Maurer Jonathan, Malburet Camille, François-Heude Marc, Guillarme Davy
Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel Servet 1, 1211, Geneva, Switzerland; School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland; mRNA Center of Excellence, Analytical Sciences, Sanofi, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France.
mRNA Center of Excellence, Analytical Sciences, Sanofi, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France.
J Chromatogr A. 2025 Jan 11;1740:465574. doi: 10.1016/j.chroma.2024.465574. Epub 2024 Dec 2.
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.
基于mRNA的疗法的迅速发展，尤其是在新冠疫情之后，使得对mRNA质量属性（包括序列完整性）进行精确表征成为必要。离子对反相液相色谱法（IP-RPLC）已被广泛认可为表征小寡核苷酸的参考方法。一些研究已经探讨了IP-RPLC在RNA分析中的应用，但尚未开发出系统的方法来评估离子对试剂（IPA）对大RNA分子分离的影响。本研究通过考察IP-RPLC对大RNA分子的分离和表征潜力来填补这一空白，特别关注优化IPA的使用以提高保留率和选择性。使用具有非常宽孔径范围的超大孔聚合物（二乙烯基苯）柱，在包括不同温度、pH值和IPA浓度的各种条件下，系统地测试了13种不同疏水性的IPA。结果表明，中等疏水性的IPA为长达6000个核苷酸的RNA种类提供了卓越的分离度。100 mM乙酸丁铵和50 mM乙酸三丙铵的优化组合实现了最佳的整体分离效果，与单独使用IPA相比，分辨率显著提高了35%。该研究还确定了RNA分离的最佳条件，包括流动相pH值为7.0、乙腈作为有机溶剂以及柱温为65°C。在第二步中，通过组合两种不同的液相色谱柱，研究了一种提高小核苷酸保留率从而分离1至6000个核苷酸的核酸的方法，这使得能够表征长度不同的体外转录mRNA并研究其完整性和片段化情况，或者监测过程中杂质（核苷酸）的存在。这些发现增强了用于评估RNA关键质量属性的分析工具箱，为可靠且高效的基于RNA的疗法的开发提供了支持。
