Analyzing Small RNA-based Therapeutics and Their Process Impurities Using Fast and Sensitive LC–HRMS

Silvia Millán-Martín discusses recent advances in the analytical characterization of small RNA-based therapeutics. Her team developed an ion-pair reversed-phase liquid chromatography–high-resolution mass spectrometry (IP-RPLC–HRMS) method using a novel combination of pentylamine and HFIP, enabling sensitive, robust analysis of antisense oligonucleotides with diverse modifications. The approach minimizes adduct formation and in-source induced artifacts while supporting regulatory compliance. This workflow offers broad applicability in biopharmaceutical development, aiding impurity profiling, quality control, and accelerated approval of RNA-based drugs.

You recently published a manuscript called Characterization of Small RNA-based Therapeutics and Their Process Impurities by Fast and Sensitive LC–HRMS (1). What was the rationale behind this research?.

At the Characterization and Comparability Laboratory (CCL) at the National Institute for Bioprocessing Research and Training (NIBRT), our goal is to develop analytical solutions to complex scientific problems that deliver utility and impact, and facilitate advances in biopharmaceutical manufacturing, with the ultimate motivation to deliver positive patient outcomes.

The number of ribonucleic acid (RNA)-based therapies have increased considerably in recent years, since the first approval in 1998 by FDA. This has also been influenced by the COVID-19 messenger ribonucleic acid (mRNA) vaccine success, and by the fact that they represent a promising approach with rapid development potential and broad applicability across diseases previously “undruggable,” such as genetic disorders, metabolic disorders, cancers, or infectious diseases, offering new hope for patients. This type of new therapeutic modality could become a cornerstone of precision medicine with the ability for tailored treatments. Their therapeutic potential and molecular complexity requires the continuous development of robust and sensitive analytical methods to help characterize their primary sequence and associated impurities to help speed their development process.

Oligonucleotides are traditionally analyzed using ion-pair reversed-phase liquid chromatography (IP-RPLC). The combination with mass spectrometry (MS) provides high sensitivity and resolution, with a wide dynamic range. Furthermore, the use of tandem MS can provide detailed information about the structure and sequence. In our recently published study (1), we focused on the use of a mild electrospray ionization (ESI) source, the heated electrospray ionization (H-ESI) source, and a new IP reagent with moderate hydrophobicity, pentylamine, which has not been reported for oligonucleotide analysis. The molecules included in this study were a selection of commercially available RNA-based small single-stranded antisense oligonucleotides (ssASOs) with different types of modifications/lengths, whose average sizes were between 14 mer to 20 mer. The selected molecules are designed with different chemical modifications from second and third generation categories, including backbone modifications, sugar modifications at the 2’ position, and base modifications, with the aim to demonstrate the applicability of the developed approach.

What is the most innovative aspect of using amine-based IP-RPLC–HRMS for analyzing ss-ASOs compared to existing methods?

As mentioned, IP-RP-HRMS is not new to RNA-based therapeutic characterization, but as new molecules are entering the market, the ability to have a general analytical approach that would cover the detailed characterization of the different types of ssASOs molecules bearing different types of modification within their structure is required.

On the other hand, most MS sources are harsher than the mild H-ESI source selected in the present study and create source-induced impurities that are not in the original sample.

Another important aspect is the interest in using more environmentally friendly reagents in accordance with the principles of green chemistry, following EU REACH regulation. Highly hydrophobic ion pairing agents, which were used previously, such as hexylamine (HA), octylamine (OA), or dibutylamine (DBA), should be avoided.

Our developed IP-RP-HRMS workflow focused on the evaluation of an alternative moderate ion pairing for small RNA-based therapeutics combined with hexafluoroisopropanol (HFIP) fluoroalcohol. Low concentration of pentylamine (15 mM) offered good resolution and retention of the impurities chromatographically, whilst the moderate hydrophobicity allowed the removal of the amine adducts with mild in-source collision energies without having to go to extreme conditions that would induce in-source fragmentation of impurities not present in the original sample. On the other hand, the addition of HFIP at low concentration (60 mM) improved MS sensitivity by improving ESI and acting as a counter ion to facilitate oligonucleotide retention on the reversed-phase stationary phase.

