Learn how the Waters ACQUITY QDa Detector is a powerful tool for mass detection in monitoring peptides in HPLC or UPLC assays, in biopharmaceutical late development and quality control. http://www.waters.com/qdabiopharm
I would like to discuss today some exciting work we’ve done recently around enabling the use of mass detection for routine peptide monitoring in the Biopharmaceutical development, production and QC environment and the benefits associated with doing so. I’ll also discuss how easy it is to add this capability to your existing Empower Software-based chromatography workflows for either HPLC or UPLC, and how even people with no mass spec experience can begin collecting mass data right away.
I’ll start by discussing what we’ve been hearing from many of our customers in the biopharmaceutical industry about their challenges and unmet needs. And as the main point of this presentation is to show how our innovative, easy-to-use, compact and affordable ACQUITY QDa mass detector can address many of these needs, I’ll provide a brief overview of the ACQUITY QDa Detector and its core attributes. Then I’ll transition into a review of data and results we recently generated, which clearly show how a Waters system with the ACQUITY QDa Detector can strengthen monitoring and improve overall productivity.
So lets start with what we’ve been hearing from many of our biopharmaceutical customers…
From these quotes it is clear that many researchers in biopharmaceutical laboratories have indicated a desire to utilize mass detection more, especially for routine monitoring - when you largely know what you’re looking for – and that they’d like for this work to be done by traditional analytical support labs that support development, manufacturing and QC, as opposed to core MS lab resources, thus enabling more streamlined workflows and improved productivity overall.
Along these lines they have expressed a desire for robust, easy-to-use and affordable instruments that can be easily added to their current LC optical workflows, and can generate GMP compliant mass information.
It is widely understood that high end mass spectrometry is critically important for research, discovery and advanced bio-characterization work, but when it comes to routine mass monitoring they can be overkill…. too big, too complex, and too expensive. Based on our recent work and the data we’ll review in this presentation, we will demonstrate how the ACQUITY QDa Mass Detector is just the kind of robust, compact and affordable tool that these customers have been looking for.
By way of introduction, the ACQUITY QDa mass detector is pioneering product that exemplifies our focused innovation strategy here at Waters. Launched in the the fall of 2013, it has seen remarkable uptake across multiple industries and is officially the most successful product introduction in the company’s history, which is saying a whole lot!
Our development goal was to give analytical chemists/chromatographers with no mass spec experience the ability to collect mass information in the same way they currently collect optical information, by providing a compact, robust and affordable mass detector that’s as easy to use as a PDA detector, and that seamlessly integrates into our existing UPLC and HPLC 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 HPLC or UPLC system today can literally add this unit to their existing stack and begin collecting mass data right away.
Here you can see how the ACQUITY QDa detector is designed to slot right into an existing stack. It can be easily added to all existing Waters ACQUITY UPLC systems and Alliance HPLC systems running with Empower 2 (FR5) or Empower 3. The ACQUITY QDa requires minimal training to operate, and qualification of the instrument is currently available meaning this mass detection capability can be added to chromatographic systems throughout your organization – in both non-regulated and regulated environments. The QDa has also been designed to work with both 110 and 220 V power making integration that much easier as a special electrical hookup in North America is not required. Plus it is incredibly easy to use and requires very minimal maintenance, as we will show on the next few slides.
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 Graphical User Interface (GUI) within Empower for the set-up, data viewing and reporting are all identical to that of a PDA detector, so anyone running Empower today would have no problem working with the ACQUITY QDa. The interface on this slide shows how users program the instrument to collect mass data. Similar to a PDA, users can define their mass range, whether they want positive, negative or both ionization modes, and in addition, they can define particular masses of interest they wish to monitor by using the selected ion recording (SIR) feature.
The ACQUITY QDa performs automated calibration and resolution with every start up ensuring the data collected is always accurate and precise, so you don’t have to worry about it. 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 - a factor which has been and continues to be a challenge when introducing mass spectrometry to new settings. With the ACQUITY QDa, we have addressed these issues and made mass detection approachable and accessible to a great many more end users.
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 we designed these parts to be disposable.
On the left you can see how easy it is to remove the sample aperture (little black piece that looks like a top hat). 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. No cutting or assembly required by the end user.
So in summary, the ACQUITY QDa Mass Detector is yet another pioneering, innovative product from Waters that has experienced tremendous success since it’s launch in in the fall of 2013. 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 fuelling productivity gains in many industrial workflows, including those of pharmaceutical and biopharmaceutical development, production and QC.
Now that you have some background on the ACQUITY QDa, I’ll now transition our discussion to its use for peptide analysis.
