2015 Waters Corporation 27
0.0E+00 50 100
G0F
Fluore
scen
ce P
eak
Are
aFA
2 F
luore
scen
ce P
eak
Are
a
RapiFluor-MS Reagent Concentration (mM)
54 mM
72 mM
108 mM
4 9 min
Patent Pending
Revolutionizing N-Glycan Sample PrepRobustness: Optimized Labeling
� Labeling optimized for high yield and minimization of artifacts
BPI
10x zoom
FA2+2 Labels
FA2G1+2 Labels
Overlabeled~1% 0.3
0.4
(FA2 +
1 L
abel
/ F
A2 +
2 L
abel
s)
% Overlabeled
Minimization of artifacts+
Maximized yield
Not OptimizedHigh Ionic StrengthpH 8.8180 mM Reagent
©2015 Waters Corporation 28
0
0.1
0.2
0 50 100
(FA2 +
1 L
abel
/ F
A2 +
2 L
abel
s)
10x zoom
Overlabeled≤0.1%
Optimized50 mM HEPESpH 7.936 mM Reagent
BPI
A2
RapiFluor-MS Reagent Concentration (mM)
Patent Pending
Revolutionizing N-Glycan Sample PrepRobustness: High Yield
2E+6
FA2
N-Glycan Yield
(entire workflow)
~73%
y = 113033x - 11366R² = 11E+6
Ex 2
65 n
m /
Em
425 n
m)
©2015 Waters Corporation 29
0E+0
3 4 5 6 7 8 9 10 11 12 13
FA2Rep #1
1.6 pmolRep #2
1.7 pmol
100% Theoretical Yield = 2.3 pmol
0E+0
2E+5
4E+5
6E+5
8E+5
0 5 10
Peak A
rea
(Flu
ore
scence
-Ex 2
65 n
m /
Em
425 n
m)
RapiFluor-MS Label
(pmol)
1.5x107 pg IgG1 pmol
150,000 pg
2 pmol glycan1 pmol IgG
0.45 pmol FA21 pmol total
glycan pool
10 µL injection400 µL
sample
prepared
X X X X = 2.3 pmol
Revolutionizing N-Glycan Sample PrepRobustness: High Yield with Minimal Bias
Step Yield Testing to Confirm Minimal BiasIntact mass analysis / Subunit LC-MS
©2015 Waters Corporation 30
FA2Rep #1
1.6 pmolRep #2
1.7 pmol
100% Theoretical Yield = 2.3 pmol
Deglycosylation Complete � Intact mass analysis / Subunit LC-MS� Gel shift assays
Labeling >95% � Released glycan profile vs subunit derived glycan information
SPE ~74% � Recovery measurements� Glycan profile before vs after SPE
GlycoWorks RapiFluor-MS N-Glycan Kit
~73% Yield
GlykoPrep® Rapid N-Glycan Preparation
with 2-AB~35% Yield
1
2
3
GlykoPrep is a copyright of Prozyme. These values may not be representative of all applications.
Revolutionizing N-Glycan Sample PrepRobustness: Stable Glycan Derivatives
80000
100000
120000
140000
160000
180000
200000
0 Hours
72 Hours
Fluorescence
Fluore
scen
ce P
eak
Are
as
Initial
Intial
3 Days10x zoom
©2015 Waters Corporation 31
� No significant change in the glycan FLR signal even after 3 days at 10˚C� Stable glycan derivatives
0
20000
40000
60000
80000
Fluore
scen
ce P
eak
Are
as
A2
FA2
FA2G
1
FA2G
1(i
so)
FA2G
2
FA2G
2Sg1
FA2G
2G
a2
10 30 min
10 30 min
10x zoom
After 3 days10˚C
SummaryPart I� Preparation of labeled N-glycans (from
glycoprotein to analysis ready sample) in 30 minutes
� Unprecedented sensitivity for labeled N-
glycans
� Complete deglycosylation producing unbiased results
� Simple, streamlined protocol
RapiFluor-MS
©2015 Waters Corporation 32
� Simple, streamlined protocol
� Accurate profiling based on robust SPE for neutral to tetrasialylated N-glycans