What was the biggest challenge you faced during the development and optimization of this LC–HRMS method, particularly in balancing chromatographic separation with MS sensitivity?

Adduct formation from used amine or trace metal impurities (such as sodium and potassium) is probably the most common challenge during the characterization of small RNA-based therapeutics by LC–HRMS. Amine ion-pairing agents and metal adducts create a quantitation problem because of multiple split signals, which should be minimized as much as possible or even totally avoided. Ion-pairing reagent concentration should be reduced, especially when using strong IP reagents. Moderate IP reagents are therefore recommended. High-purity solvents and reagents must be used and, if possible, a dedicated LC–MS system is also recommended. In-source generated impurities are also a critical aspect that must be studied and avoided. It is recommended to use a mild ESI source and optimize source parameters, such as in-source collision energy (SID), to find a balance between removing adducts and preventing unwanted fragmentation, and source and gas temperatures. Mobile phase pH is critical too, and it is recommended to be kept between pH 9 and pH 10.

The combination of amine ion pairing and fluoroalcohol was first described by Apffel et al. (2) as the best option for balancing chromatographic separation and MS sensitivity. Several studies (3–6) have evaluated the concentrations of various alkylamines and fluoroalcohols, both of which will cause ion suppression during electrospray ionization at elevated concentrations (>100 mM). Over the last decade, efforts have focused on improving chromatographic resolution and improving MS sensitivity. IP-RPLC–MS separation of oligonucleotides is strongly influenced by the sequence of the oligonucleotide under investigation. The MS sensitivity depends on the hydrophobicity of nucleotide bases, alkylamine/HFIP concentrations, and adduct formation. Optimal MS signal has been observed when using low concentrations (5–30 mM) of alkylamines buffered with 50–100 mM HFIP. Interestingly, there does not appear to be any universal best combination of alkylamine and fluoroalcohol for LC–MS experiments, and this aspect should be optimized for each specific situation, depending on the question to be answered.

The aim of our study was the use of a general method that could be applied to different ssRNA-based molecules, of different lengths and with different types of modifications. Although full chromatographic resolution would be ideal, a compromise between analysis time was considered too. In the present study (1), an optimized gradient of 27 min was selected to cover all the different types of small ssRNA-based molecules from 14 mer to 21 mer and corresponding low-level impurities. The used molecules bearing multiple impurities resulted in complex elution profiles, and although optimal chromatographic separation of all detected impurities is desirable, this is not mandatory when using HRMS. As demonstrated in this work, high-resolution accurate mass spectrometry can accurately and sensitively identify closely eluting species.

How do you see this method advancing routine characterization of RNA-based drugs and their process impurities in both research and regulatory settings?

Driven by drug development needs and regulatory requirements, critical quality attributes (CQAs) of oligonucleotide therapeutics, such as purity, impurity profile, and sequence, must be characterized to ensure process consistency, product quality, safety, and efficacy. The proposed method will advance the characterization of small ssRNA-based therapeutics as the intact analysis allowed for the detection and quantification of full-length products with different sizes and purity values, and the detection of multiple impurities and degradants, even without being fully chromatographically resolved, with high sensitivity. Accurate masses were obtained by performing deconvolution of MS1 data using the intact mass analysis workflow while complementary MS/MS analysis and data processing supported the findings by sequencing experiments. This approach would support an initial discovery phase during process development. Once the molecule is fully characterized, a more targeted approach could be attained in a compliant and automated high throughput fashion by using an effective chromatographic data system (CDS), which allows for good laboratory practice (GLP)-compliant data handling and reporting.

The developed method can streamline manufacturing processes and support quality assurance (QA) as it shows a comprehensive characterization of the target molecule, and a wide range of impurities, product variants, or degradation products. It would also help to provide the evidence needed for regulatory bodies to assess safety and efficacy, facilitating the approval of new RNA-based therapeutics.

