Fast and powerful solution for released N-glycan monitoring with mass confirmation
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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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Fast and powerful solution for released N-glycan monitoring with mass confirmation
- 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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©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. ©2015 Waters Corporation 22 Empower Software Integration: GMP Compliant Reporting with Mass >
- 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. ©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. ©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. ©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. ©2015 Waters Corporation 27 ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm Reimagine glycan analysis: www.waters.com/glycans
Hinweis der Redaktion
- Hello to all of you and welcome to a presentation about innovative technology solutions from Waters! We’re really excited to share with you a fast and powerful new solution we have developed for released N-glycan monitoring, and that we think will greatly benefit scientists like you working in biopharmaceutical development, production and QC environments. Building upon our core ACQUITY UPLC Systems and industry-leading Empower chromatography data software (CDS), the released N-glycan monitoring solution we’re talking about is made possible by two highly innovative new products from Waters – the first being our groundbreaking new GlycoWorks RapiFluor-MS N-Glycan Kit that we launched at the WCBP conference in January (2015), and the second being our ACQUITY QDa Mass Detector, which is another revolutionary product we introduced in the Fall of 2013. As part of this presentation I’ll take a little time to introduce you to both these products, so you understand what makes them so unique and powerful, and then we’ll focus on how RapiFluor-MS labeling reagent combines with our ACQUITY UPLC H-Class Systems, UPLC glycan columns, and Empower Software to make for a very fast and powerful new solution for N-glycan monitoring on a routine basis. RapiFluor-MS delivers a 16x faster sample preparation protocol compared to traditional labeling techniques, and it’s 8x faster than other “instant” labeling techniques. In addition, it provides faster chromatographic runs and more accurate results backed up by mass confirmation, and all within a GMP-compliant ready workflow. Sound interesting? Let’s jump in.
- To begin, here is a basic outline of the presentation… We’ll start by first sharing some feedback we’ve been hearing from biopharma customers about the challenges they face today when doing routine glycan analysis. And as our new RapiFluor-MS N-Glycan Kit and the ACQUITY QDa mass detector are key components to our overall solution to address these challenges, we’ll provide a brief introductory overview of both these products. Next, we’ll look at data that our scientific team has generated, which demonstrate the enhanced speed, performance and productivity that can be achieved when utilizing our new solution. And finally, we’ll summarize what we discussed and demonstrate that putting our new glycan monitoring solution into practice will not only bring you greater speed, but greater confidence in your glycan assignments for better decision making and lower overall risk, and that it can significantly improved productivity as well.
- So let’s start with what we’ve been hearing from biopharmaceutical developers and manufacturers like yourself… The quotes on this slide clearly indicate a level of frustration out there with regard to current released N-glycan methods, especially with regard to the long sample prep time, but also with regard to the data sometimes not providing enough clarity. So what if you could reduce sample prep time from a day down to just 30 minutes? And what if you could capture both fluorescent data and mass data in a single run, such that you can gain greater confidence in your results with increased sensitivity and mass confirmation? And what if you could do all of this in a GMP compliant manner using your existing workflow, and you could speed up sample runs by 50% or more? Well, that’s just what you can do with our new released N-glycan monitoring solution and I’ll show you data that backs this up. But first let me take a few moments to introduce you to the two products that make this new solution possible: our brand new RapiFluor-MS N-Glycan Kit and our ACQUITY QDa detector.
- The key enabler for our new solution to N-glycan monitoring is our recently launched RapiFluor-MS reagent. To develop this reagent, we revisited Waters’ expertise in rapid, fluorescence labeling of amino acids For 20 years, Waters has been providing a solution for amino acid labeling that uses the novel reagent, known as AccQ•Fluor. This molecule has 3 very important chemical characteristics; It has a rapid tagging reactive group, and It has an efficient fluorophore for high sensitivity fluorescence detection, and To facilitate modern N-glycan analyses, AccQ Fluor was functionalized so as to have a third chemical property, an MS enhancing tertiary amine charge tag. This has produced a novel labeling reagent that we call RapiFluor-MS.
- Beyond our new label, the GlycoWorks RapiFluor-MS N-Glycan Kit also includes a new and proprietary enzyme mixture which greatly simplifies the deglycosolation process and reduces deglycosolation time from 16+ hours down to less than 15 minutes!!! Yes - you heard that correctly. In fact, with the RapiFluor-MS N-Glycan Kit you can complete your entire sample prep in just 30 minutes! This remarkable improvement not only enhances productivity on the sample prep side, but also on the detection side, by enabling more rapid methods (explained later in the presentation) and by enabling both fluorescent detection and mass detection within a single GMP compliant workflow, thereby increasing certainty especially with overlapping peaks, and reducing the amount of follow-on analysis that may be needed.
- Comparing FLR and MS sensitivity to 3 typical labels used on the market. FLR is in red, and MS in blue.
- Because the RapiFluor-MS label has high proton affinity, derivatized glycans preferentially adopt uniquely high charge states during positive ion mode electrospray ionization. As shown with the mass spectra on the left, the predominant charge state for a small neutral glycan, such as an FA2 glycan, labeled with RapiFluorMS is 2+, although it increases to 3+ for larger molecular weight glycans, such as an FA2BG2S2, disialylated glycan. Notice that these species when labeled with 2-AB adopt only 1+ and 2+ charge states, respectively.
