Separation of Long-stranded RNAs by RP-HPLC Using an Octadecyl-based Column with Super-wide Pores

Overview

Journal Anal Sci

Publisher Springer

Specialty Chemistry

Date 2022 Dec 24

PMID 36566342

Authors

Tomomi Kuwayama

Makoto Ozaki

Motoshi Shimotsuma

Tsunehisa Hirose

Affiliations

Soon will be listed here.

Abstract

Messenger ribonucleic acids (mRNAs) have been used in vaccines for various diseases and are attracting attention as a new pharmaceutical paradigm. The purification of mRNAs is necessary because various impurities, such as template DNAs and transcription enzymes, remain in the crude product after mRNA synthesis. Among the various purification methods, reversed-phase high-performance liquid chromatography (RP-HPLC) is currently attracting attention. Herein, we optimized the pore size of the packing materials, the mobile phase composition, and the temperature of the process; we also evaluated changes in the separation patterns of RNA strands of various lengths via RP-HPLC. Additionally, single-stranded (50-1000 nucleotides in length) and double-stranded (80-500 base pairs in length) RNAs were separated while their non-denatured states were maintained by performing the analysis at 60 °C using triethylammonium acetate as the mobile phase and octadecyl-based RNA-RP1 with super-wide pores (> 30 nm) as the column. Furthermore, impurities in a long-stranded RNA of several thousand nucleotides synthesized by in vitro transcription were successfully separated using an RNA-RP1 column. The columns used in this study are expected to separate various RNA strands and the impurities contained in them.

Citing Articles

Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography.

Silva-Santos A, Rosa S, Marques M, Azevedo A, Prazeres D ACS Omega. 2024; 9(21):22619-22624.

PMID: 38826531 PMC: 11137683. DOI: 10.1021/acsomega.3c10533.

Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

Camperi J, Lippold S, Ayalew L, Roper B, Shao S, Freund E Anal Chem. 2024; 96(9):3886-3897.

PMID: 38377434 PMC: 10918618. DOI: 10.1021/acs.analchem.3c05539.

Neoantigen vaccine nanoformulations based on Chemically synthesized minimal mRNA (CmRNA): small molecules, big impact.

Imani S, Tagit O, Pichon C NPJ Vaccines. 2024; 9(1):14.

PMID: 38238340 PMC: 10796345. DOI: 10.1038/s41541-024-00807-1.

Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity.

Gilar M, Doneanu C, Gaye M Anal Chem. 2023; 95(38):14308-14316.

PMID: 37696042 PMC: 10535021. DOI: 10.1021/acs.analchem.3c02552.

References

Jansen Y, Kruse V, Corthals J, Schats K, Van Dam P, Seremet T . A randomized controlled phase II clinical trial on mRNA electroporated autologous monocyte-derived dendritic cells (TriMixDC-MEL) as adjuvant treatment for stage III/IV melanoma patients who are disease-free following the resection of macrometastases. Cancer Immunol Immunother. 2020; 69(12):2589-2598. PMC: 11027452. DOI: 10.1007/s00262-020-02618-4. View

Huang Z, Jayaseelan S, Hebert J, Seo H, Niu L . Single-nucleotide resolution of RNAs up to 59 nucleotides by high-performance liquid chromatography. Anal Biochem. 2013; 435(1):35-43. PMC: 5577504. DOI: 10.1016/j.ab.2012.12.011. View

Wagner B, Schuster S, Boyes B, Shields T, Miles W, Haynes M . Superficially porous particles with 1000Å pores for large biomolecule high performance liquid chromatography and polymer size exclusion chromatography. J Chromatogr A. 2017; 1489:75-85. PMC: 5360106. DOI: 10.1016/j.chroma.2017.01.082. View

McCarthy S, Gilar M, Gebler J . Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA. Anal Biochem. 2009; 390(2):181-8. DOI: 10.1016/j.ab.2009.03.042. View