How does the choice of ion-pairing reagent (hydrophobic vs. moderately hydrophobic amines) influence chromatographic resolution, ion suppression, and adduct formation when separating modified-RNA oligonucleotides? What are the trade-offs between achieving full chromatographic resolution of ss-ASOs and prioritizing sensitivity in high-resolution MS detection, especially for low-abundance impurities?

The impact of IP reagents on the selectivity and sensitivity during LC–MS analysis of oligonucleotides has been extensively studied in the literature (6,7–11); however, there is no clear consensus on the best choice because it is very much sequence-dependent and conditioned by the question to be answered. Existing literature (9,12,13) shows the use of a variety of ion pairings with different degrees of attributed strength determined by hydrophobic/hydrophilic characteristics, sometimes driven by the number of carbon atoms contained.

Hydrophobic alkylamines act as more efficient IP reagents because of their increased strength of adsorption to the stationary phase compared to less hydrophobic IP reagents. However, they also generate a decrease in MS signal intensity because of the formation of ion-pair adducts that are difficult to control or eliminate and have long-lasting environmental impacts. It is assumed that weak IP agents form a weaker interaction with the hydrophobic ligands of the stationary phase. Thus, analyte retention is mainly driven by hydrophobic interactions between the analytes and the stationary phase ligands, and this may be a good option for oligonucleotides bearing different types of modifications within the sequence. The use of moderate IP reagents is recommended to minimize or avoid adduct formation. The concentration for each specific IP reagent must be studied and optimized as high concentration values (>100 mM) can cause ion suppression during electrospray ionization.

When using HRMS, full chromatographic resolution, although always desired, is not mandatory as HRMS will be able to detect and identify low-abundance species that may coelute with the full-length product (FLP) or with other major impurities or degradation products. As a general principle, less-than-ideal chromatographic resolution may be acceptable if it enables higher sensitivity and accuracy. However, in cases of greater sample complexity, chromatographic conditions should also be optimized as needed to ensure comprehensive coverage of all impurities.

To what extent can IP-RP-ultrahigh-pressure liquid chromatography (UHPLC) conditions be tuned to differentiate sequence-related impurities (shortmers, deletions, or base-modified variants) vs. non-sequence-related degradants?

Chromatographically, some impurities, such as N-x shortmers from 5’ end truncation, result in signals eluting at decreasing retention times before the FLP. 5’ truncation can happen during production, while 3’ truncation occurs during storage or purification steps. The latter would be considered a degradation product instead of an impurity. N-x shortmers resulting from 3’ also elute at decreasing retention times before the FLP too. However, not all impurities or degradation products can be fully chromatographically resolved if the sample complexity is high. Still, the use of HRMS allows for their characterization with high accuracy and sensitivity. The most common impurity found was the presence of the phosphodiester (PO) in phosphorothioate (PS) oligonucleotides, followed by N-x impurities. PO impurity elutes very close to the FLP. Single-base losses were observed in low abundance for some of the studied molecules and they eluted under the main chromatographic peak too. Finally, addition sequences, mostly restricted to N+1 and N+2, eluted after the FLP.

It is also important to point out that regulatory bodies now claim that MS is required for detailed impurity characterization when full chromatographic resolution using other types of detectors, such as UV, is not possible.

How do common backbone (phosphorothioate vs. phosphodiester), sugar (2’-O modifications), and base modifications affect retention behavior and ionization efficiency during LC–HRMS analysis?

Backbone, sugar, and nucleobase modifications of small RNA-based therapeutics such as ASOs can impact their chromatographic separation and mass spectrometry ionization by altering polarity, charge, or hydrophobicity.

