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Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides
Journal Pre-proofs Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides Brooke Koshel, Robert Birdsall, Weibin Chen PII: DOI: Reference:
S1570-0232(19)31378-9 https://doi.org/10.1016/j.jchromb.2019.121906 CHROMB 121906
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
12 September 2019 21 November 2019 24 November 2019
Please cite this article as: B. Koshel, R. Birdsall, W. Chen, Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121906
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Title: Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides Authors: Brooke M. Koshel, Robert E. Birdsall, Weibin Chen Affiliation: Waters Corporation 34 Maple Street Milford, MA 01757 Author E-mail(s): Brooke Koshel, [email protected] Robert Birdsall, [email protected] Weibin Chen, [email protected]
Corresponding author: Brooke Koshel E-mail: [email protected]
Title: Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides Authors: Brooke M. Koshel, Robert E. Birdsall, Weibin Chen Abstract Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in onedimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dyeconjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the twodimensional analysis can be detected at low nanogram levels, comparable to results achieved in a onedimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques. Keywords: two-dimensional liquid chromatography, mass spectrometry, oligonucleotides, ion-pair reversed-phase, heart-cut, purity 1. Introduction Fluorescently-labelled synthetic oligonucleotides are useful in several biological applications, most commonly including traditional and real-time quantitative PCR, DNA sequencing, and various genetic assays [1]. There are many fluorophores available that emit in the visible spectrum, and they can be readily conjugated for high sensitivity assays that lend themselves to straightforward detection techniques. Because assay performance can be impacted by primer and probe purity, high purity oligonucleotides are generally desired.
Oligonucleotides are synthesized through a series of coupling steps that incorporate nucleotides in a sequential manner until the desired sequence is achieved [2]. Although coupling efficiencies of approximately 99 percent can be achieved, as oligonucleotide length increases, the yield decreases in an exponential fashion according to the number of coupling steps. Furthermore, although the chemistry to couple the fluorophore to the oligonucleotide is well understood, coupling efficiency is dependent on which fluorophore is used, where the post-synthesis addition of these fluorophores can result in a significant decrease in yield compared to un-labeled oligonucleotides having the same sequence [3]. Removing the resulting impurities is critical for assay performance, as poor quality primers and probes have been shown to have undesirable results such as amplification of unwanted sequences in PCR and increased false positive and false negative assignments in genotyping [4]. Oligonucleotide separation and purification is commonly carried out through electrophoresis or ion exchange and reversed-phase chromatographic techniques. Anion exchange chromatography (AEX) is a charge-based technique where the separation generally results in shorter sequences eluting earlier than longer sequences, thus making this technique suitable for resolution of n-x failed sequences [5]. Reversed-phase purification (based on hydrophobicity differences), can be useful for separation of oligonucleotides containing a protecting group from those that have had the protecting group cleaved during synthesis, but ion-pair reversed-phase chromatography (IP-RP) is a far more common technique. By incorporating ion-pairing agents in to the mobile phase, the desired resolution can be achieved by taking advantage of negatively charged oligonucleotides in addition to any differences in their hydrophobicity. Triethylammonium acetate (TEAA) has historically been the most employed ion-pairing agent for optical-based separations because of its separation efficiency across various RNA and DNA oligonucleotide species [5]. While features of the oligonucleotide such as its length and sequence are known to impact retention, selectivity and resolution can be optimized by making changes to the mobile phase composition or gradient [6]. Triethylamine (TEA) and other ion-pairing agents having short alkyl chains or those exhibiting low adsorptive properties are considered to be “weak” ion-pairing agents, while ion-pairing agents with longer alkyl chains or those exhibiting higher affinity to reversed phase stationary phases are considered “strong” ion-pairing agents. There are a number of reports in literature that investigate various alternative ion-pairing agents or counter ions to improve chromatography as well as mass spectrometry sensitivity [7]–[9]. In addition to these studies, alternative approaches to method optimization beyond choosing the best ion-pairing agent and gradient have proven beneficial in the separation of oligonucleotides. Levin and coworkers demonstrated a platform method for analyzing several siRNA compounds through using mobile phases containing a combination of weak and strong ion-pairing agents which was shown to improve impurity profiling through better peak shape and resolution [10]. Oligonucleotides containing modifications, including those having fluoro-conjugates, further complicate the separation mechanism, as the presence of a fluorophore as well as the structure of the fluorophore impact retention, and determination of an appropriate ion-pairing agent and the effect it has on the separation also requires evaluation [11]–[14]. The aforementioned TEA is a relatively weak ion-pairing agent, and as a result, hydrophobic effects impact separation quality. These same hydrophobic effects
can be minimized by using stronger ion-pairing agents. Anacleto and colleagues took advantage of this principle by applying a two-step purification technique that utilized two different ion-pairing agents to separate fluorophore-conjugated oligonucleotides that differ in hydrophobicity and length variants through the use of weak ion-pairing and strong ion-pairing, respectively [14]. Their work first used TEAA to best separate the probe of interest from the most undesirable synthesis-related impurity. Tetrabutylamine, a stronger ion-pairing agent, was then used in a polishing step to remove additional truncated species from the product in a length-based separation. While this analysis resulted in high product purity, offline fractionation was required. Online methods can be preferred to fractionation because they eliminate the need for manual sample handling and analysis can often be completed more efficiently. In addition, online methods have the benefit of being directly coupled to mass spectrometry (MS). The incorporation of mass information facilitates unambiguous direct identification of impurities resulting from the synthesis process, bypassing the need for fractionation or extraneous sample handling procedures. It can thus be envisioned that by incorporating two-dimensional liquid chromatography with mass spectrometry (2DLC/MS) for the analysis of dye-conjugated oligonucleotides, that a single assay can be used to more effectively understand sample purity and support synthesis processes in the production of fluoroconjugated oligonucleotides. 2D-LC/MS technology has been used in numerous applications across the pharmaceutical industry [15]– [17]. However, fewer examples are documented in oligonucleotide analysis. Li and colleagues reported a comprehensive HILIC x IP-RP LC approach for separating dC, dT, and dA homo-oligonucleotide ladders in a 27-component mixture. Due to the limited resolution achieved in the separation, only oligonucleotides of 2-10 base pairs long could be separated [18]. More recently, Roussis and colleagues evaluated size exclusion chromatography (SEC), RP, or strong anion exchange (SAX) as first dimension chemistries coupled with IP-RP in the second dimension for MS compatibility in an effort to explore the use of 2D-LC/MS for enhancing oligonucleotide impurity analysis [19]. While not all configurations were shown to be advantageous, RP coupled to IP-RP was used to analyze monomethoxytrityl (MMT)-on and MMT-off synthesis impurities in both comprehensive and heart-cut mode to demonstrate an orthogonal separation and an alternative to off-line fractionation for optimization of synthesis and purification strategies, respectively. This same comprehensive RP x IP-RP configuration was compared to an alternative comprehensive 2D approach with IP-RP x IP-RP, where the IP-RP x IP-RP configuration ultimately suffered from longer run times and a separation that is short of the desired orthogonality. In the present work, dye-conjugated oligonucleotides are analyzed via 2D-LC/MS to address the challenges commonly encountered when a single ion-pairing agent is used to deliver optimal performance in both chromatography and mass spectrometry. Failed sequences are successfully resolved from the parent oligonucleotide with a strong ion-pairing agent, hexylammonium acetate (HAA), while the identity of impurities can be more readily confirmed after the second dimension using a weaker but MS-friendly ion-pairing agent, TEA buffered in hexafluoroisopropanol (HFIP), is coupled to a single quadrupole MS. Although coupling two reversed-phase dimensions is the most reported configuration in both comprehensive and heart-cut mode, to the best of our knowledge, this is the first time a heart-cut configuration using IP-RP – IP-RP 2D-LC application has been reported to show noted
