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©2015 Waters Corporation 1
GlycoWorks RapiFluor-MS
and the
ACQUITY QDa Mass Detector
A fast and powerful new solution
for ...
©2015 Waters Corporation 2
Presentation Outline
 Market feedback: Unmet needs in biopharma
 GlycoWorks™ RapiFluor-MS™ N-...
©2015 Waters Corporation 3
Market feedback
“We need a faster, more robust method for doing routine glycan
analysis. Our cu...
©2015 Waters Corporation 4
AccQ•Fluor
Rapid
Fluorescence
Patent Pending
RapiFluor-MS
RapiFluor-MS Reagent
Built on Waters’...
©2015 Waters Corporation 5
GlycoWorks RapiFluor-MS N-Glycan Kit
At least 16x faster than current 2AB methods
New proprieta...
©2015 Waters Corporation 6
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
...
©2015 Waters Corporation 7
FA2BG2S2
How RapiFluor-MS Helps…
Higher Charge for MS Compatibility
5.3e2
%
100
m/z
500 1000 15...
©2015 Waters Corporation 8
The ACQUITY QDa Mass Detector
A pioneering product with mass appeal
 Innovative design focused...
©2015 Waters Corporation 9
Easily Added to Existing Systems
Existing stack
Easy to deploy
Fully integrated with
Empower CD...
©2015 Waters Corporation 10
Familiar Graphical User Interface
for Ease-Of-Use and Fast Adoption
Empower GUI is identical
t...
©2015 Waters Corporation 11
Automated Start Up Provides
Robust, Reproducible Performance
 Automated resolution and calibr...
©2015 Waters Corporation 12
Disposable Sample Aperture
and Capillary for Easy Maintenance
Sample Aperture:
As simple as re...
©2015 Waters Corporation 13
ACQUITY QDa in Summary
 A pioneering product that is empowering analytical chemists
and chrom...
©2015 Waters Corporation 14
A Powerful Released N-Glycan
Monitoring Solution
What the following data demonstrates:
 The A...
©2015 Waters Corporation 15
Routine N-Glycan Detection with
Matching FLR and MS response
Minutes
5 10 15 20 25 30 35 40
Mi...
©2015 Waters Corporation 16
Routine N-Glycan Detection with
Matching FLR and MS response
Minutes
5 10 15 20 25 30 35 40
Mi...
©2015 Waters Corporation 17
IgG Glycan Profile and Structure
Confirmation Using the ACQUITY QDa
Key Takeaway: Large dynami...
©2015 Waters Corporation 18
Ions Detected with ACQUITY QDa
for IgG MS Standard
Glycan Mavg (Da) [M+H]+1
[M+2H]+2
[M+3H]+3
...
©2015 Waters Corporation 19
How About Something More Complex?
EU
0
200
400
TIC
CODA Processed
Intensity
0
2x10
6
4x106
6x1...
©2015 Waters Corporation 20
Largest Fetuin Structure Appears
in the ACQUITY QDa Mass Window
QDa Positive Scan
Apex
1161.78...
©2015 Waters Corporation 21
Empower Software Integration:
Easily Combine Optical and Mass Data
Empower integration enables...
©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
0
10
20
30
40
Minutes
2 3 4 5
10 m...
©2015 Waters Corporation 24
Enabling Accelerated Bioprocess
Development
EU
0
20
40 FLR
Intensity
0
1x106
2x106
Minutes
1 2...
©2015 Waters Corporation 25
Monitoring Key Glycan Ratios:
Keeping Tabs on Mannose 5
Time 1 Time 2 Time 3
Low Medium High
M...
©2015 Waters Corporation 26
 GlycoWorks RapiFluor-MS
N-Glycan Kit enables at least 16X
faster sample prep time
 RapiFluo...
©2015 Waters Corporation 27
ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm
Reimagine glycan analysis: www.w...
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Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

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Learn how new technologies from Waters, the RapiFluor-MS Labeling Reagent and the ACQUITY QDa Mass Detector, enable biopharmaceutical development and QC labs to monitor released N-glycans with complementary fluorescence and mass detection. http://www.waters.com/glycans

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Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

  1. 1. ©2015 Waters Corporation 1 GlycoWorks RapiFluor-MS and the ACQUITY QDa Mass Detector A fast and powerful new solution for released N-glycan monitoring
  2. 2. ©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
  3. 3. ©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 laboratories need a faster workflow with improved selectivity and sensitivity for routine glycan profiling.
  4. 4. ©2015 Waters Corporation 4 AccQ•Fluor Rapid Fluorescence Patent Pending RapiFluor-MS RapiFluor-MS Reagent Built 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
  5. 5. ©2015 Waters Corporation 5 GlycoWorks RapiFluor-MS N-Glycan Kit At 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-MS Method 30 Minutes! 15 min 5 min 10 min
  6. 6. ©2015 Waters Corporation 6 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 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* 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
  7. 7. ©2015 Waters Corporation 7 FA2BG2S2 How RapiFluor-MS Helps… Higher Charge for MS Compatibility 5.3e2 % 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 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 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 % 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 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 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-ABRapiFluor-MS Example courtesy of M.Lauber
  8. 8. ©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
  9. 9. ©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
  10. 10. ©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
  11. 11. ©2015 Waters Corporation 11 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 Graphical ACQUITY QDa monitor display Enables easy viewing and adjustment of system parameters
  12. 12. ©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
  13. 13. ©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
  14. 14. ©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
  15. 15. ©2015 Waters Corporation 15 Routine N-Glycan Detection with Matching 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-antennary structures RNase B High mannose structures Fetuin Large, complex and highly sialylated structures FLR ACQUITY QDa
  16. 16. ©2015 Waters Corporation 16 Routine N-Glycan Detection with Matching FLR and MS response Minutes 5 10 15 20 25 30 35 40 Minutes 5 10 15 20 25 30 35 40 EU 0 20 40 60 FLR Intensity 1x10 6 2x10 6 3x10 6 4x10 6 5x10 6 6x10 6 TIC Key Takeaway: Equivalent FLR and MS response all within a single run, with the added benefit of mass confirmation.
  17. 17. ©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 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
  18. 18. ©2015 Waters Corporation 18 Ions Detected with ACQUITY QDa for 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
  19. 19. ©2015 Waters Corporation 19 How About Something More Complex? EU 0 200 400 TIC CODA Processed Intensity 0 2x10 6 4x106 6x106 8x10 6 Minutes 10 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
  20. 20. ©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 50000 100000 150000 200000 250000 400 600 800 1000 1200 A3G3S4 [M+3H]3+ A complex tri-antennary tetra-sialylated glycoform Analysis on our Xevo® G2-XS QTof confirms the +3 charge state is the only charge state for this glycoform
  21. 21. ©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 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
  22. 22. ©2015 Waters Corporation 22 Empower Software Integration: GMP Compliant Reporting with Mass >
  23. 23. ©2015 Waters Corporation 23 Developing a Rapid Method for Released N-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 Trastuzumab N-Glycan Analysis
  24. 24. ©2015 Waters Corporation 24 Enabling Accelerated Bioprocess Development 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)1 896.4 m/z F(6)A2 888.4 m/z F(6)A2G(4)1 969.4 m/z F(6)A2G(4)2 1050.5 m/z A2 815.3 m/z F(6)A2G(4)2S1 865.5 m/z M5 774.1 m/z
  25. 25. ©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 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: 969.4 m/z M5 SIR: 774.1 m/z Man5: F(6)A2G(4)1 Ratio
  26. 26. ©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
  27. 27. ©2015 Waters Corporation 27 ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm Reimagine glycan analysis: www.waters.com/glycans

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