Evaluation of ion pairing reversed-phase liquid chromatography for the separation of large RNA molecules
Journal of Chromatography A, Volume 1740, 2025: Fig. 7. Chromatograms obtained for the eight RNA from the RiboRuler High Range RNA Ladder during a 20 min linear optimized gradient run. Ion-pairing agents are added in both mobile phases. Peaks have been labelled with the corresponding number of nucleotides.
The goal of this study is to advance the precise characterization of RNA, particularly for large molecules up to 6000 nucleotides, by optimizing ion-pair reversed-phase liquid chromatography (IP-RPLC). This includes a systematic evaluation of 13 ion-pairing agents (IPAs) with varying hydrophobicity, using a supermacroporous polymeric column under different conditions such as temperature, pH, and IPA concentrations. The study identifies optimal separation conditions, with moderately hydrophobic IPAs providing superior resolution and an optimized combination of butylammonium acetate and tripropylammonium acetate improving resolution by 35%.
Additionally, the research develops a method to retain and separate small nucleotides and large RNA molecules on combined LC columns, enabling the characterization of in-vitro transcribed (IVT) mRNA, assessment of integrity and fragmentation, and monitoring of process impurities. This study significantly enhances the analytical methods available for evaluating RNA quality attributes, supporting the development of mRNA-based therapeutics and addressing critical needs in RNA therapeutic production post-COVID-19.
The original article
Evaluation of ion pairing reversed-phase liquid chromatography for the separation of large RNA molecules
Jonathan Maurer, Camille Malburet, Marc François-Heude, Davy Guillarme
Journal of Chromatography A, Volume 1740, 11 January 2025, 465574
https://doi.org/10.1016/j.chroma.2024.465574
licensed under CC-BY 4.0
Selected sections from the article follow. Formats and hyperlinks were adapted from the original.
Abstract
The rapid development of mRNA-based therapeutics, especially post-COVID-19, has necessitated the precise characterization of mRNA quality attributes, including sequence integrity. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) has been widely accepted as a reference method for the characterization of small oligonucleotides. Some studies have already investigated the use of IP-RPLC for RNA, but no systematic approach has been developed to assess the impact of ion-pairing agents (IPAs) on the separation of large RNA molecules. This study addresses this gap by investigating the potential of IP-RPLC for the separation and characterization of large RNA molecules, with a specific focus on optimizing the use of IPAs to enhance retention and selectivity. Thirteen different IPAs, varying in hydrophobicity, were systematically tested using a supermacroporous polymeric (divinylbenzene) column with a very broad pore size range under various conditions, including different temperatures, pH, and IPA concentrations. The results demonstrate that moderately hydrophobic IPAs provide superior resolution for RNA species up to 6000 nucleotides. An optimized combination of 100 mM butylammonium acetate and 50 mM tripropylammonium acetate achieved the best overall separation, significantly improving resolution by 35% compared to individual IPAs. The study also identifies optimal conditions for RNA separation, including a mobile phase pH of 7.0, acetonitrile as the organic solvent, and a column temperature of 65 °C. In a second step, a solution to increase the retention of small nucleotides and thereby separate nucleic acids ranging from 1 to 6000 nucleotides allowing to characterize IVT-mRNA differing in length and study their integrity and fragmentation or monitor the presence of in-process impurities (nucleotides) was investigated by combining two different LC columns. These findings enhance the analytical toolbox for evaluating the critical quality attributes of RNA, supporting the development of reliable and efficient RNA-based therapeutics.
1. Introduction
The COVID-19 pandemic shed new lights on the use of mRNA as effective vaccines, thus accelerating new developments for prophylactic and therapeutic applications [1]. With the growing number of mRNA-based medicines in development – several hundred mRNA drugs are in the clinical pipeline worldwide – there is an urgent need to fully characterize the mRNA produced by in vitro transcription (IVT) by evaluating specific critical quality attributes (CQAs) [2].
Some of these CQAs are directly related to the sequence itself, such as integrity of mRNA (% of fragments versus intact sequence, 3′-poly(A) tail length, 5′-capping efficiency), sequence conformity, and mRNA content. Capillary gel electrophoresis (CGE) under denaturing conditions, to disrupt base-pairing interactions to improve retention and peak shapes, is commonly employed to assess these attributes [3,4]. With the aim of a more precise characterization, alternative strategies such as size exclusion chromatography (SEC), anion exchange chromatography (AEX), or ion-pairing reversed phase liquid chromatography (IP-RPLC) are being developed [[5], [6], [7]].