benefits [17]. This work utilizes C18 RP columns having the same chemistry in both the first and second dimension, with each dimension using a different ion-pairing agent, to achieve both orthogonal separation selectivity, as well as to maintain sensitive MS detection. The identical column chemistry used in each dimension demonstrates the importance of mobile phase selection, where the choice of ion-pairing agent in turn dictates whether the dominant separation mechanism is hydrophobicity or charge driven for each dimension. The use of IP-RP in both dimensions eliminates the need for a more traditional AEX separation in the first dimension, which has a higher risk of salt contamination of MS components, and thus increases instrument complexity by diverting flow prior to MS. This work demonstrates a single online IP-RP – IP-RP/MS analysis that can facilitate both separation and identification of fluoro-conjugated oligonucleotide impurity profiles; results otherwise not achievable using traditional one-dimensional techniques alone. 2. Material and methods 2.1 Oligonucleotides and reagents Dye-conjugated oligonucleotides were purchased from Integrated DNA Technologies (Skokie, IL, USA). Sequence and molecular weight information is provided in Table 1. Samples were purified by the vendor via HPLC and were supplied in lyophilized form. All samples were prepared to a stock concentration of 100 µM in LC/MS grade water (Fisher Scientific, Pittsburg, PA, USA) and further diluted to approximately 2 pmol/μL. Mobile phases were prepared gravimetrically using LC/MS grade reagents when available. Hexylamine (99% purity) purchased from Sigma-Aldrich (St. Louis, MO, USA) and acetic acid (99.8% purity) purchased from Honeywell Fluka (Morris Plains, New Jersey) were used to prepare 100 mM HAA, pH 7. Triethylamine (99.5% purity) and 1,1,1,3,3,3-hexafluoro-2-propanol (99.8% purity) purchased from Honeywell Fluka were used to prepare 15 mM TEA, 400 mM HFIP, pH 8. LC/MS grade water (Fisher Scientific), and acetonitrile and methanol (Honeywell USA) were also used in mobile phase preparation. Mobile phase concentrations were determined based on literature reports of the concentration required to maintain comparable chromatographic efficiencies between ion-pairing systems [20]. A one minute low pH re-conditioning step using 50: 50 methanol (Fisher Scientific): 0.2% formic acid (FA) v/v in LC/MS grade water (Fisher Scientific) was incorporated at the end of the first dimension gradient to mitigate metal adduct formation [21]. 2.2 2D-LC Instrumentation An ACQUITY UPLC H-Class Bio System with 2D LC Technology (Waters Corporation, Milford, MA, USA) equipped with MassLynx Software V4.1 (Waters Corporation) for data acquisition was used in all experiments. A quaternary solvent manager (QSM) and binary solvent manager (BSM) were used for the first- and second-dimension pumps, respectively. Optical data was collected at 260 nm using a tunable ultra-violet (TUV) detector (first dimension). ACQUITY UPLC Oligonucleotide BEH C18 (130 Å, 1.7 µm, 2.1 x 50 mm) columns (Waters Corporation) at 60 °C were used for both first- and second-dimension separations. Dedicated columns were used for each ion-pairing agent, as hexylamine has a strong
affinity for the particle sorbent. A 4 µL injection was used for all injections. Additional chromatographic method details are described in the corresponding figures. A complete instrument configuration with associated plumbing diagrams can be seen in Figure 1A. The 2D workflow is employed through the LC column manager, which houses two two-position six port valves. Through a series of valve switches controlled by the MassLynx software, the first- and seconddimension flow paths can be isolated or combined to perform the desired functionality. To begin, flow paths in the first and second dimension were isolated from one another, where a traditional 1D separation can be monitored by directing flow through the first-dimension column to a TUV detector. A heart-cut can be transferred to the second dimension by switching the valve positons to combine flow paths and using a stainless-steel tee for at-column dilution (ACD), which functions to dilute the organic composition so that the sample is retained and focused on the head of the second column. By switching the valve positions back to the original position, the flow paths are once again isolated from one another, and the second-dimension separation can proceed. 2.3 MS instrumentation A single quadrupole mass spectrometer (SQ Detector 2, Waters Corporation) was used to collect mass data post optical detection. MS data was collected in negative mode over a scan range of 410 – 3000 m/z. MS instrument settings were independently optimized in both HAA and TEA-HFIP. In TEA-HFIP, the following settings were used: capillary voltage 0.8 kV, cone voltage 50 V, and desolvation temperature 350 °C. The desolvation gas flow and cone gas flow were set to 700 L/Hr and 10 L/Hr, respectively. When investigating a 1D dilution series in HAA, a capillary voltage of 2.0 kV and a cone voltage of 20 V were used with all other settings remaining the same. MassLynx 4.1 (Waters Corporation) was used for data deconvolution using the embedded MaxEnt 1 algorithm to report average mass. 3. Results and discussion 3.1 1D separation of dye-conjugated oligonucleotides Many of the fluorescent tags conjugated to DNA primers and probes are extremely hydrophobic. To demonstrate the effect that these hydrophobic fluorescent tags have on a separation, the elution order of failed sequences of a Cy3 conjugated 25-mer was studied using two different ion-pairing agents. The n-1, n-2, and n-3 failed sequences (Table 1) of this oligonucleotide were synthesized and spiked in to the 25-mer sample in equal amounts. Mobile Phase A (MPA) consisted of either TEA-HFIP or HAA ion-pairing agents and Mobile phase B (MPB) was 100% LC/MS grade organic solvent (methanol or acetonitrile). RP separations more commonly use acetonitrile as the organic solvent, but as HFIP is not miscible in acetonitrile, methanol was used in this case. From Figure 2A, the n-1 and n-2 failed sequences co-elute after the main peak when TEA-HFIP is used as MPA. While further optimization of the gradient can potentially provide a marginal improvement in resolution, it is more important to note that elution order is not a length-based separation as would be predicted in a more traditional separation of unmodified oligonucleotides. Gilar and colleagues developed a model for predicting oligonucleotide retention under IP-RP conditions based
on oligonucleotide length and sequence, which lends insight to selecting initial organic composition to achieve high-throughput methods [22]. Although there have been further efforts to model elution behavior of unmodified oligonucleotides since this foundational work, no model for dye-conjugated oligonucleotides is reported. HAA has been studied as an alternative ion-pairing agent for noted benefits in a number of applications [20], [23], [24]. Hexylamine serves as a more efficient ion-pairing agent through more effective adsorption to the RP stationary phase than triethylamine because of the increased length of the alkyl chain. As a result, HAA diminishes the reversed-phase contribution from the hydrophobic dye, and the labelled oligonucleotide separation becomes AEX-like and is mainly dictated by the oligonucleotide length in HAA (Figure 2B). In our study, when using a strong ion-pairing agent such as HAA, the organic content required to elute the oligonucleotide is much higher than with a weak ion-pairing agent. In our example, 52% acetonitrile is required to elute the full-length product (this takes into account a combined system volume and column volume of 0.67 mL, which is negligible compared to the volume required to elute the peak of interest). In TEAA, 34% methanol is required to elute this same species. Under these weaker elution conditions, the dye is still retained on the stationary phase, and the resulting separation is mixed-mode driven, supported by the elution order of the failed sequences relative to the main peak (Figure 2A). It should be noted that the oligonucleotides are more retained in methanol than acetonitrile, and the percent organic required for elution will be greater in HAA in methanol than HAA in acetonitrile, which will be further evaluated in the 2D workflow. The usefulness of HAA for this application was further investigated by exploring MS analysis of failed sequences using a single quadrupole detector (SQ Detector 2). Detection limits of a Cy3-1 dilution series in 100 mM HAA were compared to results in TEA-HFIP. Since its introduction by Apffel and colleagues, HFIP is the most commonly used counter ion when mass spectrometric analysis of oligonucleotides is needed, as HFIP offers enhanced MS signal intensity [25]. Table 2 reports a dilution series of the n-1 failed sequence and the corresponding signal-to-noise ratio (SNR) as determined from the average peak intensity of the total ion chromatogram (TIC) across three injections. In TEA-HFIP mobile phase, the limit of detection (LOD) approaches a 1 ng mass load. In HAA, a substantially higher mass load of over 60 ng is barely detectable at SNR = 3, and therefore cannot be further diluted. When determining impurities via relative percentage, larger injection volumes can be made to achieve lower detection limits if required, but there are limitations to sample load volume due to peak broadening [23]. An alternative approach to detecting lower concentration limits is through the use of more sensitive instrumentation, but as the purpose of our study is for impurity confirmation and not characterization, a lower resolution quadrupole instrument is appropriate for the intended application [26]. The effects of ion suppression in HAA compared to TEA-HFIP are shown in Figure 3, which shows both the TIC and raw data from the first injection of the dilution series in the respective mobile phases. From the TIC, the analyte in HAA is barely distinguishable from the noise. From the raw data, adducts are especially evident at the lower charge states. We suspect that reagent purity also impacts MS signal by introducing additional adducts, and hexylamine is not readily available in LC/MS grade. Gong reported a
