A Xevo™ G3-based Workflow for Purity Determination, Intact Mass Measurement, and MS/MS Sequencing of Impurities Detected in Synthetic Oligonucleotides

This study evaluates how UPLC™-QToF-MS with integrated informatics can facilitate the characterization of synthetic oligonucleotides and their impurities. The combination of oligonucleotide intact mass measurement and MS/MS fragmentation-based sequencing is highly effective at confirming the expected sequence of the Full-Length Product (FLP) and identifying modified variants that can represent potential impurities. The Xevo G3 QTof System is a bench-top High Resolution Mass Spectrometer, which is operated on the compliant-ready informatics platform, waters_connect™, enabling integrated data acquisition and processing. Data independent (MS^E) and targeted MS/MS acquisitions were utilized to characterize the modified oligonucleotides. The holistic analytical and informatics workflow is suitable for both regulated and non-regulated laboratories, enabling both characterization and routine oligonucleotide quality assessments.

Benefits

• The INTACT™ Mass App provides quick and automated mass assignment of the Full-Length Product and putative identification of oligonucleotide impurities
• The CONFIRM™ Sequence App streamlines the annotation of oligo fragment ions from both DIA (MS^E) and targeted MS/MS data
• Finding sequence omissions and insertions or identifying sites of sequence modification and scrambling
• The Xevo G3 QTof MS paired with the ACQUITY™ Premier UPLC provides a sensitive, robust, and flexible analytical platform for synthetic oligonucleotide analysis capable of being deployed in both regulated and non-regulated laboratories

Many oligonucleotide therapeutics (ONTs) are chemically synthesized and exhibit a combination of synthesis artefacts and degradation products broadly classified as product-related impurities. The most popular classes of ONT molecules include antisense oligonucleotides (ASO) and interfering RNA (siRNA oligos) used to modulate gene and protein expressions, and aptamer RNAs that modulate protein and macromolecule functions.^1-2 As synthetic oligonucleotides gain increasing interest as therapeutic drug products, and with a trend to increasing structural complexity, having advanced analytical methodologies for impurity characterization is critical to ensure product safety and efficacy.

Several LC-MS workflows for oligonucleotide analysis have been recently introduced by Waters Corporation.^3-6 These workflows were optimized using a GMP-ready UPLC-Tof MS System that provides a robust and reproducible LC-MS platform, accessible to non-MS experts and supported by streamlined informatics applications that reduce the need for manual data interpretation. Previous studies show that ACQUITY Premier Technologies using Max Peak Technology can improve the chromatographic separation of synthetic oligonucleotides in terms of recovery and reproducibility.^7,8 In addition, the informatics platform used for data processing enables automated peak picking and fast deconvolution of oligonucleotide spectra, providing fast impurity assignments as well as the resulting mass accuracy and quantitative capability to support impurity analysis.^5-6

The CONFIRM Sequence waters_connect App was introduced for automated processing of both MS/MS and MS^E (data independent) fragmentation spectra for sequence verification.⁶ The sequence coverage can be viewed in a "dot-map" diagram to easily assess sequence coverage and to localize sites of modification.

In the studies described here, the Xevo G3 QTof System (Figure 1), along with two waters_connect applications (INTACT Mass and CONFIRM Sequence) were used for analysis of synthetic nucleotide impurities. The ability to detect and confirm low-level impurities was assessed.

A 21-mer synthetic siRNA oligonucleotide, containing 13 modified nucleotides, (sequence: GCC UCA GTC TGC TTC GCA CCT), was obtained from ATDBio (Southampton, UK). The oligonucleotide sequence uses colored letters to denote the modified nucleotides, while the regular (unmodified) nucleotides are black. A 2’-O-(2-methoxyethyl) - (2’-MOE) modification was attached to two guanosines (G) and one adenosine (A). Also, in addition to the attachment of the 2’-MOE functional group to one uridine (U) and six cytidines (C), the nucleobases of these two nucleotides were further modified by the attachment of a 5-Methyl (5-Me) group to produce one 2’-MOE 5-Me uridine (U) and six 2’-MOE 5-Me cytidines (C). Out of the nine cytidine residues present in the 21-mer sequence, three cytidines were modified only at the nucleobase by the addition of a 5-Me functional group (C). Finally, there were eight nucleotides left unmodified: one adenosine (A), three guanosines (G) and five deoxythymidines (T). Stock solutions were prepared in DI water at a concentration of 1 µM (or 2.4 µg/mL), from which a 10 µL volume was injected, which corresponds to loading 10 picomoles of the 21-mer oligonucleotide on-column. Figure 2 displays the structure of the synthetic oligonucleotide (21 mer).

Data acquisition and processing was performed using the waters_connect informatics platform. ESI-MS spectra of intact oligonucleotides were processed automatically using the INTACT Mass App. The oligonucleotide purity assessments were calculated based on UV response as part of the automated processing workflow. Individual MS/MS spectra and MS^E datasets were processed by the CONFIRM Sequence App using the optimum collision energy for fragmentation of each oligonucleotide precursor to sequence the Full-Length Product and product impurities.

Data Acquisition using the ACQUITY Premier UPLC with Max Peak Technology System coupled with the Xevo G3 QTof MS System

The siRNA sample (21-mer) used for this study was synthesized using a typical solid phase approach. We previously demonstrated that an ACQUITY Premier UPLC Column, featuring High Performance Surfaces Technology, improves the chromatographic performance for oligonucleotide impurities with reduced need for column conditioning, improved peak shape, and enhanced recovery of low abundance impurities, when compared to a traditional stainless-steel column.⁴ Sixteen synthesis-related impurities resolved for this siRNA oligomer are shown in the LC-UV chromatogram displayed in Figure 3. The lowest abundance impurity, according to the UV data, is the first eluting peak with an abundance of 0.05% relative to the peak area of the FLP. The ACQUITY UPLC Premier System used for this impurity assay, is a very inert system which allows for robust separation of trace-level oligonucleotide impurities.