Kis Z, Kontoravdi C, Dey A, Shattock R, Shah N . Rapid development and deployment of high-volume vaccines for pandemic response. J Adv Manuf Process. 2021; 2(3):e10060. PMC: 7361221. DOI: 10.1002/amp2.10060. View

Wetzel C, Limbach P . Mass spectrometry of modified RNAs: recent developments. Analyst. 2015; 141(1):16-23. PMC: 4679475. DOI: 10.1039/c5an01797a. View

Franklin R . Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc Natl Acad Sci U S A. 1966; 55(6):1504-11. PMC: 224351. DOI: 10.1073/pnas.55.6.1504. View

Close E, Nwokeoji A, Milton D, Cook K, Hindocha D, Hook E . Nucleic acid separations using superficially porous silica particles. J Chromatogr A. 2016; 1440:135-144. PMC: 4801196. DOI: 10.1016/j.chroma.2016.02.057. View

Freyn A, Silva J, Rosado V, Bliss C, Pine M, Mui B . A Multi-Targeting, Nucleoside-Modified mRNA Influenza Virus Vaccine Provides Broad Protection in Mice. Mol Ther. 2020; 28(7):1569-1584. PMC: 7335735. DOI: 10.1016/j.ymthe.2020.04.018. View

10.

Apffel A, Chakel J, Fischer S, Lichtenwalter K, Hancock W . Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry. Anal Chem. 2011; 69(7):1320-5. DOI: 10.1021/ac960916h. View

11.

Granados-Riveron J, Aquino-Jarquin G . Engineering of the current nucleoside-modified mRNA-LNP vaccines against SARS-CoV-2. Biomed Pharmacother. 2021; 142:111953. PMC: 8299225. DOI: 10.1016/j.biopha.2021.111953. View

12.

Qin S, Tang X, Chen Y, Chen K, Fan N, Xiao W . mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022; 7(1):166. PMC: 9123296. DOI: 10.1038/s41392-022-01007-w. View

13.

Lim M, Xia Y, Bettegowda C, Weller M . Current state of immunotherapy for glioblastoma. Nat Rev Clin Oncol. 2018; 15(7):422-442. DOI: 10.1038/s41571-018-0003-5. View

14.

Bagge J, Enmark M, Lesko M, Lime F, Fornstedt T, Samuelsson J . Impact of stationary-phase pore size on chromatographic performance using oligonucleotide separation as a model. J Chromatogr A. 2020; 1634:461653. DOI: 10.1016/j.chroma.2020.461653. View

15.

Baiersdorfer M, Boros G, Muramatsu H, Mahiny A, Vlatkovic I, Sahin U . A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA. Mol Ther Nucleic Acids. 2019; 15:26-35. PMC: 6444222. DOI: 10.1016/j.omtn.2019.02.018. View

16.

Yamauchi Y, Taoka M, Nobe Y, Izumikawa K, Takahashi N, Nakayama H . Denaturing reversed phase liquid chromatographic separation of non-coding ribonucleic acids on macro-porous polystyrene-divinylbenzene resins. J Chromatogr A. 2013; 1312:87-92. DOI: 10.1016/j.chroma.2013.09.021. View

17.

Fritz H, Belagaje R, Brown E, Fritz R, Jones R, Lees R . High-pressure liquid chromatography in polynucleotide synthesis. Biochemistry. 1978; 17(7):1257-67. DOI: 10.1021/bi00600a020. View

18.

Lukavsky P, Puglisi J . Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA. 2004; 10(5):889-93. PMC: 1370578. DOI: 10.1261/rna.5264804. View

19.

Fountain K, Gilar M, Gebler J . Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom. 2003; 17(7):646-53. DOI: 10.1002/rcm.959. View

20.

Baden L, El Sahly H, Essink B, Kotloff K, Frey S, Novak R . Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2020; 384(5):403-416. PMC: 7787219. DOI: 10.1056/NEJMoa2035389. View