Over the next several slides we will show various LC optical applications where the ACQUITY QDa was added as an orthogonal mass detector and discuss our findings. We will show that the ACQUITY QDa is able to detect and monitor peptides over a wide molecular weight range and how the addition of mass detection allows one to monitor and quantify peptides with greater specificity. We’ll also show how the ACQUITY QDa expands the sensitivity currently available with optical only workflows, and demonstrate how the addition of mass detection allows scientists to selectively detect and monitor co-eluting species. And of key importance to many customers, we’ll also demonstrate that this mass detector works very well with both TFA and FA based separations.
To start, here is an example of a mixture of synthetic peptide standards which span a wide mass range. You see from the table that this mixture contains peptides from about 900 Da to almost 3000 Da. The masses highlighted in green are the multiply charge species detected for each of these peptides. We see that the total ion chromatogram (TIC) for the separation of this mixture is quite good, and the mass spectra, in this case for the largest peptide in this mixture (melittin) exhibits 3 charge states of the peptide.
Experimental: LC Conditions: LC System: ACQUITY UPLC® H-Class Detectors: ACQUITY UPLC® TUV Absorption Wavelength: 215 nm Vials: Total Recovery vial: 12x32 mm glass, screw neck, cap, nonslit (p/n 6000000750cv) Column: Waters ACQUITY UPLC CSH 130 C18 column (2.1 x 100 mm, 1.7m) Column Temperature: 60 °C Sample Temperature: 4 °C Injection Volume: 10 µL
Mobile phase: A: H2O, 0.1 % TFA B: Acetonitrile, 0.1 % TFA C: H2O, 0.1 % FA D: Acetonitrile, 0.1 % FA
Time Flow A B C D Initial 0.300 19 1 76 4 2.00 0.300 19 1 76 4 22.00 0.300 11 9 44 36 25.00 0.300 3 17 12 68 28.00 0.300 3 17 12 68 28.01 0.300 19 1 76 4 30.00 0.300 19 1 76 4
QDa Settings: Sample rate: 2 points/sec Mass range: 200 – 1250 Da. Cone voltage: 7 V Capillary voltage: 0.5 kV Probe Temperature: 600 °C
Since most of you are likely not interested in mixtures of unrelated peptides, we next analyzed a tryptic map of a monoclonal antibody - Trastuzumab. As shown, we analyzed this digest with both TFA and FA and in each case we were able to detect the resulting peptides from the digest. While there is a difference in absolute intensity, the use of both TFA and FA based eluents are compatible with the ACQUITY QDa. Naturally the question from this data centers on how many of the peptides can we see and over which mass range for each experiment.
To answer that question, we interrogated the TFA and FA data sets to determine the range of peptides we observed and over which charge states. What we see here is a list of the peptides observed for the heavy chain of the mAb comparing the results obtained in both TFA and FA based eluents. As shown, all of the peptides are seen in both experiments and we have highlighted either in blue or green the peptides seen in each experiment. Overall, slightly more charge states can be seen using FA as the acid modifier, but regardless, all peptides are well detected using FA or TFA as the acid modifier.
Similar to the data on the previous slide, here we show the peptides associated with the light chain of the same mAb. Again we see all of the peptides with each modifier while the charge states in some cases may be slightly different. From the data on the past two slides we show that the ACQUITY QDa is compatible with both TFA and FA based separations and that in most cases, multiple charge states are seen for each peptide.
Digging into this a little deeper, here we show the largest tryptic peptide from the Trastuzumab digest. While this peptide is quite large, nearly 7000 Da, we are able to observe 6 different charge states within the ACQUITY QDa mass analysis window.
Another natural question is the accuracy with which we are detecting the charge states of these peptides. We investigated the mass error for monitoring the peptides in a tryptic peptide map and found that for all peptides the error was well within the specification of the QDa, which is +/- 0.2 Da. In fact, as we show on the bottom right, greater than 90% fall within 0.15 Da.
If we move now into some specific applications of the ACQUITY QDa for monitoring peptides, lets look first at a fairly common issue which is a partial co-elution. In this case we have a pair of peptides which are partially co-eluting. If we rely on UV integration we have a potential issue in that we may over or underestimating the species of interest making quantification difficult. With the mass detector, however, we can selectively monitor each of the peptides by using their unique mass adding a new dimension of specificity. This specificity leads to a more accurate determination of the abundance of each species.
If we now look at the worst case of a full co-elution, where the components appear as a single peak via UV detection, we clearly see the benefit of the ACQUITY QDa Detector. As we show here, in a single experiment we can collect the UV and mass data. In this experiment we collected a total ion chromatogram to ensure we detected all of the ions, and in addition we collected data for a selected ion within the same experiment. With the addition of mass data you expand your ability to determine if you have co-eluting species and can selectively monitor those that are known.
