Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection
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Transcript of Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection
©2015 Waters Corporation 1
GlycoWorks RapiFluor-MS
and the
ACQUITY QDa Mass Detector
A fast and powerful new solution
for released N-glycan monitoring
©2015 Waters Corporation 2
Presentation Outline
Market feedback: Unmet needs in biopharma
GlycoWorks™ RapiFluor-MS™ N-Glycan Kit
and the ACQUITY® QDa® Detector
Powerful N-glycan profiling with mass confirmation
Greater speed, confidence and productivity
Reimagine glycan analysis
©2015 Waters Corporation 3
Market feedback
“We need a faster, more robust method for doing routine glycan
analysis. Our current sample prep method takes a day and a
half, and our results are not as sensitive, or as clear cut as we’d
like them to be.”
“It’s critical that we monitor the glycan profile of our protein
therapeutic and ensure that it remains consistent as we move
through development, production and final release.”
“Sometimes we see unexpected peaks or shoulders in our
fluorescent trace and the only way to obtain mass data is to
send samples to our core mass spec lab.”
The bottom line: Biopharmaceutical laboratoriesneed a faster workflow with improved selectivity and
sensitivity for routine glycan profiling.
©2015 Waters Corporation 4
AccQ•Fluor
RapidFluorescence
Patent Pending
RapiFluor-MS
RapiFluor-MS ReagentBuilt on Waters’ Biopharm Applications Knowledge
From Waters’ expertise in rapid,
fluorescence labeling of amino acids
Enhanced chemical properties
for glycan analysis:
Rapid tagging
Efficient fluorescence
Enhanced ionization efficiency
©2015 Waters Corporation 5
GlycoWorks RapiFluor-MS N-Glycan KitAt least 16x faster than current 2AB methods
New proprietary reagent mix reduces de-glycosylation time from 16+ hours down to 15 minutes!
Novel label reduces labeling time to just 5 minutes, and dramatically improves FLR and MS sensitivity
Samples ready for analysis in 30 minutes. Can also be dried down for later analysis
>1 work day
Conventional 2AB Method
Pre-Labeling, Clean-Up + Drying
3 hours
Drying
2 hours
Deglycosolation
16+ hours
Labeling
3 hours
Clean-Up
0.1 hours
RapiFluor-MSMethod
30 Minutes!
15 min
5 min
10 min
©2015 Waters Corporation 6
Unparalleled FLR and MS Sensitivity
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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
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30.0*
7.0*
RapiFluor-MS vs Instant: 2x ↑ in FLR and 1000x ↑ in MSRapiFluor-MS vs Traditional: 14x ↑ in FLR and 160x ↑ in MSRapiFluor-MS vs PCA: 14x ↑ in FLR and 3x ↑ in MS
©2015 Waters Corporation 7
FA2BG2S2
How RapiFluor-MS Helps…Higher Charge for MS Compatibility
5.3e2
m/z500 1000 1500 2000
%
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m/z500 1000 1500 2000
%
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m/z500 1000 1500 2000
%
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m/z500 1000 1500 2000
%
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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
m/z500 1000 1500 2000
%
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m/z500 1000 1500 2000
%
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m/z500 1000 1500 2000
%
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m/z500 1000 1500 2000
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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
m/z500 1000 1500 2000
%
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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
m/z500 1000 1500 2000
%
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m/z500 1000 1500 2000
%
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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
500 2000 m/z
500 2000 m/z
500 2000 m/z
2.9e4
1.2e5
3.5e62+
3+
2+ 2+
1+
FA2
2-ABRapiFluor-MS
Example courtesy of M.Lauber
©2015 Waters Corporation 8
The ACQUITY QDa Mass Detector A pioneering product with mass appeal
Innovative design focused on ease of
use for chromatographers
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 UPLC® Systems for
ease-of-use and GMP compliance
www.waters.com/qda
©2015 Waters Corporation 9
Easily Added to Existing Systems
Existing stack
Easy to deploy
Fully integrated with Empower CDS Software
Minimal training required
110/220V operation
Minimal maintenance
ACQUITY QDa
Existing stack +1upgrade
©2015 Waters Corporation 10
Familiar Graphical User Interface for Ease-Of-Use and Fast Adoption
Empower GUI is identical to that of a FLR for setup, data viewing and reporting Very little training needed
Quick addendum to current lab SOP
©2015 Waters Corporation 11
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
Graphical ACQUITY QDamonitor display Enables easy viewing and adjustment of system parameters
©2015 Waters Corporation 12
Disposable Sample Aperture and Capillary for Easy Maintenance
Sample Aperture:
As simple as replacing a detector lamp
Capillary:
No cutting or assembly required
©2015 Waters Corporation 13
ACQUITY QDa in Summary
A pioneering product that is empowering analytical chemists
and chromatographers everywhere with the power of mass
detection
Brings greater insight into every peak, enhanced monitoring
and streamlined workflows for improved productivity
Easy to deploy, simple to use and maintain, compact, robust
and affordable!
www.waters.com/qdabiopharm
©2015 Waters Corporation 14
A Powerful Released N-Glycan Monitoring Solution
What the following data demonstrates:
The ACQUITY QDa Detector detects a broad range of
RapiFluor-MS labeled N-glycans across a large dynamic range
The ACQUITY QDa seamlessly integrates with UPLC workflows
using Empower Software, enabling GMP-compliant reporting
with mass data
The ACQUITY QDa and RapiFluor-MS combination enables
faster methods and higher throughput N-glycan profiling for
improved process development, optimization and monitoring
©2015 Waters Corporation 15
Routine N-Glycan Detection with Matching 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-antennary structures
RNase BHigh mannose structures
FetuinLarge, complex and highly sialylatedstructures
FLR
ACQUITYQDa
©2015 Waters Corporation 16
Routine N-Glycan Detection with Matching FLR and MS response
Minutes5 10 15 20 25 30 35 40
Minutes5 10 15 20 25 30 35 40
EU
0
20
40
60
FLR
Inte
nsity
1x106
2x106
3x106
4x106
5x106
6x106
TIC
Key Takeaway:Equivalent FLR and MS response all within a single run, with the added benefit of mass confirmation.