- Now let me take a few moments to tell you about the ACQUITY QDa detector, another pioneering product that exemplifies our innovation strategy here at Waters. Launched in 2013, it has seen remarkable uptake across multiple industries and is considered one of the most successful product introductions in the company’s history, which is saying a lot! Our goal from the outset was to allow analytical chemists to collect mass information in a similar way to how they currently collect optical information, by providing a compact, robust, and highly affordable mass detector that’s as easy to use as a PDA detector, and that seamlessly integrates into existing Empower based UPLC systems. That is exactly what we did. The instrument control, data acquisition and reporting functions are all fully integrated into our industry leading Empower Chromatography Data Software (Empower 2 FR5 and Empower 3), so anyone using an Empower based UPLC system today can literally add this unit to their existing stack and begin collecting mass data right away.
- Here you see how the ACQUITY QDa Detector is designed to slot right into an existing stack. It is easily added to all existing ACQUITY UPLC systems running with Empower 2 (FR5) or Empower 3, which enables the data to be captured and reported in a GMP compliant manner. The QDa has been designed to work with both 110 and 220V power, so importantly in North America it does not require any special electrical hook-up. It also requires minimal maintenance, and requires minimal training to operate, all of which makes it incredibly easy to implement.
- (Note: GUI = Graphical User Interface) As previously indicated, adding an ACQUITY QDa to an existing stack and running it is no more difficult than that of a PDA detector. In fact the GUI interfaces within Empower Software for the set-up, data viewing and reporting are all identical to that of a FLR detector. The interface on this slide shows how users program the instrument to collect mass data. Users can easily select the mass range to be scanned, what ion detection mode to use (positive, negative or both), and through the advanced tab, one can easily create a list of target analytes they wish to monitor by using selected ion recording (SIR).
- The ACQUITY QDa performs automated calibration and resolution with every start-up, ensuring data collected is accurate and precise every time the instrument is used. In addition, the electrospray (ESI) interface has been optimized to preserve the resolution of your separation. The pre optimized source greatly limits the amount of “tuning” necessary to get good results, a factor which has been and continues to be a key challenge when introducing mass spectrometry to new settings. With the ACQUITY QDa, we have addressed these issues and made it much easier for chromatographers and analytical chemists to start generating their own mass data.
- As mentioned previously, the ACQUITY QDa is incredibly robust and requires minimal maintenance, which is often not the case with mass spectrometry equipment. There are just two components that periodically need to be replaced - the sample aperture, and the capillary assembly - and to make replacement as easy as possible to maintain, we designed these parts to be disposable. On the left you can see how it is to remove the sample aperture. Once removed it is discarded and replaced with a new aperture, and the entire process can be completed in under 10 minutes. On the right is a pre-cut and assembled capillary assembly, which can easily be replaced in under a minute. S No cutting or assembly required on the part of the end user.
- So in summary, the ACQUITY QDa is yet another pioneering, innovative product from Waters that has experienced tremendous success since it’s market introduction. As simple to deploy as a PDA, easy to use and maintain, highly compact and affordable – the ACQUITY QDa is a groundbreaking product that is expanding and strengthening the capabilities of analytical chemists by allowing them to collect mass data routinely for greater confidence and insight in their separations, and that is fueling productivity gains in many industrial workflows, including those of pharmaceutical and biopharmaceutical development, production and QC.
- So now that you know more about these two innovative products, let me walk you through some released N-glycan data we recently generated, which shows: how well the QDa detects RapiFluor-MS labeled N-glycans; how the QDa fully integrates with our industry leading Empower CDS software, making it a very simple add-on to your existing chromatographic workflow – allowing you to capture, annotate and report mass data in a GMP compliant manner; and how the QDa and RapiFluor-MS combination enables much faster and higher throughput N-glycan profiling on a routine basis, which can speed up process development and optimization and improve productivity overall.
- So let’s look first at N-glycan detection with the RapiFluor-MS label using both a fluorescence detector and the QDa mass detector. Represented on this slide are the combined chromatograms of released N-glycans from three different samples chosen to reflect the wide range of glycans typically seen and monitored in biotherapeutic development and production settings. These are 1) IgG, which includes simple bi-antennary glycan structures, 2) Rnase B, which includes many high mannose structures, and 3) Bovine Fetuin, which includes many larger, more complex and highly sialylated glycan structures. As you can see, both chromatograms show equivalent detection responses, and all the peaks in one are seen in the other.
- The FLR trace is on top, and the TIC trace is on the bottom. Analytical chemists now have the ability to detect and monitor glycoforms not just by retention time and fluorescent signal, but my mass data as well, and that all of this data can be captured simultaneously in a single run, simply by adding the ACQUITY QDa as an orthogonal mass detector to existing Empower based chromatographic workflows.