The effectiveness of these methods is often significantly influenced by advancements in column technology. Indeed, large RNAs require low-binding materials like high-performance surface (HPS) coating, PEEK, or any other kind of bioinert columns due to their sensitivity to adsorption. Moreover, pore size plays a critical role for proper analysis, since large RNA can reach up to 3000 kDa, and are not adequately analyzed with columns having traditional pore sizes of 100–300 Å, not matching RNA hydrodynamic radii. This results in suboptimal performance, because the accessibility of the stationary phase and the diffusion are reduced, limiting the interactions and negatively impacting the mass transfer and kinetic efficiency [[8], [9], [10]]. Finally, newly developed stationary phases, such as divinylbenzene-based columns and supermacroporous polymer resin offer enhanced performance for large RNA and exhibit exceptional resistance to harsh pH and temperature conditions. The integration of these innovations is essential for achieving effective separation and developing robust methods for large RNA analysis [11].
In this context, ultra-wide pore SEC columns were suggested as a good alternative to CGE [9,12]. However, SEC suffers several drawbacks, such as susceptibility to unwanted physicochemical interactions requiring low-adsorption columns, its filtering-only mechanism, and other technical and physical limitations due to filtration effects and shear forces [13]. As an alternative, AEX utilizes the negatively charged phosphate groups in the RNA structure to retain RNA on positively charged stationary phases. Accordingly, AEX appears as a very good choice for the separation of large RNA. Despite this, only few applications are reported, mainly on small oligonucleotides (ONs), as larger RNA often displaying broad peaks, poor resolution and non-negligible carry-over effects. This could be due to AEX's non-denaturing conditions, which allow RNA to retain its secondary structure conformation, thus decreasing accessibility to the phosphate groups. Additionally, AEX, as SEC, are poorly compatible with mass spectrometry (MS), compared to other techniques due to the presence of non-volatile salts in the mobile phase [14]. However, this is not a real issue for intact mRNA analysis, since MS analysis of such molecules is not readily feasible with electrospray ionization due to the large size of mRNA molecules and the multiple charging states and adducts that generate highly complex convoluted spectra and very limited sensitivity.
On the other hand, IP-RPLC offers a great potential, since it takes profit of the RNA negatively charged backbone that complexes with the positively charged alkylamine ion-pairing agent (IPA) added to the mobile phase [15]. The RNA-ion pair complex displays an increased affinity for the stationary phase compared to the naked RNA, enhancing retention on a hydrophobic column, which is further improved by harsh denaturing conditions. This makes possible the separation of RNA species based on size and sequence, with the possibility to tune the retention by varying the IPA used. While IP-RPLC has been extensively studied for small ONs analysis [[16], [17], [18], [19], [20], [21], [22], [23]], its application for large RNA analysis remains largely underexplored [14]. Only a few applications have been reported, mostly using triethylammonium acetate (TEA) as IPA [15,[24], [25], [26], [27], [28], [29]]. Other authors advocate for the use of a mixture of IPAs, such as the combination of TEA with propylamine [30], or with dibutylammonium acetate [31]. While combining IPAs appears to be a promising strategy for enhancing resolution, there is currently no systematic published study to validate this approach, and there has been no comprehensive study on the behavior of large RNAs in IP-RPLC conditions.
To address this gap, we systematically explored the effect of thirteen IPA with different hydrophobicities on the retention and selectivity of RNAs, using the recently commercialized Thermo DNAPac RP column. The ion-pairing agents were evaluated alone or in combination, at temperatures varying from 45 to 85 °C, with pH from 6 to 8, and concentrations from 25 mM to 200 mM. In a second step, we further investigated a solution to increase the retention of small nucleotides. Finally, our optimized method successfully separated oligonucleotides containing between 1 and 6000 units, expanding the toolbox to comprehensively evaluate CQAs of newly produced RNA.
2. Experimental
2.3. UHPLC instrumentation, columns and experimental conditions
The chromatographic measurements were performed on an ACQUITY UPLC H-class system (Waters, Milford, MA, USA), equipped with a 10 µl flow through needle injector, a quaternary solvent manager (QSM) equipped with a 380 µl mixing chamber, a column manager module, and an ACQUITY photodiode array detector. UV was set at a wavelength of 260 nm. Data acquisition and instrument control were performed by Empower 3 software (Waters). Resolution (Rs) was calculated using the European Pharmacopeia equation using peak widths at half height (1).(1)
\(Rs = \frac{1.18(Rt_2- Rt_1)}{(W_2 + W_1)}\)
Where Rt2 and Rt1 are the retention times of the peak 2 and 1, respectively, and W2 + W1 the sum of peak widths at 50% peak height.