PS backbone is formed by replacing an oxygen with a sulfur atom, increasing hydrophobicity and potentially affecting retention behavior. Replacing the oxygen with the sulfur in the phosphodiester backbone introduces a chiral center at the phosphorus atom. This leads to the formation of diastereomers, which are different structural isomers that can cause peak splitting and reduced resolution in chromatography. Fully PS oligonucleotides would consist of millions of diastereomers, and only partial diastereomeric separation could be achieved. In some cases, it is beneficial to suppress the diastereomeric separation when the interest is on FLP and N-x separation of PS oligonucleotides, which is the case for IP-RP-HPLC using the combination of amines and fluoroalcohols as counter ions. However, it is a regulatory requirement to estimate the diastereoisomeric profile distribution, and in a quality control (QC) environment, batch-to-batch reproducibility must be assessed as differences in the profile may have an impact on product stability and safety. Emerging trends in this regard would use multidimensional separations by coupling techniques like hydrophilic interaction liquid chromatography (HILIC) to IP-RP or anion-exchange chromatography (AEX) (14).

Sugar modifications introduce new functional groups at the 2’ position of the ribose such as 2’-O-methyl (2-Ome), 2’-O-methoxyethyl (2-MOE), which increase the steric bulk around the sugar ring and can introduce or enhance hydrophobicity, potentially altering interaction with the stationary phase in reversed-phase chromatography and influencing retention times.

Base modifications, such as 5-methylcytosine, increase hydrophobicity, affecting retention primarily through changes in the oligonucleotide’s overall hydrophobic character and potentially influencing ionization because of alterations in the base’s electronic properties.

What separation or source-optimization strategies are most effective in minimizing in-source conversion artifacts (PO to PS changes) without compromising throughput?

As mentioned earlier, source parameters such as source-induced dissociation energy, source temperature, and gas temperature could create in-source impurities, so those parameters should be studied and optimized for the studied molecules. In the present work, chromatographic parameters and MS-optimized parameters were kept the same for a variety of small ssRNA-based therapies of different lengths and of different types of modifications, so throughput was not compromised.

How can the developed IP-RP-UHPLC–HRMS workflow be adapted for process-related impurity profiling in a QC or manufacturing environment, where robustness and speed are critical?

According to regulatory bodies, suitable state-of-the-art analytical methods with appropriate limits of detection (LOD) and limits of quantitation (LOQ) for the detection of CQAs and impurities are required, and, when possible, capable of resolving impurities from the parent molecule and from each other. However, it is acknowledged that full resolution of all individual product- or process-related impurities are usually not technically achievable with a single method due to sample complexity. In this regard, mass spectrometry is a suitable analytical tool for structure elucidation of oligonucleotides and impurity profiling (15).

The developed method could be applicable during manufacturing and QC environments, after a complete validation according to regulatory requirements, to ensure accuracy and reproducibility, including specificity, linearity, accuracy, precision, and LOD/LOQ, some of which have been evaluated for the studied molecules. The applicability of the method to other type of small RNA-based molecules, such as ssDNA-based therapeutics, could also be explored.

References

(1) Millán-Martín, S.; Guapo, F.; Carillo, S.; et al. Characterisation of Small RNA-based Therapeutics and Their Process Impurities by Fast and Sensitive Liquid Chromatography High Resolution Mass Spectrometry. JPBA 2026, 268, 117097. DOI: 10.1016/j.jpba.2025.117097

(2) Apfell, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Analysis of Oligonucleotides by HPLC−Electrospray Ionization Mass Spectrometry. Anal. Chem 1997, 69 (7) 1320–1325. DOI: 10.1021/ac960916h

(3) Liu, R.; Ruan, Y.; Liu, Z.; Gong, L. The Role of Fluoroalcohols as Counter Anions for Ion-pairing Reversed-phase Liquid Chromatography/High-resolution Electrospray Ionization Mass Spectrometry Analysis of Oligonucleotides. Rapid Commun. Mass Spectrom. 2019, 33 (7), 697–709. DOI: 10.1002/rcm.8386

(4) Gong, L. Comparing Ion-pairing Reagents and Counter Anions for Ion-pair Reversed-phase Liquid Chromatography/Electrospray Ionization Mass Spectrometry Analysis of Synthetic Oligonucleotides. Rapid Commun. Mass Spectrom. 2015, 29 (24), 2402–2410. DOI: 10.1002/rcm.7409