GlycoWorks RapiFluor-MS N-Glycan Kit
Part II
GlycoWorks RapiFluor-MS N-Glycan Kit
©2015 Waters Corporation 33
GlycoWorks RapiFluor-MS N-Glycan Kit
@ Work
Conventional 2-ABGlycan from 10 µg of Protein1.5 Day Sample Prep
Glycan Characterization Using UPLC/FLR/Xevo G2-XS QTof
©2015 Waters Corporation 34
RapiFluor-MSGlycan from 0.1 µg Protein<1 hr Sample Prep 100x
Sensitivity
10 20 min
1: TOF MS ES+BPI
6.93e6
Example courtesy of Y. Yu
CharacterizationCharacterization
High Resolution LC-MS(/MS)
Conventional 2-ABGlycan from 10 µg of Protein1.5 Day Sample Prep
1130.921, 2+
Glycan Characterization Using UPLC/FLR/Xevo G2-XS
FA2G2Ga1
©2015 Waters Corporation 35
1: TOF MS ES+BPI
6.93e6
RapiFluor-MSGlycan from 0.1 µg Protein<1 hr Sample Prep 100x
Sensitivity
Example courtesy of Y. Yu
10 20 min
800 1000 1200 1400 1600800 1600 m/z
CharacterizationCharacterization
High Resolution LC-MS(/MS)
Glycan CharacterizationEnhanced Electrospray Ionization
3.47e6 2.89e42.9e43.5e62+ 1+
FA2
2-ABRapiFluor-MS
©2015 Waters Corporation 36
FA2BG2S25.3e2
500 1000 1500 2000
500 1000 1500 2000
1.15e5
500 1000 1500 2000500 2000 m/z
500 2000 m/z
500 2000 m/z
500 2000 m/z
1.2e5
3+
2+ 2+
FA2QTofCollision Induced Dissociation
Glycan CharacterizationInformation-Rich MS/MS Spectra
©2015 Waters Corporation 37
� Contiguous series of glycosydic bond fragments(Near exclusively containing label)
� MS/MS spectra additionally contain some cross ring fragmentation
Intact mAb Mass Check Standardanti-citrinin murine monoclonal IgG1FLR
MS
Glycan CharacterizationDetailing the N-Glycan Profile of a mAb
Rapid Preparation
DeglycosylationLabeling
SPE
30 min
2.1E+6
0.0E+6
8.4E+6
©2015 Waters Corporation 38
0.0E+6
Peak No.
Glycan NameObs Mass Mass Error Approximate
(M+H)+ (ppm) Relative%
Intact mAb Mass Check Standardanti-citrinin murine monoclonal IgG1FLR
MS
Glycan CharacterizationDetailing the N-Glycan Profile of a mAb
©2015 Waters Corporation 39
F: FucoseA: Antennary G: GalactoseB: BisectingGa: Alpha-galactose
Sg: N-glycolylneuraminic acid
iso: Structural isomer
1 A1 1425.5837 -3.8 0.09
2 FA1 1571.6446 -1.5 0.17
3 A2 1628.6676 -0.5 1.21
4 FA2 1774.7256 -0.4 46.02
5 A2G1 1790.7194 -1.1 0.66
FA1G1 1733.6976 -1.3 <0.04
6 A2G1(iso) 1790.7194 -1.1 0.30
7 FA2G1 1936.7772 -1.0 18.59
8 FA2G1(iso) 1936.7772 -1.0 21.51
9 FA2G1B 2139.8558 -1.3 0.04
10 FA2G1B (iso) 2139.8558 -1.3 0.21
11 FA2G2 2098.828 -1.9 8.03
12 FA2G1Ga1 2098.828 -1.9 0.67
13 FA2G2Ga1 2260.877 -3.4 1.08
14 FA2G2Sg1 2405.9174 -2.0 0.18
15 FA2G2Sg1(iso) 2405.9174 -2.0 0.06
16 FA2G2Ga2 2422.9322 -2.2 0.87
17 FA2G2GaSg1 2567.9694 -2.2 0.20
18 FA2G2Ga1Sg1(iso) 2567.9694 -2.2 0.11
Intact mAb Mass Check Standardanti-citrinin murine monoclonal IgG1FLR
MS FA2G2?
Glycan CharacterizationDetailing the N-Glycan Profile of a mAb
©2015 Waters Corporation 40
Zoom 10x
?