similar result, where the acetate salt of his ion-pairing agent yielded more adducts than bicarbonate or HFIP salts [27]. The MaxEnt 1 algorithm was used to deconvolute data over the same number of scans, and the response is approximately three orders of magnitude lower for HAA than TEA-HFIP with the same sample load. The increased MS sensitivity when using HFIP-based ion-pairing buffers provides a means to identify and quantify low-level impurities based on mass. This can be seen in the plot shown in Figure 4, where a high degree of linearity was observed throughout the working range selected from Table 2. Interestingly, although hexylamine has been shown to provide comparable peak intensity in ESI of oligonucleotides when using HFIP as the counter ion (versus acetate) when compared to TEA-HFIP, the chromatography does not offer this same performance [8]. Li et al. report notable improvements in chromatographic resolution and retention across RNA, DNA, and MDNA when comparing TEA-HFIP to HA-HFIP [7]. Our work further demonstrates the importance of balancing chromatography and MS performance to achieve the desired result. Because HAA is a more efficient ion-pairing agent, it provides a length-based separation for optical detection, but due to the hydrophobicity of the dye conjugate, the reversed phase mechanism dominates the ion-pairing mechanism for a weak ion-pairing agent such as TEA-HFIP. It also becomes apparent that HAA does not provide adequate sensitivity for this application. This lends itself to a 2D-LC/MS solution where HAA in the first dimension yields a length-based separation, and by selectively heart-cutting impurities, a second dimension can be used in TEA-HFIP to provide MS detection and identity confirmation. 3.2 2D heart-cutting configuration, events tab To begin method development, the pressure tolerance of the first-dimension column was evaluated using different flow rates. This is a necessary step of method development due to the configuration of our 2D system, which couples the first and second dimension columns in serial when the flow paths are combined during the heart-cut (Figure 1A). For this reason, the flow rate used in each dimension must be amenable to the pressure tolerance of the instrument and columns. Alternative plumbing configurations or trap columns can be used to eliminate or alleviate the pressure increase across the first-dimension column, but these options were not explored as pressure restrictions were not exceeded in this study. In addition to pressure restrictions, there also needs to be consideration for strong solvent effects when determining flow rate. The n-1 impurity elutes at an organic concentration that is too high to retain the analyte on the second-dimension column. By incorporating ACD, aqueous-based mobile phase (MPA from the second dimension) can be introduced through a fluidic tee using a second pump. The first-dimension separation was run at 0.1 mL/min and the second dimension was run at 0.25 mL/min, which effectively serves as a 3.5-fold dilution of the organic composition. This dilution factor reduces the organic content to re-focus the analyte on the second-dimension column prior to separation in the second dimension. The first-dimension separation in 100 mM HAA was optimized prior to coupling to the second dimension. Methanol was used as the organic solvent for both dimensions in all 2D separations, as HFIP is not soluble in acetonitrile. Although ACD is employed, there are immiscibility issues upon contact
when acetonitrile is used in the first dimension. When HAA is used in combination with methanol, a greater percent organic composition is needed to elute the analytes than with acetonitrile. A gradient from 66% - 76% B over 20 minutes was used to separate Cy3 from the spiked in n-1 impurity (Cy3-1) in the first dimension. The n-1 impurity elutes in approximately the middle of the gradient at 70% methanol. When the n-1 impurity started to elute, the valve switching events were set up to take the heart cut over 0.5 minutes (Figure 1B). As the primary purpose of the second dimension is impurity confirmation, a rapid screening gradient (20 – 70% B over 10 minutes) can be used without further optimization. A hold step was employed after the heart-cut and before the start of the seconddimension gradient to wash and refocus the sample. Figure 5 demonstrates this proof of concept using the Cy3-conjugated 25-mer with the n-1 impurity spiked in at approximately 10 percent by optical peak area. Figure 5A shows the TUV trace, which was used to determine the heart-cut window for the n-1 peak to be selectively sent to the second dimension. The corresponding second dimension figure shows the TIC of the n-1 impurity (Figure 5B). The TIC was used to confirm the mass of the n-1 peak. 3.3 2D of Cy3-conjugated 25-mer To demonstrate the applicability of this method for a more realistic sample, the Cy3-conjugated 25-mer was analyzed without the addition of the n-1 impurity spike-in using the same configuration as described above. Oligonucleotides can generally be purified via standard desalting or HPLC, although the manufacturer requires Cy3-conjugated oligonucleotides to be purified via HPLC. Selecting a method of purification can also be dependent on the intended use of the oligonucleotide. Because the target application of these dye conjugated oligonucleotides requires high purity, HPLC purification maintains assay quality. From Figure 6, the n-1 impurity could be successfully identified at 2.4 percent (percentage determined from the TUV trace). This result agrees with results reported from the 1D dilution series (Table 2), where the lower limit of detection approached 1.5 percent. In terms of sample concentration, these values align with the expected limit of detection of oligonucleotides with the instrumentation used, but more importantly, sensitivity is not lost between the 1D and 2D assays. To detect more extensive impurity types with greater sensitivity, high-resolution MS is required [26]. 4. Conclusion This work demonstrates the ability to couple two IP-RP methods for 2D-LC/MS analysis of oligonucleotides in an effort to achieve desirable performance in both chromatographic separation and mass spectrometric detection. Because the same column chemistry was used in both the first and second dimension and does not give rise to selectivity differences, this work explored changes in mobile phase composition to control the separation mechanism. HAA was used in the first dimension to achieve a length-based separation of a Cy3-conjugated 25-mer and its failed sequences. By selectively heartcutting the n-1 impurity, TEA-HFIP could be introduced in the second dimension for identity confirmation using a single quadrupole detector. This method could be used to successfully confirm low level impurities, which was not achievable using a one-dimensional technique. Funding
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Table 1. Oligonucleotide sequence and MW information. Table 2. Limits of detection of Cy3-1 in TEA-HFIP and HAA. The Cy3-1 failed sequence was prepared at a stock concentration of 100 µM and further diluted to 2.22 pmol/µL, corresponding to a 62.68 ng mass load (4 µL injection). A 2-fold dilution series was carried out until the analyte could no longer be detected. The reported signal-to-noise (SNR) is the average peak intensity of the total ion chromatogram (TIC) across three injections. Method conditions in HAA: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% acetonitrile. Method conditions in TEA-HFIP: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol. The same gradient conditions were used in both HAA and TEAHFIP. Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 1. 2D system configuration. 1A) Two two-position six port valves housed in a column manager enable heart-cut functionality as depicted in the schematic. When the left and right valves are in Position 1, the first- and seconddimension flow paths are isolated from one another. This allows for separation in the first dimension (top panel), and as the analyte of interest begins to elute, the left and right valves are switched to Position 2 to selectively transfer the analyte to the second-dimension (middle panel) for analysis. Atcolumn dilution (ACD) works to focus the analyte at the head of the second-dimension column by mixing aqueous mobile phase from the second-dimension pump with the eluent from the first-dimension column, thus reducing the organic content that was required to elute the analyte in the first-dimension. The second-dimension separation begins when the flow paths are once again isolated from one another (bottom panel). System component abbreviations: quaternary solvent manager (QSM); sample manager with flow-through needle (SM-FTN); tunable ultraviolet detector (TUV); binary solvent manager (BSM); photodiode array detector (PDA); and mass spectrometer (MS). 1B) A 0.5 minute heart-cut window from 9.50 to 10.00 minutes was used to transfer the analyte of interest to the second-dimension by creating a timed event within the software. This timed event switches both valves from Position 1 (isolated flow paths) to Position 2 (combined flow paths). Figure 2. Comparing optical detection of a Cy3 and its failed sequences in TEA-HFIP and HAA. 2A) Due to the hydrophobicity imparted by the dye conjugate, n-1 and n-2 failed sequences elute after the main peak when a weak ion-pairing agent is used. Method conditions: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 28 – 38% B in 7.5 minutes at 0.2 mL/min. (Peak height is normalized.) 2B) When using a more efficient ion-pairing agent, the reversed-phase contribution from the hydrophobic dye is minimized to result in a separation largely dictated by oligonucleotide length. Method conditions: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% acetonitrile; Gradient conditions: 30 – 60% B in 7.5 minutes at 0.2 mL/min. (Peak height is normalized.) Figure 3. Comparison of MS data for Cy3-1 in TEA-HFIP (3A) and HAA (3B). A 62.68 ng mass load (4 µL injection) of Cy3-1 was used to compare MS response. When the total ion chromatogram (TIC) and raw data are placed on the same scale, an enhanced MS signal can be seen in TEA-HFIP compared to HAA, where the analyte response is barely distinguishable from the noise.