Several chromatographic columns were employed in this work, namely Thermo DNAPac RP 2.1×100 mm 4 µm column, which is a supermacroporous column made of 4μm divinylbenzene polymer resin with a broad range of pore sizes, specifically designed for the separation of small and large oligonucleotides [11], and Waters ACQUITY BEH C18 300 Å columns of 2.1×5, 20, and 100 mm.
The physico-chemical properties of IPAs used in this study are summarized in Table 1. The mobile phases were always composed of an equimolar concentration of IPA and acetic acid. In this study, hexafluoro-2-propanol (HFIP) was not considered as an alternative to acetic acid, since HFIP is typically used to boost ESI-MS sensitivity and it is not appropriate to analyze large mRNA. Mobile phase A was composed of pure water or 30% acetonitrile, depending on the solubility of the IPAs in the mobile phase. The pH was measured in the mobile phase A (in absence of organic solvent). For the initial screening, pH was adjusted to 7.0 (± 0.1) by adding acetic acid. The mobile phase B was composed of 75% to 95% acetonitrile, depending on the IPAs hydrophobicity. The mobile phase B was supplemented with the same concentrations of IPA and acetic acid as in mobile phase A, to prevent the formation of an IPA gradient. The flow rate was set at 0.4 mL/min and column temperature at 65 °C. A generic gradient having a composition range of 45% ACN in 20 minutes was always used to have reliable comparison of retention. In a second instance, the gradient conditions (initial and final compositions) were optimized for each IPA, while the gradient time was kept at 20 minutes.
Ion-pairing agents, particularly those with higher hydrophobicity, can exhibit poor solubility in water. To enhance solubility, it is advisable to add a small percentage of an organic solvent to the mobile phase, with sonication further helping dissolution. Also, a thorough column equilibration is needed prior to analysis, allowing sufficient time for the ion-pairing agent to bind to the stationary phase. We used a minimum equilibration time of 10 column volumes. Finally, mobile phases containing IPAs are stable for several weeks, but to maintain homogeneity, we shook the bottles briefly each day.
IPAs are persistent and can remain in columns and tubing for weeks. To avoid cross-contamination, it is advisable to dedicate columns solely for use with IPAs. Additionally, traditional washes are ineffective at removing IPAs; therefore, we used a specific wash protocol between each change of mobile phase conditions: a 45-minute wash at 65 °C, at 0.2 mL/min using a 1:1:1:1 mixture of isopropanol, methanol, acetonitrile, and water, with 0.1% pentafluoropropionic acid, followed by a 10-minute wash with 100% acetonitrile. This cleaning method efficiently removes positively charged IPAs, as demonstrated in Figures S1 and S2, where a ladder and a surrogate analyte (ibuprofen) were used, respectively. It resulted in clean chromatograms and reliable analyses. When this cleaning procedure was implemented, we did not experience anymore loss of sensitivity and resolution, or baseline drift when changing the nature of IPA. While we cannot prove that our washing procedure completely removes the remaining IPAs out of the system, we can assume with high confidence that their impact on RNA retention is extremely limited. This wash mixture is also highly effective for cleaning IPAs from mobile phase bottles, particularly for hydrophobic agents like dihexylamine that tend to adsorb onto glass surfaces.
4. Conclusion
The objective of this study was to assess the effects of different IPAs on RNA separation under IP-RPLC conditions and to develop a method capable of separating nucleic acids ranging from 1 to 6000 nucleotides. To identify the optimal conditions, 13 IPAs were tested using a Thermo DNAPac RP column of 2.1×100mm. The results demonstrated that moderately hydrophobic IPAs at concentrations between 100 and 200 mM are the most effective additives for enhancing RNA retention and resolution. Additionally, combining a weakly hydrophobic IPA with a moderately hydrophobic one further improved resolution, by taking advantage of the complementary properties of the IPAs. The best resolution was achieved using 100 mM BA and 50 mM TPA in both mobile phases, allowing effective separation of RNA across a wide range of sizes. However, under these conditions, nucleoside triphosphates exhibited poor retention, which is problematic, since these molecules are used as starting material in IVT RNA production. To address this, we combined the Thermo DNAPac RP column of 2.1×100mm with a Waters Acquity BEH C18 300 Å of 2.1×20 mm as a post-column. This setup achieved good retention and resolution of a ladder composed of 22 nucleic acids ranging from 1 to 6000 nucleotides, resulting in an efficient and robust method for evaluating RNA integrity as well as for monitoring IVT reactions at the core of RNA-based therapeutics.