Glycan CharacterizationDetailing the N-Glycan Profile of a mAb
Relative Abundance8.0%
©2015 Waters Corporation 41
Prominent 528 m/z ion and loss of GlcNAc
Relative Abundance0.7%
Glycan CharacterizationDetailing the N-Glycan Profile of a mAb
Intact mAb Mass Check Standardanti-citrinin murine monoclonal IgG1FLR
MS FA2G2?FA2G1Ga1
©2015 Waters Corporation 42
Zoom 10x
?FA2G1Ga1
30.0010.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
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
Fluore
scen
ce (
rela
tive
)
Glycan MonitoringTransferability – UPLC / HPLC
©2015 Waters Corporation 43
30.00
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
Total Run Time = 121.3 minutes
Fluore
scen
ce (
rela
tive
)
Time (minutes)
� Comparable sensitivity and resolution – 3x sample load / 2x increase in time
� Transfer between labs with different LC equipment capabilities
Glycan MonitoringUPLC/FLR/ACQUITY QDa Mass Detector
Detection across a broad range of glycan species:
IgGSimple bi-antennerystructures
RNase BHigh mannose structures
Rountine Analysis with Mass Detection
FLR
©2015 Waters Corporation 44
5 10 15 20 25 30 35
Minutes5 10 15 20 25 30 35
High mannose structures
FetuinLarge, complex structures
Example courtesy of S. McCarthy
MonitoringMonitoring
Intermediate Technology UPLC-FLR
UPLC-Mass Detection
3x
3x
QDa
Glycan MonitoringUPLC/FLR/ACQUITY QDa Mass Detector
FLR
©2015 Waters Corporation 45
QDa
2
6
7
11
14
Labeled Glycans of IgG Pooled from Human Serum 400 pmol
FLR
Peak GlycanApprox
RT (min)
Approx Rel
Abund (%)
1 A2 11.8 0.7
2 FA2 12.8 20.9
3 FA2B 13.9 4.3
4 A2G1 14.2 1.0
5 A2G1 14.5 0.4
6 FA2G1 15.1 19.4
7 FA2G1 15.5 10.0
8 FA2BG1 15.9 5.0
9 FA2BG1 16.3 0.5
10 A2G2 16.8 0.9
11 FA2G2 17.7 14.8
12 FA2BG2 18.2 1.7
FA2BG2S2
FA2
GlycoWorks RapiFluor-MS StandardsRapiFluor-MS Glycan Performance Test Standard
©2015 Waters Corporation 46
1
3
45
8
91012
13 15 1617
MS(BPI)
5
12 FA2BG2 18.2 1.7
13 FA2G1S1 18.9 2.8
14 FA2G2S1 20.8 9.6
15 FA2BG2S1 21.5 2.4
16 FA2G2S2 23.6 2.2
17 FA2BG2S2 24.0 2.6
� System suitability� Method familiarization� Benchmarking� Troubleshooting
LC Calibration with Labeled Dextran
12
14
16
18
20
6
7
8910 11 12 13
14151617
n
Lo
g M
ol W
t
4.8
4.9
5.0
5.1
5.2
4
5
6
7
8
9
10
Cubic Spline Fitting
Calibration Curve
GU
Valu
e
©2015 Waters Corporation 47
EU
0
2
4
6
8
10
Retention Time (min)
5 10 15 20 25
3 45 L
og
Mo
l W
t
4.4
4.5
4.6
4.7
Retention Time (min)
5 10 15 20
0
1
2
3
5 6 7 8 9 10 11 12
Cubic Spline Fitting
Retention Time (min)
Lo
g M
ol W
t
4.8
4.9
5.0
5.1
5.2
12
14
16
18
20
4.44 min
4
5
6
7
8
9
10
Cubic Spline Fitting
Calibration Curve
GU
Valu
e
LC Calibration with Labeled Dextran
©2015 Waters Corporation 48
Lo
g M
ol W
t
4.4
4.5
4.6
4.7
Retention Time (min)
5 10 15 20
EU
0
2
4
6
8
10
12
Retention Time (min)
5 10 15 20 25
5.89GU
0
1
2
3
5 6 7 8 9 10 11 12
Cubic Spline Fitting
Retention Time (min)
5.85GU
5.95GU
25
30
35
Multiple sites reporting onone glycan profile
Significant deviations in RTs
8
9
10
11
12
Cubic Spline Fit Type
Ret
ention T
ime
(min
)
GU
Val
ue
LC Calibration with Labeled Dextran
©2015 Waters Corporation 49
10
15
20
0 5 10 15 20 25 30
Glycan Profile Component
RSDmax = 8.505
6
7
0 5 10 15 20 25 30
RSDmax = 0.49
Ret
ention T
ime
(min
)
GU
Val
ue
Glycan Profile Component Glycan Profile Component
� GU values improve the robustness of data reporting
LC Calibration with Labeled Dextran