Deconvoluted data shows the MS response in HAA is approximately three orders of magnitude lower than in TEA-HFIP for the same sample load. Method conditions in HAA: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% acetonitrile. Method conditions in TEA-HFIP: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol. The same gradient conditions were used in both HAA and TEA-HFIP. Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 4. Dynamic range of Cy3-1 in TEA-HFIP. The Cy3-1 failed sequence was prepared at a stock concentration of 100 µM and further diluted to 2.22 pmol/µL, corresponding to a 62.68 ng mass load (4 µL injection). A two-fold dilution series was carried out until the analyte could no longer be detected. The reported peak area is from the average total ion chromatogram (TIC) peak area across three injections. Method conditions: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 5. Evaluation of 2D system configuration using Cy3 and spiked in Cy3-1 impurity. 5A) Optical (UV) data showing separation of n-1 impurity from the full-length product. The n-1 impurity spike-in is at approximately 10 percent as determined from the integrated peak areas in the optical trace. Using this data to determine the heart-cut window, the n-1 peak is selectively transferred to the second-dimension for mass confirmation. Method conditions in first-dimension: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% methanol; Gradient conditions: 66 – 76% B in 20 minutes at 0.1 mL/min. 5B) Total ion chromatogram (TIC) of the n-1 impurity selected from the first-dimension. An isocratic hold at initial conditions is employed for 14 minutes during the heart-cut and transfer of analyte to the second dimension. The broad peak at ~12.5 minutes is attributed to mobile phase transfer from the first dimension passing through the second-dimension column. Total run time is 30 minutes to incorporate a column wash at the end of the second-dimension separation. Method conditions in second-dimension: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 6. 2D Cy3-1 impurity analysis of Cy3. A 62.68 ng mass load (4 µL injection) of Cy3 was separated to determine if the n-1 impurity resulting from synthesis could be detected. From the optical (UV) trace, the n-1 impurity was determined to be present at approximately 2.4 percent as determined from peak area integration. The deconvolution inset shows that peak identity can be confirmed at this level, which is consistent with detection limits achieved from the 1D dilution series. Method conditions in first-dimension: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% methanol; Gradient conditions: 66 – 76% B in 20 minutes at 0.1 mL/min. Method conditions in second-dimension: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min.
A) 15 mM TEA, 400 mM HFIP, pH 8 B) Column manager Time (min)
Event
Action
Initial
Left Valve
Position 1
Initial
Right Valve
Position 1
9.49
Left Valve
Position 2
9.50
Right Valve
Position 2
10.00
Left Valve
Position 1
10.00
Right Valve
Position 1
General tab
Events tab
A) 15 mM TEA, 400 mM HFIP, pH 8 B) Column manager Time (min)
Event
Action
Initial
Left Valve
Position 1
Initial
Right Valve
Position 1
9.49
Left Valve
Position 2
9.50
Right Valve
Position 2
10.00
Left Valve
Position 1
10.00
Right Valve
Position 1
General tab
Events tab
A) 15 mM TEA, 400 mM HFIP, pH 8 4.0x10
8
TIC 4.0x10
6
Raw data 2.0x10
7
1.6x10
7
1.2x10
7
8.0x10
6
4.0x10
6
7855.2 Da
2.0x10
8
1.0x10
8
0.0 4
5
6
7
3.0x10
6
2.0x10
6
-3 -9 -7 -8 -6 -5
1.0x10
6
Intensity (counts)
8
Intensity (counts)
Intensity (counts)
-4 3.0x10
0.0 7000
0.0 500 1000 1500 2000 2500 3000
8
m/z m/z
Retention time (min)
Deconvoluted data
7500
8000
8500
9000
Mass (Da) (Da) Mass
B) 100 mM HAA, pH 7
3.0x10
8
2.0x10
8
1.0x10
8
TIC 4.0x10
6
3.0x10
6
2.0x10
6
1.0x10
6
Deconvoluted data
Raw data
Intensity (counts)
8
Intensity (counts)
Intensity (counts)
4.0x10
-5 7.00
7.25
7.50
7.75
Retention Time Retention time (min) (min)
8.00
-4
-3 -6 0.0 500 1000 1500 2000 2500 3000 m/z
m/z
5.0x10
4
4.0x10
4
3.0x10
4
2.0x10
4
1.0x10
4
0.0 7000
7853.5 Da
7500
8000 Mass (Da)
8500
Mass (Da)
9000
7
2.0x10
7
Peak area
1.6x10
7
1.2x10
R2 = 0.9977
6
8.0x10
6
4.0x10
0.0 0
10
20
30
40
Mass load (ng)
50
60
AU AU AU
A) 1D TUV 0.06 0.05 0.04 0.03 0.02 0.01 0.00
n n-1
0
5
10
15
20
Intensity AU(counts)
Retention time (min)
B) 2D TIC n-1 7854.0 m/z
8
1.5x10
8
1.0x10
7
5.0x10
0.0 0
5
10
Retention time (min)
15
20
0.005 0.004 0.003
n-1 Deconvolution
n
Intensity (counts)
A AU AU
1D TUV
Heart-cut window
n-1
0.002 0.001 0.000 (min) Retention Time time (min)
15
7.0x10
3
6.0x10
3
5.0x10
3
4.0x10
3
3.0x10
3
2.0x10
3
1.0x10
3
0.0 6000
n-1 7853.6 m/z
7000
8000
9000
Mass (Da) Mass (Da)
10000
Oligonucleotide Cy3 Cy3-1 Cy3-2 Cy3-3
Sequence (5' - 3') Cy3-TTT GAC TTA GAC TTA GAC TTA GTT T -T -TT -TTT
MW (Da) 8158.6 7854.4 7550.2 7246.0
Sample conc. (pmol/ μL) 2.22 1.11 0.56 0.28 0.14 0.069 0.035
Mass load (ng) 62.68 31.34 15.67 7.83 3.92 1.96 0.98
Impurity (%) 50 25 12.5 6.25 3.13 1.56
MS SNR HAA 3.34 -
MS SNR TEA-HFIP 125.64 67.76 36.01 19.2 8.93 4.7 2.09
Highlights:
Dye-conjugated oligonucleotides impact retention by imparting hydrophobicity IP – RP can achieve an AEX-like result by using a strong IP agent Selecting an IP agent requires a balance of chromatographic and MS performance 2D coupling of strong and weak IP agents can be used to improve detection limits
Abstract Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in onedimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dyeconjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the twodimensional analysis can be detected at low nanogram levels, comparable to results achieved in a onedimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.
Author Statement Brooke M. Koshel: Investigation, Writing – Original Draft Robert E. Birdsall: Writing – Reviewing and Editing, Supervision Weibin Chen: Writing – Reviewing and Editing, Supervision
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S1570-0232(19)31378-9 https://doi.org/10.1016/j.jchromb.2019.121906 CHROMB 121906
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
12 September 2019 21 November 2019 24 November 2019
Please cite this article as: B. Koshel, R. Birdsall, W. Chen, Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121906
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© 2019 Published by Elsevier B.V.