When we look at the improved specificity provided by the ACQUITY QDa, a natural application of this is in the quantification of peaks of interest. Shown here are linear ranges for each detector and mode of acquisition when using both UV and mass detection. What we see is that we have expanded the linear dynamic range of the experiment by using mass detection. By using the total ion chromatogram, or incorporating selected ion recording you can greatly expand the linear range your experiment can cover.
If we look more closely at a particular example, in this case monitoring an oxidized peptide with our ACQUITY UPLC Charged Surface Hybrid (CSH) column with FA modifier we see the advantage of using the ACQUITY QDa in terms of the level of sensitivity possible. While each peptide will have its own associated ionization efficiency, near the limit of quantification the data obtained will be similar to that shown here. In this case we are able to achieve reliable detection of an impurity at very low levels with impressive linearity. This highlights the utility of the ACQUITY QDa for detection and quantification of low level peptide impurities.
When we look at quantification of species, often the request is for quantification of related species. In the example shown here we have the T21 peptide from trastuzumab and its oxidized form. While the T21 peptide is clearly visible in the UV trace, the oxidized species is not clearly defined. In the TIC trace each species is seen, however it is not clear at first glance that each of the peaks is homogeneous. By using selected ion recording (SIR) we can clearly distinguish each of the two species, and in this experiment, do a relative quantification although as we have just discussed we could do an absolute measurement as well.
Another key application in which the use of the ACQUITY QDa is useful is in the monitoring of complimentary domain region (CDR) peptides. These particular peptides are important as they govern whether the antibody will interact with its specific antigen. Shown here are the CDR peptides for trastuzumab monitored with the ACQUITY QDa. In this case we extracted a chromatogram which represents the masses of each of the CDR containing peptides within our peptide map.
As we just said, monitoring the CDR peptides is important and is easily accomplished with the QDa. In this particular case, trastuzumab has a known asparagine in the CDR region which is susceptible to deamidation. In addition, deamidation is known to create two different products, aspartic acid (annotated as D in the figure) and iso-aspartic acid (annotated as iso in figure). As we show the QDa is able to monitor both deamidated forms as well as the non modified form. In this particular case, one of the deamidated forms elutes with several other species making the selectivity of the QDa a benefit.
It is VERY IMPORTANT to note here that we are able to monitor these deamidated species because they are well resolved from the unmodified peptide. If the deamidated speceis were co-eluted with its unmodified form we would lose the ability to monitor these components with the QDa due to the mass difference between these species and the resolution of the QDa. .
In summary, we have shown you that the ACQUITY QDa Detector is fit for purpose for many peptide based analyses. The instrument can detect peptides over a wide molecular weight range and allows you to selectively monitor variants and modified peptides. We have also shown how the ACQUITY QDa can allow you to monitor species which may be below optical detector sensitivity. The ACQUITY QDa adds selectivity to monitoring species and we have shown that it is compatible with both TFA and FA eluents.
In closing… When incorporated into Empower based chromatographic workflows, whether you’re using an HPLC or UPLC system, the ACQUITY QDa Mass Detector provides enhanced specificity, selectivity and quantification for greater productivity in biotherapeutic development, production and QC.
Let’s look at a case study of a biopharm lab using the ACQUITY QDa Detector.
Mobile phase solvents: A: H2O, 0.1 % TFA B: Acetonitrile, 0.1 % TFA C: H2O, 0.1 % FA D: Acetonitrile, 0.1 % FA
Detectors: ACQUITY UPLC TUV Absorption Wavelength: 215 nm Column: Waters ACQUITY UPLC Peptide CSH C18 130 Å 1.7 µm column, 2.1x100 mm Column Temperature: 65 °C Sample Temperature: 5 °C Injection Volume: 10 µL
ACQUITY QDa Settings: Sample rate: 5 points/sec Mass range: 300 – 1250 Da. Cone voltage: 10 V Capillary voltage: 1.5 kV Probe Temperature: 500 °C
Informatics for data collection & processing: Empower 3 SR2 Software
To see if the ACQUITY QDa Mass Detector was detecting all the peptides from the digest, we ran samples from the same batch of LysC digest on a Waters SYNAPT MS System as well, and compared the results. As seen on the slide and the next three slides, there were no peaks that the SYNAPT detected that were not also detected using the ACQUITY QDa.
Note: the SYNAPT was paired with an ACQUITY UPLC I-Class System, which has a smaller dwell volume compared to the ACQUITY UPLC H-Class system used with the ACQUITY QDa Detector. To properly align the peaks for comparison purposes, the SYNAPT data was offset by 2.4 minutes.
In this 90-120 minute time span we highlight the largest peptide form the LysC digest (K7). The peptide is clearly detected by the ACQUITY QDa and the corresponding mass spectra shows a minimum of five charge states are detected for this peptide.