- On this slide we show the ability of the QDa to detect RapiFluor-MS labeled N-glycans across a wide dynamic range. Shown on the right side are the mass spectra for the two peaks that represent the most abundant and least abundant glycoforms in this IgG glycan profile (FA2 and A2G1b respectively). Note, RPA% is calculated based on peak areas from the fluorescent trace.
- On this slide we list all of the released N-glycans from an IgG MS standard, and the charge state by which the QDa detects each glycoform.
- For a more complex set of glycoforms we looked at Bovine Fetuin glycans, which include some the largest, most complex and highly sialylated glycoforms that can be associated with protein therapeutics. As shown, all peaks on the FLR trace are seen with equivalent response levels on the TIC trace using the QDa - further demonstrating the ability of the QDa to accurately detect large and complex glycoforms tagged with the RapiFluor-MS label, and underscoring its utility as a robust glycan monitoring tool.
- Here we look at the mass spectra for one of the largest and most complex glycoforms in the Fetuin profile. The A3G3S4 glycan is a large tri-antennary tetra-sialylated glycoform, and as shown, its +3 charge state clearly appears within the ACQUITY QDa mass window. To determine if other charge states are perhaps not being seeing with the ACQUITY QDa, we analyzed the same glycoform using our Xevo G2-XS QTof mass spectrometer, which confirmed only the +3 charge state exists for this glycoform.
- With the QDa fully integrated into Empower, both optical and mass data can now be captured within a single run, and as shown on this slide, it’s easy to annotate fluorescent chromatograms with peak names and corresponding mass data.
- Empower is the leading Chromatography Data Software (CDS) in the industry because of it’s ease-of-use, intuitive functionality, and its comprehensive GMP compliant ready data analysis and reporting capabilities. And with it now being fully integrated with the QDa, all the mass data captured can be combined with optical data and presented in a consolidated GMP compliant report, making it a great solution for biotherapeutic development, production and QC settings.
- On this slide we show a 55 minute chromatographic method that has been compressed down to a 10 minute method. You can see there is a loss in chromatographic resolution with the 10 minute method, but if you know what you are looking for and have developed an SIR list (as seen on the next slide), you retain the ability to confirm glycan structures via mass. This can come in handy when doing process development and optimization work and are having to evaluate many different samples/permutations (clone selection, various bioreactor conditions, etc…). The quick 10 minute SIR method in this case enables a higher throughput workflow enabling you to evaluate a wide range of samples more quickly. Materials and Methods: C: ACQUITY UPLC BEH Amide column, 1.7 m, 2.1 mm x 50 mm F: 0.8 mL/min T: 60 C A: Acn B: 50 mM AF, pH 4.5 Gradient for 10 minute method: T F %ACN 0 0.8 75 6 0.8 54 6.25 0.4 0 7 0.4 0 7.25 0.4 0 8 0.8 75 10 0.8 75 Gradient for 55 minute method: T F %ACN 0 0.4 75 35 0.4 54 36.5 0.2 0 39.5 0.2 0 43.1 0.2 75 47.6 0.4 75 55 0.4 75
- To illustrate this, here we see the 10 minute method with both the fluorescent chromatogram and the SIR overlay, along with the individual SIR’s for each of the targeted glycans being monitored. The key point here is that with the QDa added to your workflow, you not only gain mass confirmation for unambiguous glycan assignments, but you increase productivity as well. With peak identities confirmed via mass, you don’t need as much chromatographic resolution and can run faster methods, thus increasing throughput and accelerating bioprocess development work. (clone selection, bioreactor conditions, etc…)
- Increases in Mannose 5 are generally considered undesirable for a host of reasons. Here we simulate an increasing amount of the M5 Mannose structure over time relative to another glycoform: F(6)A2G(4)1. In the table report you can see how the ratio increases with each successive increment in time. This just demonstrates another way the QDa can improve monitoring capabilities and productivity.
- To summarize, our new Glycoworks RapiFluor-MS N-glycan kit represents a tremendous breakthrough in released N-glycan analysis and monitoring. Not only are sample prep times cut down from a day and a half to under 30 minutes, but glycan assignments can now be confirmed via orthogonal mass detection within a single workflow. This not only lowers risk by strengthening confidence in glycan assignments, but the use of mass detection also enables much faster and higher throughput methods, which can significantly increase productivity, especially during process development and optimization work. We’ve shown that the QDa accurately detects a wide range of RapiFluor-MS labeled N-glycans, from simple to highly complex, with excellent sensitivity and selectivity, and across a wide dynamic range. And again, the unique ability to set up SIR lists for monitoring can facilitate higher throughput analysis and strengthen monitoring, especially during process development and optimization. With RapiFluor-MS and the QDa you can now carry out fast, powerful N-glycan profiling on a routine basis, with unrivaled sensitivity and mass confirmation, all within a single workflow, in your own lab, without having to rely upon external MS resources. Thank you!
- Reimagine glycan analysis with RapiFluor-MS released N-glycan label and the ACQUITY QDa Detector: www.waters.com/glycans www.waters.com/glycans More info on the ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm www.waters.com/waters/nav.htm?cid=134837932 Details on the ACQUITY QDa Detector: www.waters.com/qda www.waters.com/waters/nav.htm?cid=134761404