Title: Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides Authors: Brooke M. Koshel, Robert E. Birdsall, Weibin Chen Affiliation: Waters Corporation 34 Maple Street Milford, MA 01757 Author E-mail(s): Brooke Koshel, [email protected] Robert Birdsall, [email protected] Weibin Chen, [email protected]
Corresponding author: Brooke Koshel E-mail: [email protected]
Title: Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides Authors: Brooke M. Koshel, Robert E. Birdsall, Weibin Chen Abstract Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in onedimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dyeconjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the twodimensional analysis can be detected at low nanogram levels, comparable to results achieved in a onedimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques. Keywords: two-dimensional liquid chromatography, mass spectrometry, oligonucleotides, ion-pair reversed-phase, heart-cut, purity 1. Introduction Fluorescently-labelled synthetic oligonucleotides are useful in several biological applications, most commonly including traditional and real-time quantitative PCR, DNA sequencing, and various genetic assays [1]. There are many fluorophores available that emit in the visible spectrum, and they can be readily conjugated for high sensitivity assays that lend themselves to straightforward detection techniques. Because assay performance can be impacted by primer and probe purity, high purity oligonucleotides are generally desired.
Oligonucleotides are synthesized through a series of coupling steps that incorporate nucleotides in a sequential manner until the desired sequence is achieved [2]. Although coupling efficiencies of approximately 99 percent can be achieved, as oligonucleotide length increases, the yield decreases in an exponential fashion according to the number of coupling steps. Furthermore, although the chemistry to couple the fluorophore to the oligonucleotide is well understood, coupling efficiency is dependent on which fluorophore is used, where the post-synthesis addition of these fluorophores can result in a significant decrease in yield compared to un-labeled oligonucleotides having the same sequence [3]. Removing the resulting impurities is critical for assay performance, as poor quality primers and probes have been shown to have undesirable results such as amplification of unwanted sequences in PCR and increased false positive and false negative assignments in genotyping [4]. Oligonucleotide separation and purification is commonly carried out through electrophoresis or ion exchange and reversed-phase chromatographic techniques. Anion exchange chromatography (AEX) is a charge-based technique where the separation generally results in shorter sequences eluting earlier than longer sequences, thus making this technique suitable for resolution of n-x failed sequences [5]. Reversed-phase purification (based on hydrophobicity differences), can be useful for separation of oligonucleotides containing a protecting group from those that have had the protecting group cleaved during synthesis, but ion-pair reversed-phase chromatography (IP-RP) is a far more common technique. By incorporating ion-pairing agents in to the mobile phase, the desired resolution can be achieved by taking advantage of negatively charged oligonucleotides in addition to any differences in their hydrophobicity. Triethylammonium acetate (TEAA) has historically been the most employed ion-pairing agent for optical-based separations because of its separation efficiency across various RNA and DNA oligonucleotide species [5]. While features of the oligonucleotide such as its length and sequence are known to impact retention, selectivity and resolution can be optimized by making changes to the mobile phase composition or gradient [6]. Triethylamine (TEA) and other ion-pairing agents having short alkyl chains or those exhibiting low adsorptive properties are considered to be “weak” ion-pairing agents, while ion-pairing agents with longer alkyl chains or those exhibiting higher affinity to reversed phase stationary phases are considered “strong” ion-pairing agents. There are a number of reports in literature that investigate various alternative ion-pairing agents or counter ions to improve chromatography as well as mass spectrometry sensitivity [7]–[9]. In addition to these studies, alternative approaches to method optimization beyond choosing the best ion-pairing agent and gradient have proven beneficial in the separation of oligonucleotides. Levin and coworkers demonstrated a platform method for analyzing several siRNA compounds through using mobile phases containing a combination of weak and strong ion-pairing agents which was shown to improve impurity profiling through better peak shape and resolution [10]. Oligonucleotides containing modifications, including those having fluoro-conjugates, further complicate the separation mechanism, as the presence of a fluorophore as well as the structure of the fluorophore impact retention, and determination of an appropriate ion-pairing agent and the effect it has on the separation also requires evaluation [11]–[14]. The aforementioned TEA is a relatively weak ion-pairing agent, and as a result, hydrophobic effects impact separation quality. These same hydrophobic effects
can be minimized by using stronger ion-pairing agents. Anacleto and colleagues took advantage of this principle by applying a two-step purification technique that utilized two different ion-pairing agents to separate fluorophore-conjugated oligonucleotides that differ in hydrophobicity and length variants through the use of weak ion-pairing and strong ion-pairing, respectively [14]. Their work first used TEAA to best separate the probe of interest from the most undesirable synthesis-related impurity. Tetrabutylamine, a stronger ion-pairing agent, was then used in a polishing step to remove additional truncated species from the product in a length-based separation. While this analysis resulted in high product purity, offline fractionation was required. Online methods can be preferred to fractionation because they eliminate the need for manual sample handling and analysis can often be completed more efficiently. In addition, online methods have the benefit of being directly coupled to mass spectrometry (MS). The incorporation of mass information facilitates unambiguous direct identification of impurities resulting from the synthesis process, bypassing the need for fractionation or extraneous sample handling procedures. It can thus be envisioned that by incorporating two-dimensional liquid chromatography with mass spectrometry (2DLC/MS) for the analysis of dye-conjugated oligonucleotides, that a single assay can be used to more effectively understand sample purity and support synthesis processes in the production of fluoroconjugated oligonucleotides. 2D-LC/MS technology has been used in numerous applications across the pharmaceutical industry [15]– [17]. However, fewer examples are documented in oligonucleotide analysis. Li and colleagues reported a comprehensive HILIC x IP-RP LC approach for separating dC, dT, and dA homo-oligonucleotide ladders in a 27-component mixture. Due to the limited resolution achieved in the separation, only oligonucleotides of 2-10 base pairs long could be separated [18]. More recently, Roussis and colleagues evaluated size exclusion chromatography (SEC), RP, or strong anion exchange (SAX) as first dimension chemistries coupled with IP-RP in the second dimension for MS compatibility in an effort to explore the use of 2D-LC/MS for enhancing oligonucleotide impurity analysis [19]. While not all configurations were shown to be advantageous, RP coupled to IP-RP was used to analyze monomethoxytrityl (MMT)-on and MMT-off synthesis impurities in both comprehensive and heart-cut mode to demonstrate an orthogonal separation and an alternative to off-line fractionation for optimization of synthesis and purification strategies, respectively. This same comprehensive RP x IP-RP configuration was compared to an alternative comprehensive 2D approach with IP-RP x IP-RP, where the IP-RP x IP-RP configuration ultimately suffered from longer run times and a separation that is short of the desired orthogonality. In the present work, dye-conjugated oligonucleotides are analyzed via 2D-LC/MS to address the challenges commonly encountered when a single ion-pairing agent is used to deliver optimal performance in both chromatography and mass spectrometry. Failed sequences are successfully resolved from the parent oligonucleotide with a strong ion-pairing agent, hexylammonium acetate (HAA), while the identity of impurities can be more readily confirmed after the second dimension using a weaker but MS-friendly ion-pairing agent, TEA buffered in hexafluoroisopropanol (HFIP), is coupled to a single quadrupole MS. Although coupling two reversed-phase dimensions is the most reported configuration in both comprehensive and heart-cut mode, to the best of our knowledge, this is the first time a heart-cut configuration using IP-RP – IP-RP 2D-LC application has been reported to show noted
