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

Editor's Notes

• #2: 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.
• #3: 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.
• #4: 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.
• #5: 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.
• #6: 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.
• #7: Comparing FLR and MS sensitivity to 3 typical labels used on the market. FLR is in red, and MS in blue.
• #8: 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.
• #9: 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.
• #10: 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.
• #11: (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).
• #12: 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.
• #13: 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.
• #14: 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.
• #15: 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.
• #16: 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.
• #17: 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.
• #18: 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.
• #19: 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.
• #20: 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.
• #21: 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.
• #22: 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.
• #23: 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.
• #24: 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
• #25: 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…)
• #26: 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.
• #27: 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!
• #28: 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