©2015 Waters Corporation 17
IgG Glycan Profile and Structure Confirmation Using the ACQUITY QDa
Key Takeaway: 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
©2015 Waters Corporation 18
Ions Detected with ACQUITY QDafor IgG MS Standard
Glycan Mavg (Da) [M+H]+1 [M+2H]+2 [M+3H]+3
A2 1628.6 1629.6 815.3 543.9
FA2 1774.7 1775.7 888.4 592.6
FA2B 1977.9 1978.9 990.0 660.3
A2G1a 1790.7 1791.7 896.4 597.9
A2G1b 1790.7 1791.7 896.4 597.9
FA2G1a 1936.9 1937.9 969.4 646.6
FA2G1b 1936.9 1937.9 969.4 646.6
FA2BG1a 2140.1 2141.1 1071.0 714.4
FA2BG1b 2140.1 2141.1 1071.0 714.4
A2G2 1952.9 1953.9 977.4 652.0
FA2G2 2099.0 2100.0 1050.5 700.7
FA2BG2 2302.2 2303.2 1152.1 768.4
FA2G1S1 2228.1 2229.1 1115.1 743.7
FA2G2S1 2390.3 2391.3 1196.1 797.8
FA2BG2S1 2593.5 2594.5 1297.7 865.5
FA2G2S2 2681.5 2682.5 1341.8 894.8
FA2BG2S2 2884.7 2885.7 1443.4 962.6
©2015 Waters Corporation 19
How About Something More Complex?
EU
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400
TICCODA Processed
Inte
nsity
0
2x106
4x106
6x106
8x106
Minutes10 15 20 25 30 35 40
FLR
Bovine Fetuin RapiFluor-MS Labeled N-Glycans (~30 pmol)
Key Takeaway:Demonstrates the upper limit of detection, and the ability to detect large, complex multi-antennary structures
©2015 Waters Corporation 20
Largest Fetuin Structure Appears in the ACQUITY QDa Mass Window
QDa Positive Scan
Apex
1161.78
1167.43
1169.09
1174.72
0
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A3G3S4[M+3H]3+
A complex tri-antennary tetra-sialylatedglycoform
Analysis on our Xevo® G2-XS QTofconfirms the +3 charge state is the only charge state for this glycoform
©2015 Waters Corporation 21
Empower Software Integration:Easily Combine Optical and Mass Data
Empower integration enables annotation of peaks with both names and m/z data
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
©2015 Waters Corporation 22
Empower Software Integration:GMP Compliant Reporting with Mass
>
©2015 Waters Corporation 23
Developing a Rapid Method for Released N-Glycan Analysis
EU
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Minutes2 3 4 5
10 minute
Method
EU
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Minutes5 10 15 20
55 minute
Method
Trastuzumab N-Glycan Analysis
©2015 Waters Corporation 24
Enabling Accelerated Bioprocess Development
EU
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20
40 FLR
Inte
nsity
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1x106
2x106
Minutes1 2 3 4 5 6
SIR Overlay
Trastuzumab N-Glycan AnalysisRapiFluor-MS labeled glycans - 10 minute method In
tensity
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Inte
nsity
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Inte
nsity
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1x106
2x106
Inte
nsity
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Inte
nsity
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nsity
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nsity
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1 2 3 4 5 6
A2G(4)1896.4 m/z
F(6)A2888.4 m/z
F(6)A2G(4)1969.4 m/z
F(6)A2G(4)21050.5 m/z
A2815.3 m/z
F(6)A2G(4)2S1865.5 m/z
M5774.1 m/z
©2015 Waters Corporation 25
Monitoring Key Glycan Ratios:Keeping Tabs on Mannose 5
Time 1 Time 2 Time 3
Low Medium High
Mean 0.58 0.91 1.16
StDev 0.03 0.05 0.04
% RSD 5.44 5.47 3.85
Inte
nsity
0
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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: 969.4 m/z
M5SIR: 774.1 m/z
Man5: F(6)A2G(4)1 Ratio
©2015 Waters Corporation 26
GlycoWorks RapiFluor-MS
N-Glycan Kit enables at least 16X
faster sample prep time
RapiFluor-MS labeled N-glycans
are well observed by the
ACQUITY QDa mass detector
Empower Software supports GMP
compliant workflows
The ACQUITY QDa Detector
enables greater selectivity, faster
methods and mass confirmation
for increased confidence and
productivity
In Summary
Fast and powerful released N-glycan monitoring with the confidence of mass confirmation
RapiFluor-MS+
ACQUITY QDa
www.waters.com/qdabiopharm
©2015 Waters Corporation 27
ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm
Reimagine glycan analysis: www.waters.com/glycans