benefits [17]. This work utilizes C18 RP columns having the same chemistry in both the first and second dimension, with each dimension using a different ion-pairing agent, to achieve both orthogonal separation selectivity, as well as to maintain sensitive MS detection. The identical column chemistry used in each dimension demonstrates the importance of mobile phase selection, where the choice of ion-pairing agent in turn dictates whether the dominant separation mechanism is hydrophobicity or charge driven for each dimension. The use of IP-RP in both dimensions eliminates the need for a more traditional AEX separation in the first dimension, which has a higher risk of salt contamination of MS components, and thus increases instrument complexity by diverting flow prior to MS. This work demonstrates a single online IP-RP – IP-RP/MS analysis that can facilitate both separation and identification of fluoro-conjugated oligonucleotide impurity profiles; results otherwise not achievable using traditional one-dimensional techniques alone. 2. Material and methods 2.1 Oligonucleotides and reagents Dye-conjugated oligonucleotides were purchased from Integrated DNA Technologies (Skokie, IL, USA). Sequence and molecular weight information is provided in Table 1. Samples were purified by the vendor via HPLC and were supplied in lyophilized form. All samples were prepared to a stock concentration of 100 µM in LC/MS grade water (Fisher Scientific, Pittsburg, PA, USA) and further diluted to approximately 2 pmol/μL. Mobile phases were prepared gravimetrically using LC/MS grade reagents when available. Hexylamine (99% purity) purchased from Sigma-Aldrich (St. Louis, MO, USA) and acetic acid (99.8% purity) purchased from Honeywell Fluka (Morris Plains, New Jersey) were used to prepare 100 mM HAA, pH 7. Triethylamine (99.5% purity) and 1,1,1,3,3,3-hexafluoro-2-propanol (99.8% purity) purchased from Honeywell Fluka were used to prepare 15 mM TEA, 400 mM HFIP, pH 8. LC/MS grade water (Fisher Scientific), and acetonitrile and methanol (Honeywell USA) were also used in mobile phase preparation. Mobile phase concentrations were determined based on literature reports of the concentration required to maintain comparable chromatographic efficiencies between ion-pairing systems [20]. A one minute low pH re-conditioning step using 50: 50 methanol (Fisher Scientific): 0.2% formic acid (FA) v/v in LC/MS grade water (Fisher Scientific) was incorporated at the end of the first dimension gradient to mitigate metal adduct formation [21]. 2.2 2D-LC Instrumentation An ACQUITY UPLC H-Class Bio System with 2D LC Technology (Waters Corporation, Milford, MA, USA) equipped with MassLynx Software V4.1 (Waters Corporation) for data acquisition was used in all experiments. A quaternary solvent manager (QSM) and binary solvent manager (BSM) were used for the first- and second-dimension pumps, respectively. Optical data was collected at 260 nm using a tunable ultra-violet (TUV) detector (first dimension). ACQUITY UPLC Oligonucleotide BEH C18 (130 Å, 1.7 µm, 2.1 x 50 mm) columns (Waters Corporation) at 60 °C were used for both first- and second-dimension separations. Dedicated columns were used for each ion-pairing agent, as hexylamine has a strong
affinity for the particle sorbent. A 4 µL injection was used for all injections. Additional chromatographic method details are described in the corresponding figures. A complete instrument configuration with associated plumbing diagrams can be seen in Figure 1A. The 2D workflow is employed through the LC column manager, which houses two two-position six port valves. Through a series of valve switches controlled by the MassLynx software, the first- and seconddimension flow paths can be isolated or combined to perform the desired functionality. To begin, flow paths in the first and second dimension were isolated from one another, where a traditional 1D separation can be monitored by directing flow through the first-dimension column to a TUV detector. A heart-cut can be transferred to the second dimension by switching the valve positons to combine flow paths and using a stainless-steel tee for at-column dilution (ACD), which functions to dilute the organic composition so that the sample is retained and focused on the head of the second column. By switching the valve positions back to the original position, the flow paths are once again isolated from one another, and the second-dimension separation can proceed. 2.3 MS instrumentation A single quadrupole mass spectrometer (SQ Detector 2, Waters Corporation) was used to collect mass data post optical detection. MS data was collected in negative mode over a scan range of 410 – 3000 m/z. MS instrument settings were independently optimized in both HAA and TEA-HFIP. In TEA-HFIP, the following settings were used: capillary voltage 0.8 kV, cone voltage 50 V, and desolvation temperature 350 °C. The desolvation gas flow and cone gas flow were set to 700 L/Hr and 10 L/Hr, respectively. When investigating a 1D dilution series in HAA, a capillary voltage of 2.0 kV and a cone voltage of 20 V were used with all other settings remaining the same. MassLynx 4.1 (Waters Corporation) was used for data deconvolution using the embedded MaxEnt 1 algorithm to report average mass. 3. Results and discussion 3.1 1D separation of dye-conjugated oligonucleotides Many of the fluorescent tags conjugated to DNA primers and probes are extremely hydrophobic. To demonstrate the effect that these hydrophobic fluorescent tags have on a separation, the elution order of failed sequences of a Cy3 conjugated 25-mer was studied using two different ion-pairing agents. The n-1, n-2, and n-3 failed sequences (Table 1) of this oligonucleotide were synthesized and spiked in to the 25-mer sample in equal amounts. Mobile Phase A (MPA) consisted of either TEA-HFIP or HAA ion-pairing agents and Mobile phase B (MPB) was 100% LC/MS grade organic solvent (methanol or acetonitrile). RP separations more commonly use acetonitrile as the organic solvent, but as HFIP is not miscible in acetonitrile, methanol was used in this case. From Figure 2A, the n-1 and n-2 failed sequences co-elute after the main peak when TEA-HFIP is used as MPA. While further optimization of the gradient can potentially provide a marginal improvement in resolution, it is more important to note that elution order is not a length-based separation as would be predicted in a more traditional separation of unmodified oligonucleotides. Gilar and colleagues developed a model for predicting oligonucleotide retention under IP-RP conditions based
on oligonucleotide length and sequence, which lends insight to selecting initial organic composition to achieve high-throughput methods [22]. Although there have been further efforts to model elution behavior of unmodified oligonucleotides since this foundational work, no model for dye-conjugated oligonucleotides is reported. HAA has been studied as an alternative ion-pairing agent for noted benefits in a number of applications [20], [23], [24]. Hexylamine serves as a more efficient ion-pairing agent through more effective adsorption to the RP stationary phase than triethylamine because of the increased length of the alkyl chain. As a result, HAA diminishes the reversed-phase contribution from the hydrophobic dye, and the labelled oligonucleotide separation becomes AEX-like and is mainly dictated by the oligonucleotide length in HAA (Figure 2B). In our study, when using a strong ion-pairing agent such as HAA, the organic content required to elute the oligonucleotide is much higher than with a weak ion-pairing agent. In our example, 52% acetonitrile is required to elute the full-length product (this takes into account a combined system volume and column volume of 0.67 mL, which is negligible compared to the volume required to elute the peak of interest). In TEAA, 34% methanol is required to elute this same species. Under these weaker elution conditions, the dye is still retained on the stationary phase, and the resulting separation is mixed-mode driven, supported by the elution order of the failed sequences relative to the main peak (Figure 2A). It should be noted that the oligonucleotides are more retained in methanol than acetonitrile, and the percent organic required for elution will be greater in HAA in methanol than HAA in acetonitrile, which will be further evaluated in the 2D workflow. The usefulness of HAA for this application was further investigated by exploring MS analysis of failed sequences using a single quadrupole detector (SQ Detector 2). Detection limits of a Cy3-1 dilution series in 100 mM HAA were compared to results in TEA-HFIP. Since its introduction by Apffel and colleagues, HFIP is the most commonly used counter ion when mass spectrometric analysis of oligonucleotides is needed, as HFIP offers enhanced MS signal intensity [25]. Table 2 reports a dilution series of the n-1 failed sequence and the corresponding signal-to-noise ratio (SNR) as determined from the average peak intensity of the total ion chromatogram (TIC) across three injections. In TEA-HFIP mobile phase, the limit of detection (LOD) approaches a 1 ng mass load. In HAA, a substantially higher mass load of over 60 ng is barely detectable at SNR = 3, and therefore cannot be further diluted. When determining impurities via relative percentage, larger injection volumes can be made to achieve lower detection limits if required, but there are limitations to sample load volume due to peak broadening [23]. An alternative approach to detecting lower concentration limits is through the use of more sensitive instrumentation, but as the purpose of our study is for impurity confirmation and not characterization, a lower resolution quadrupole instrument is appropriate for the intended application [26]. The effects of ion suppression in HAA compared to TEA-HFIP are shown in Figure 3, which shows both the TIC and raw data from the first injection of the dilution series in the respective mobile phases. From the TIC, the analyte in HAA is barely distinguishable from the noise. From the raw data, adducts are especially evident at the lower charge states. We suspect that reagent purity also impacts MS signal by introducing additional adducts, and hexylamine is not readily available in LC/MS grade. Gong reported a