(5) Studzinska, S.; Rola, R.; Buszewski, B. The Impact of Ion-pairing Reagents on the Selectivity and Sensitivity in the Analysis of Modified Oligonucleotides in Serum Samples by Liquid Chromatography Coupled with Tandem Mass Spectrometry. JPBA 2017, 138, 146–152. DOI: 10.1016/j.jpba.2017.02.014

(6) Guimaraes, G. J.; Bartlett, M. G. The Critical Role of Mobile Phase pH in the Performance of Oligonucleotide Ion-Pair Liquid Chromatography–Mass Spectrometry Methods. Fut. Sci. OA 2021, 7 (10), FSO753. DOI: 10.2144/fsoa-2021-0084

(7) Sharma, V. K.; Glick, J.; Vouros, P. Reversed-phase Ion-pair Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry for Separation, Sequencing and Mapping of Sites of Base Modification of Isomeric Oligonucleotide Adducts Using Monolithic Column. J. Chrom. A 2012, 1245, 65–74. DOI: 10.1016/j.chroma.2012.05.003

(8) Gong, L.; McCullagh, J. S. O. Comparing Ion-pairing Reagents and Sample Dissolution Solvents for Ion-pairing Reversed-phase Liquid Chromatography/Electrospray Ionization Mass Spectrometry Analysis of Oligonucleotides. Rapid Commun. Mass Spectrom. 2013, 28 (4), 339–350. DOI: 10.1002/rcm.6773

(9) Basiri, B.; Murph, M. M.; Bartlett, M. G. Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides. J. Am. Soc. Mass Spectrom. 2017, 28 (8), 1647–1656. DOI: 10.1007/s13361-017-1671-6

(10) Roussis, S. G.; Cedillo, I.; Rentel, C. Automated Determination of Early Eluting Oligonucleotide Impurities Using Ion-pair Reversed-phase Liquid Chromatography High Resolution-Mass Spectrometry. Anal. Biochem. 2020, 595, 113623. DOI: 10.1016/j.ab.2020.113623

(11) Rentel, C.; Gaus, H.; Bradley, K.; et al. Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair-HPLC-MS. Nucleic Acid Ther. 2022, 32 (3), 206–220. DOI: 10.1089/nat.2021.0056

(12) Roussis, S. G.; Pearce, M.; Rentel, C. Small Alkyl Amines as Ion-pair Reagents for the Separation of Positional Isomers of Impurities in Phosphate Diester Oligonucleotides. J. Chrom. A 2019, 1594, 105–111. DOI: 10.1016/j.chroma.2019.02.026

(13) Li, N.; El Zahar, N. M.; Saad, J. G.; Van der Hage, E. R. E.; Bartlett, M. G. Alkylamine Ion-pairing Reagents and the Chromatographic Separation of Oligonucleotides. J. Chrom. A 2018, 1580, 110–119. DOI: 10.1016/j.chroma.2018.10.040

(14) Chen, T.; Tang, S.; Fu, Y.; Napolitano, J. G.; Zhang, K. Analytical Techniques for Characterizing Diastereomers of Phosphorothioated Oligonucleotides. J. Chrom. A 2022, 1678, 463349. DOI: 10.1016/j.chroma.2022.463349

(15) EMA guidelines on the development and manufacture of oligonucleotides, EMA/CHMP/CVMP/QWP/262313/202424; https://www.ema.europa.eu/en/documents/scientific-guideline/draft-guideline-development-manufacture-oligonucleotides_en.pdf

Silvia Millán-Martín is a senior research scientist at the National Institute for Bioprocessing Research and Training (NIBRT) in Dublin, Ireland. She collaborates with Thermo Fisher Scientific to develop innovative applications for the characterization of biopharmaceuticals. Her work encompasses a broad range of chromatographic techniques, ensuring comprehensive analytical solutions for the scientific and biopharmaceutical industries. Her extensive background in analytical chemistry and her dedication to advancing biopharmaceutical characterization make her a leading expert in her field. In recent years, her work has focused on the multi-attribute method (MAM) approach and the application of advanced LC–MS platforms to support the development of new therapeutic modalities.