The Xevo G3 QTof MS System has been purposefully developed with capabilities optimized for biopharmaceutical applications: 1) improved ion transmission of low mass labile species; 2) enhanced quantitative capabilities with wide inter and intra-spectrum dynamics range for comprehensive Quan/Qual analysis; 3) new ion optics for improved robustness and reproducibility. When combined with waters_connect informatics, the platform is well suited for product characterization in Development, or for more routine assays in regulated Development, Manufacturing and Quality organizations.

The combined ESI-MS spectrum recorded for the lowest abundance impurity (peak 1 from Figure 3, a 6-mer phosphorylated impurity) is displayed in Figure 4. The corresponding doubly and triply charged precursors are highlighted in this spectrum indicating the ability of the Xevo G3 instrument to detect trace-level impurities.

Sensitive transmission of precursors produced by lower-abundance oligonucleotide impurities ultimately improves the fragmentation through collision-induced-dissociation for both targeted MS/MS or DIA (MS^E) acquisition modes. As shown in Figure 5, a high sequence coverage (83%) was obtained from the MS/MS fragmentation of the doubly charged precursor (m/z=1120.7) of the 5’-phosphorylated 6-mer (identified as peak 1 in Figure 3) when the data was analyzed with the CONFIRM Sequence app. The combination of intact mass measurement and MS/MS sequencing data synergize to provide both semiquantitative data and strong confidence in impurity assignment.

Automated Mass Assignments with the INTACT Mass app

Calculating the molecular weight and the relative abundance of impurities compared to the FLP peak area (based on the TUV and/or the TIC trace) is performed for each oligo impurity detected above a specified threshold. The INTACT Mass App software was used to process the IP-RP UPLC-UV-MS chromatograms in an untargeted fashion, using automated BayesSpray^TM deconvolution to calculate the neutral monoisotopic mass for all components that meet criteria set in the data processing method (RT window and the ion count thresholds).
The untargeted mass analysis can be converted into a targeted analysis simply by updating a target list in the processing method that includes the oligo identification and the expected accurate monoisotopic mass (neutral mass). The INTACT Mass results shown in Figure 6 are presented as three panels, including the deconvolved spectrum (panel A – top), the raw ESI-MS spectrum showing a wide variety of charge states (panel B – middle) and the mock spectrum used for assessing the fidelity of the deconvolution process (panel C – bottom). A portion of the table containing the INTACT Mass processing result is displayed (Figure 7). Out of sixteen impurities (total number of impurities detected in this sample), nine impurities shown in this table along with the FLP, were putatively assigned with mass accuracies better than 10 ppm.

Sequence Confirmation of oligonucleotide impurities using the CONFIRM Sequence App

The automated annotation of data independent fragmentation spectra (high energy MS^E) as well as annotation of targeted MS/MS spectra were accomplished using the waters_connect CONFIRM Sequence App. Multiple MS/MS and/or MS^E spectra can be analyzed against one or more oligonucleotide sequences.

The Synthetic Library, which stores the oligonucleotide sequences, was populated with the 21-mer extensively modified siRNA oligonucleotide (Figure 8). The [M-4H]^-4 precursor was selected for MS/MS fragmentation using the quadrupole and fragmented in the collision cell of a Xevo G3 QTof MS using an optimized fixed collision voltage (40 V). The result obtained following data processing with the CONFIRM Sequence App - (Figure 9) shows complete (100%) sequence coverage for the FLP. This “dot map” coverage view displays the 21-mer sequence from the 5’ to the 3’ end, with assigned fragment ions displayed above and below the dots (individual nucleotides).

In addition to the FLP, targeted MS/MS was performed on six out of the sixteen oligonucleotide impurities with TUV peak area at or above 0.05% of the FLP peak area. Data from these analyses are summarized in Table I, including nucleotide sequence, elemental composition, neutral monoisotopic mass, most abundant precursors, optimized collision energies, sequence coverage (from a single MS/MS spectrum as well as from two combined MS/MS spectra) and impurity abundance relative to the FLP (as the TUV area percentage). The lowest abundant impurity, measured at 0.05% was identified as 5’-phosphorylated GCA CCT, according to the ESI-MS and MS/MS spectra shown in Figures 4 and 5. The overall combined sequence coverage resulted from the analysis of MS/MS fragmentation spectra of the doubly and triply charged precursors of this impurity is 89.5%, as indicated in Table I.

• The ability to identify and confirm low-level impurities is critical to effectively characterize oligonucleotide therapeutics and monitor the quality of product batches.
• The synergy of intact mass analysis and fragmentation-based sequencing, enables holistic impurity profiling and targeted confirmation of impurity assignments, enabling confidence in synthetic production processes for this important class of therapeutics.
  
• The automated assignment of putative impurities detected in synthesized oligonucleotides with both UV data and the Xevo G3 QTof MS detector was accomplished using the waters_connect INTACT Mass App with typical mass errors below 10 ppm.
  
• The MS/MS spectra recorded for six oligonucleotide impurities along with the MS/MS spectra recorded for the 21-mer extensively modified FLP were acquired on this same system and processed using the CONFIRM Sequence App to verify their expected sequences.
  
• The capability of the CONFIRM Sequence App to achieve high sequence coverage (83.3%) of lower abundance impurities (~ 0.05% relative abundance) was confirmed.
  

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