similar result, where the acetate salt of his ion-pairing agent yielded more adducts than bicarbonate or HFIP salts [27]. The MaxEnt 1 algorithm was used to deconvolute data over the same number of scans, and the response is approximately three orders of magnitude lower for HAA than TEA-HFIP with the same sample load. The increased MS sensitivity when using HFIP-based ion-pairing buffers provides a means to identify and quantify low-level impurities based on mass. This can be seen in the plot shown in Figure 4, where a high degree of linearity was observed throughout the working range selected from Table 2. Interestingly, although hexylamine has been shown to provide comparable peak intensity in ESI of oligonucleotides when using HFIP as the counter ion (versus acetate) when compared to TEA-HFIP, the chromatography does not offer this same performance [8]. Li et al. report notable improvements in chromatographic resolution and retention across RNA, DNA, and MDNA when comparing TEA-HFIP to HA-HFIP [7]. Our work further demonstrates the importance of balancing chromatography and MS performance to achieve the desired result. Because HAA is a more efficient ion-pairing agent, it provides a length-based separation for optical detection, but due to the hydrophobicity of the dye conjugate, the reversed phase mechanism dominates the ion-pairing mechanism for a weak ion-pairing agent such as TEA-HFIP. It also becomes apparent that HAA does not provide adequate sensitivity for this application. This lends itself to a 2D-LC/MS solution where HAA in the first dimension yields a length-based separation, and by selectively heart-cutting impurities, a second dimension can be used in TEA-HFIP to provide MS detection and identity confirmation. 3.2 2D heart-cutting configuration, events tab To begin method development, the pressure tolerance of the first-dimension column was evaluated using different flow rates. This is a necessary step of method development due to the configuration of our 2D system, which couples the first and second dimension columns in serial when the flow paths are combined during the heart-cut (Figure 1A). For this reason, the flow rate used in each dimension must be amenable to the pressure tolerance of the instrument and columns. Alternative plumbing configurations or trap columns can be used to eliminate or alleviate the pressure increase across the first-dimension column, but these options were not explored as pressure restrictions were not exceeded in this study. In addition to pressure restrictions, there also needs to be consideration for strong solvent effects when determining flow rate. The n-1 impurity elutes at an organic concentration that is too high to retain the analyte on the second-dimension column. By incorporating ACD, aqueous-based mobile phase (MPA from the second dimension) can be introduced through a fluidic tee using a second pump. The first-dimension separation was run at 0.1 mL/min and the second dimension was run at 0.25 mL/min, which effectively serves as a 3.5-fold dilution of the organic composition. This dilution factor reduces the organic content to re-focus the analyte on the second-dimension column prior to separation in the second dimension. The first-dimension separation in 100 mM HAA was optimized prior to coupling to the second dimension. Methanol was used as the organic solvent for both dimensions in all 2D separations, as HFIP is not soluble in acetonitrile. Although ACD is employed, there are immiscibility issues upon contact
when acetonitrile is used in the first dimension. When HAA is used in combination with methanol, a greater percent organic composition is needed to elute the analytes than with acetonitrile. A gradient from 66% - 76% B over 20 minutes was used to separate Cy3 from the spiked in n-1 impurity (Cy3-1) in the first dimension. The n-1 impurity elutes in approximately the middle of the gradient at 70% methanol. When the n-1 impurity started to elute, the valve switching events were set up to take the heart cut over 0.5 minutes (Figure 1B). As the primary purpose of the second dimension is impurity confirmation, a rapid screening gradient (20 – 70% B over 10 minutes) can be used without further optimization. A hold step was employed after the heart-cut and before the start of the seconddimension gradient to wash and refocus the sample. Figure 5 demonstrates this proof of concept using the Cy3-conjugated 25-mer with the n-1 impurity spiked in at approximately 10 percent by optical peak area. Figure 5A shows the TUV trace, which was used to determine the heart-cut window for the n-1 peak to be selectively sent to the second dimension. The corresponding second dimension figure shows the TIC of the n-1 impurity (Figure 5B). The TIC was used to confirm the mass of the n-1 peak. 3.3 2D of Cy3-conjugated 25-mer To demonstrate the applicability of this method for a more realistic sample, the Cy3-conjugated 25-mer was analyzed without the addition of the n-1 impurity spike-in using the same configuration as described above. Oligonucleotides can generally be purified via standard desalting or HPLC, although the manufacturer requires Cy3-conjugated oligonucleotides to be purified via HPLC. Selecting a method of purification can also be dependent on the intended use of the oligonucleotide. Because the target application of these dye conjugated oligonucleotides requires high purity, HPLC purification maintains assay quality. From Figure 6, the n-1 impurity could be successfully identified at 2.4 percent (percentage determined from the TUV trace). This result agrees with results reported from the 1D dilution series (Table 2), where the lower limit of detection approached 1.5 percent. In terms of sample concentration, these values align with the expected limit of detection of oligonucleotides with the instrumentation used, but more importantly, sensitivity is not lost between the 1D and 2D assays. To detect more extensive impurity types with greater sensitivity, high-resolution MS is required [26]. 4. Conclusion This work demonstrates the ability to couple two IP-RP methods for 2D-LC/MS analysis of oligonucleotides in an effort to achieve desirable performance in both chromatographic separation and mass spectrometric detection. Because the same column chemistry was used in both the first and second dimension and does not give rise to selectivity differences, this work explored changes in mobile phase composition to control the separation mechanism. HAA was used in the first dimension to achieve a length-based separation of a Cy3-conjugated 25-mer and its failed sequences. By selectively heartcutting the n-1 impurity, TEA-HFIP could be introduced in the second dimension for identity confirmation using a single quadrupole detector. This method could be used to successfully confirm low level impurities, which was not achievable using a one-dimensional technique. Funding
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Table 1. Oligonucleotide sequence and MW information. Table 2. Limits of detection of Cy3-1 in TEA-HFIP and HAA. The Cy3-1 failed sequence was prepared at a stock concentration of 100 µM and further diluted to 2.22 pmol/µL, corresponding to a 62.68 ng mass load (4 µL injection). A 2-fold dilution series was carried out until the analyte could no longer be detected. The reported signal-to-noise (SNR) is the average peak intensity of the total ion chromatogram (TIC) across three injections. Method conditions in HAA: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% acetonitrile. Method conditions in TEA-HFIP: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol. The same gradient conditions were used in both HAA and TEAHFIP. Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 1. 2D system configuration. 1A) Two two-position six port valves housed in a column manager enable heart-cut functionality as depicted in the schematic. When the left and right valves are in Position 1, the first- and seconddimension flow paths are isolated from one another. This allows for separation in the first dimension (top panel), and as the analyte of interest begins to elute, the left and right valves are switched to Position 2 to selectively transfer the analyte to the second-dimension (middle panel) for analysis. Atcolumn dilution (ACD) works to focus the analyte at the head of the second-dimension column by mixing aqueous mobile phase from the second-dimension pump with the eluent from the first-dimension column, thus reducing the organic content that was required to elute the analyte in the first-dimension. The second-dimension separation begins when the flow paths are once again isolated from one another (bottom panel). System component abbreviations: quaternary solvent manager (QSM); sample manager with flow-through needle (SM-FTN); tunable ultraviolet detector (TUV); binary solvent manager (BSM); photodiode array detector (PDA); and mass spectrometer (MS). 1B) A 0.5 minute heart-cut window from 9.50 to 10.00 minutes was used to transfer the analyte of interest to the second-dimension by creating a timed event within the software. This timed event switches both valves from Position 1 (isolated flow paths) to Position 2 (combined flow paths). Figure 2. Comparing optical detection of a Cy3 and its failed sequences in TEA-HFIP and HAA. 2A) Due to the hydrophobicity imparted by the dye conjugate, n-1 and n-2 failed sequences elute after the main peak when a weak ion-pairing agent is used. Method conditions: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 28 – 38% B in 7.5 minutes at 0.2 mL/min. (Peak height is normalized.) 2B) When using a more efficient ion-pairing agent, the reversed-phase contribution from the hydrophobic dye is minimized to result in a separation largely dictated by oligonucleotide length. Method conditions: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% acetonitrile; Gradient conditions: 30 – 60% B in 7.5 minutes at 0.2 mL/min. (Peak height is normalized.) Figure 3. Comparison of MS data for Cy3-1 in TEA-HFIP (3A) and HAA (3B). A 62.68 ng mass load (4 µL injection) of Cy3-1 was used to compare MS response. When the total ion chromatogram (TIC) and raw data are placed on the same scale, an enhanced MS signal can be seen in TEA-HFIP compared to HAA, where the analyte response is barely distinguishable from the noise.