� Dextran – no amine to modified with rapid tagging reagents
� Reductive amination with analogs is disadvantageous- different physicochemical properties due to different linkage- FLR wavelengths / basicity / pKas / polarity / HILIC retention
Reductive AminationWith an Analog
RapiFluor-MS Labeled N-Glycans
Dextran
Novel Dextran Ladder
DextranGlycan
©2015 Waters Corporation 50
235 nm
240 nm
245 nm
250 nm
255 nm
260 nm
265 nm
270 nm
275 nm
280 nm
285 nm
290 nm
295 nm
λExcitation,Max=265 nm λEmission,Max=425 nm Ex 265 nm
Em 425 nm
400 500 nm 400 500 nm 400 500 nm
x x265 nm
325 nm
370 nm
x
400 450 500
265 nm
315 nm
365 nm
Ex 265 nmEm 425 nm
xEx 370 nmEm 480 nm
DextranGlycan
50 µg of a Novel Dextran Ladder
GlycoWorks RapiFluor-MS StandardsRapiFluor-MS Dextran Calibration Ladder
FLR 813
9 1011 12
76
5
Ethanolamino Urea Linkage
RapiFluor-MS LabelDextran
� FLR properties match RapiFluor-MS labeled glycans � Tuned for HILIC Retention
1415
1617
©2015 Waters Corporation 51
4.00 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
MS(BPI)
GU 3
54
Patent Pending
1617 18
4 38 min
GlycoWorks Mobile Phase Concentrate
� High purity mobile phases are critical to successful glycan LC-MS
� LC-MS grade mobile phase concentrate– Eliminate concerns
LC-MS Grade
©2015 Waters Corporation 52
– Eliminate concerns on NH4 HCOOH purity and glass-ware cleanliness
– 10 mL concentrate simply poured into a 1L bottle of LC-MS water
Adduct Formation• Na• K• KCl / NaCl
Not LC-MS Grade
SummaryPart II
� N-glycan characterization facilitated by combining RapiFluor-MS labeling and high sensitivity QTof MS
� RapiFluor-MS enhances ESI of glycans, producing high charge state, protonated ions
� RapiFluor-MS enables the possibility of QDa mass detection during routine
GlycoWorks RapiFluor-MS N-Glycan Kit
RapiFluor-MS
©2015 Waters Corporation 53
QDa mass detection during routine analyses
� In addition to the GlycoWorks RapiFluor-MS N-glycan kit, new glycan standards and reagents have been developed to support analytical workflows, such as GU value assignment
RapiFluor-MS N-Glycan Kit
Ethanolamino Urea Linkage
RapiFluor-MS LabelDextran
Calibration Ladder
� “Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent” Waters Application Note 720005275EN. January 2015.
� “Robustness of RapiFluor-MS N-Glycan Sample Preparation and HILIC Analysis” Waters
Application Note in preparation. March 2015.
� Manuscript in preparation
Useful Literature
Posters
Application Notes / Publications
©2015 Waters Corporation 54
WCBP 2015 / P-206-W“Routine Monitoring of N-Glycans Using a Novel Labeling Reagent with Fluorescence and Mass Detection”
WCBP 2015 / P-216-W“Rapid Preparation of N-Glycans Using a Novel Fluorescence and MS Active Labeling Reagent”
WCBP 2015 / P-115-T“Developing a Scientific Library for UPLC/FLR/MS Analysis of Released N-glycans Labeled with a Novel
www.waters.com/Glycans www.waters.com/RapiFluor-MS
Posters
Thank You for Attending!
� Post-Event Home Page: http://www.waters.com/Feb24
� 30% Introductory Offer On GlycoWorks RapiFluor-MS
– Full Webinar Recording of Today’s Session w/PDF Slide Deck
– Compilation of TODAY’S KEY Literature, Brochures etc…
� For Questions and to Submit your Ideas for our Next Topic
©2015 Waters Corporation 55
� For Questions and to Submit your Ideas for our Next Topic
– Please eMail - mychemrep@waters.com
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