Here we show mass spectra from two samples of the same peptide generated from two separate digests – one using Trypsin and the other LysC, which were separated with different mobile phases (TFA and FA) on different days, and as can be seen the results are very consistent. This just further underscores that the ACQUITY QDa is a robust mass detector that generates consistent results over time and across different samples and chromatographic conditions.
Here we were asked to determine the relative quantification of a peptide (H21) and its oxidized form (H21-Ox). In this optimal scenario both species were chromatographically well resolved and there was a good delta between their M+2 m/z values. As such both SIR’s were included within a single channel. As shown, there is excellent agreement between the ACQUITY QDa generated results and those of the customer using a high resolution QTof instrument from another vendor.
In this scenario we are looking at a native peptide and its two deamidated forms, which have a very small delta in m/z relative to the native peptide. In this case running SIR’s for all species in a single channel might be problematic as there would likely be ion overlap between the indicative SIR’s (362.4 vs. 362.7, and 543.1 vs. 543.6 m/z), which could skew the results. One way to handle this is to run these SIR’s on two separate channels (enabled through Empower), and to then calculate the peak area for the non-deamidated SIR in channel 1, and the two deamidates species in channel 2 and sum them for the total peak area, then divide each individual peak area by the summed area to arrive at the relative percent of each species. This kind of “cross-channel” processing is also fully supported in Empower 3.0 Softwrae and higher.
In this scenario, we again are looking for the relative percent of native and deamidated species of a peptide (H37), in this case using only one charge state. Instead of using two SIR channels however, we are using one channel, but have created two time windows and set the SIR scan in each to be different. This just represents another way to obtain the desired results, which works perfectly fine so long as you have good chromatographic resolution between the component species you are looking to quantify.
With little expectation of being able to see anything, we were challenged to see if the ACQUITY QDa might be useful for mAb sub-unit analyses. We were provided with an IdeS digest of the mAb Humira. Both we and the customer were surprised to see the ACQUITY QDa clearly detected both FC and Fab sub-units.
UPLC Configuration and Settings: Detectors: ACQUITY UPLC TUV Absorption Wavelength: 215 nm Column: Waters ACQUITY UPLC BEH C4 130 Å 1.7 µm column, 2.1x100 mm Column Temperature: 80 °C Sample Temperature: 5 °C Injection Volume: 3 µL
QDa Settings: Sample rate: 5 points/sec Mass range: 300 – 1250 Da. Cone voltage: 10 V Capillary voltage: 1.5 kV Probe Temperature: 500 °C
Informatics for data collection & processing: Empower 3 SR2 Software
Using a focused gradient on the FC fragment for greater component resolution we were able to zoom in and see multiple charge states under each of the FC peaks, opening the possibility the ACQUITY QDa Detector could also be used to strengthen monitoring of mAb subunits and/or intact proteins as well.
Finally, we were asked to run a Trypsin digest of a mAB (later revealed to be Herceptin (trastuzumab)) and were provided with the masses (middle column) for a list of glycopeptides (left column). We were asked to detect these using the ACQUITY QDa and to calculate the relative percent for each if possible. Having run the sample previously on the SYNAPT MS, we were able to confirm the identity of each of these glycopeptides, and subsequently ran SIRs on the ACQUITY QDa using the M+3 charge state for each (shown in right column), which we knew existed based on our SYNAPT MS data. All glycoforms were detected with the ACQUITY QDa, however, some were at the lower range of detection, and percent areas at those levels was skewed by noise. Using peak areas for the three most abundant species (G0F, G1F and G2F), we determined their relative percentage between them, and as shown their was excellent agreement between the percent distribution calculated by us and by the customer. (*Note: percentages from customer were adjusted to reflect only the three most abundant species for comparison purposes.)
In summary, we have shown you that the ACQUITY QDa Detector is fit for purpose for many peptide based analyses. The instrument can detect peptides over a wide molecular weight range and allows you to selectively monitor variants and modified peptides. We have also shown how the ACQUITY QDa can allow you to monitor species which may be below optical detector sensitivity. This Waters mass detector adds selectivity to monitoring species and we have shown that it is compatible with both TFA and FA eluents.
In closing… When incorporated into Empower Software chromatographic workflows for HPLC or UPLC, the ACQUITY QDa Detector provides enhanced specificity, selectivity and quantification for greater productivity in biotherapeutic development, production and QC.
Details on the ACQUITY QDa Detector: www.waters.com/qda www.waters.com/waters/nav.htm?cid=134761404
More info on the ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm www.waters.com/waters/nav.htm?cid=134837932
Adding Mass Detection to Monitor Peptides in Biopharmaceutical Development & QC