Deconvoluted data shows the MS response in HAA is approximately three orders of magnitude lower than in TEA-HFIP for the same sample load. Method conditions in HAA: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% acetonitrile. Method conditions in TEA-HFIP: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol. The same gradient conditions were used in both HAA and TEA-HFIP. Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 4. Dynamic range of Cy3-1 in TEA-HFIP. The Cy3-1 failed sequence was prepared at a stock concentration of 100 µM and further diluted to 2.22 pmol/µL, corresponding to a 62.68 ng mass load (4 µL injection). A two-fold dilution series was carried out until the analyte could no longer be detected. The reported peak area is from the average total ion chromatogram (TIC) peak area across three injections. Method conditions: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 5. Evaluation of 2D system configuration using Cy3 and spiked in Cy3-1 impurity. 5A) Optical (UV) data showing separation of n-1 impurity from the full-length product. The n-1 impurity spike-in is at approximately 10 percent as determined from the integrated peak areas in the optical trace. Using this data to determine the heart-cut window, the n-1 peak is selectively transferred to the second-dimension for mass confirmation. Method conditions in first-dimension: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% methanol; Gradient conditions: 66 – 76% B in 20 minutes at 0.1 mL/min. 5B) Total ion chromatogram (TIC) of the n-1 impurity selected from the first-dimension. An isocratic hold at initial conditions is employed for 14 minutes during the heart-cut and transfer of analyte to the second dimension. The broad peak at ~12.5 minutes is attributed to mobile phase transfer from the first dimension passing through the second-dimension column. Total run time is 30 minutes to incorporate a column wash at the end of the second-dimension separation. Method conditions in second-dimension: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min. Figure 6. 2D Cy3-1 impurity analysis of Cy3. A 62.68 ng mass load (4 µL injection) of Cy3 was separated to determine if the n-1 impurity resulting from synthesis could be detected. From the optical (UV) trace, the n-1 impurity was determined to be present at approximately 2.4 percent as determined from peak area integration. The deconvolution inset shows that peak identity can be confirmed at this level, which is consistent with detection limits achieved from the 1D dilution series. Method conditions in first-dimension: Mobile phase A: 100 mM HAA, pH 7; Mobile phase B: 100% methanol; Gradient conditions: 66 – 76% B in 20 minutes at 0.1 mL/min. Method conditions in second-dimension: Mobile phase A: 15 mM TEA, 400 mM HFIP, pH 8; Mobile phase B: 100% methanol; Gradient conditions: 20 – 70% B in 10 minutes at 0.25 mL/min.
A) 15 mM TEA, 400 mM HFIP, pH 8 B) Column manager Time (min)
Event
Action
Initial
Left Valve
Position 1
Initial
Right Valve
Position 1
9.49
Left Valve
Position 2
9.50
Right Valve
Position 2
10.00
Left Valve
Position 1
10.00
Right Valve
Position 1
General tab
Events tab
A) 15 mM TEA, 400 mM HFIP, pH 8 B) Column manager Time (min)
Event
Action
Initial
Left Valve
Position 1
Initial
Right Valve
Position 1
9.49
Left Valve
Position 2
9.50
Right Valve
Position 2
10.00
Left Valve
Position 1
10.00
Right Valve
Position 1
General tab
Events tab
A) 15 mM TEA, 400 mM HFIP, pH 8 4.0x10
8
TIC 4.0x10
6
Raw data 2.0x10
7
1.6x10
7
1.2x10
7
8.0x10
6
4.0x10
6
7855.2 Da
2.0x10
8
1.0x10
8
0.0 4
5
6
7
3.0x10
6
2.0x10
6
-3 -9 -7 -8 -6 -5
1.0x10
6
Intensity (counts)
8
Intensity (counts)
Intensity (counts)
-4 3.0x10
0.0 7000
0.0 500 1000 1500 2000 2500 3000
8
m/z m/z
Retention time (min)
Deconvoluted data
7500
8000
8500
9000
Mass (Da) (Da) Mass
B) 100 mM HAA, pH 7
3.0x10
8
2.0x10
8
1.0x10
8
TIC 4.0x10
6
3.0x10
6
2.0x10
6
1.0x10
6
Deconvoluted data
Raw data
Intensity (counts)
8
Intensity (counts)
Intensity (counts)
4.0x10
-5 7.00
7.25
7.50
7.75
Retention Time Retention time (min) (min)
8.00
-4
-3 -6 0.0 500 1000 1500 2000 2500 3000 m/z
m/z
5.0x10
4
4.0x10
4
3.0x10
4
2.0x10
4
1.0x10
4
0.0 7000
7853.5 Da
7500
8000 Mass (Da)
8500
Mass (Da)
9000
7
2.0x10
7
Peak area
1.6x10
7
1.2x10
R2 = 0.9977
6
8.0x10
6
4.0x10
0.0 0
10
20
30
40
Mass load (ng)
50
60
AU AU AU
A) 1D TUV 0.06 0.05 0.04 0.03 0.02 0.01 0.00
n n-1
0
5
10
15
20
Intensity AU(counts)
Retention time (min)
B) 2D TIC n-1 7854.0 m/z
8
1.5x10
8
1.0x10
7
5.0x10
0.0 0
5
10
Retention time (min)
15
20
0.005 0.004 0.003
n-1 Deconvolution
n
Intensity (counts)
A AU AU
1D TUV
Heart-cut window
n-1
0.002 0.001 0.000 (min) Retention Time time (min)
15
7.0x10
3
6.0x10
3
5.0x10
3
4.0x10
3
3.0x10
3
2.0x10
3
1.0x10
3
0.0 6000
n-1 7853.6 m/z
7000
8000
9000
Mass (Da) Mass (Da)
10000
Oligonucleotide Cy3 Cy3-1 Cy3-2 Cy3-3
Sequence (5' - 3') Cy3-TTT GAC TTA GAC TTA GAC TTA GTT T -T -TT -TTT
MW (Da) 8158.6 7854.4 7550.2 7246.0
Sample conc. (pmol/ μL) 2.22 1.11 0.56 0.28 0.14 0.069 0.035
Mass load (ng) 62.68 31.34 15.67 7.83 3.92 1.96 0.98
Impurity (%) 50 25 12.5 6.25 3.13 1.56
MS SNR HAA 3.34 -
MS SNR TEA-HFIP 125.64 67.76 36.01 19.2 8.93 4.7 2.09
Highlights:
Dye-conjugated oligonucleotides impact retention by imparting hydrophobicity IP – RP can achieve an AEX-like result by using a strong IP agent Selecting an IP agent requires a balance of chromatographic and MS performance 2D coupling of strong and weak IP agents can be used to improve detection limits
Abstract Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in onedimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dyeconjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the twodimensional analysis can be detected at low nanogram levels, comparable to results achieved in a onedimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.
Author Statement Brooke M. Koshel: Investigation, Writing – Original Draft Robert E. Birdsall: Writing – Reviewing and Editing, Supervision Weibin Chen: Writing – Reviewing and Editing, Supervision
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: