mRNA and Oligonucleotides purification with chromatography
RNA based therapeutics represent a powerful new tool for gene therapy. In RNA production, impurities such as nucleotide triphosphates, abortive transcripts, and DNA template can affect down-stream applications by triggering immune responses. Gel-based methods of purification, such as preparative denaturing polyacrylamide gel electrophoresis (PAGE) and agarose gels, are limited by poor resolution of RNA size and potential chemical modifications from reagents such as formaldehyde.
To date, high performance liquid chromatography (HPLC) remains a staple method for purification of messenger RNA (mRNA) and RNA oligonucleotides. Presented here are two HPLC methods, a size exclusion-based method for the purification of long mRNA, and a reverse-phase method for the purification of oligonucleotides.
HPLC Purification of mRNA with Size Exclusion Chromatography
Figure 1. mRNA application by RNA-SEC-100.
Analyze and purify mRNA up to 5,000 bases !
Figure 2. Molecular weight determination for mRNA up to 5,000 bases
Analysis of Nucleotides by Reverse-Phase Chromatography
Application of ΦX174 HaeIII digest fragments using wide pore C18 column
>> Information on COSMOSIL RNA-RP1 (under construction）
Single base resolution at 40 nucleotides
Fast RNA analysis Using IP-RP HPLC with a Super-Wide Pore Stationary Phase
Presented here is the rapid analysis of large RNAs using Ion-Pair Reversed-Phase (IP-RP) chromatography with a Super-Wide Pore stationary phase. This method significantly reduces analysis time to less than 4 minutes, while maintaining high resolution and sensitivity. The Super-Wide Pore stationary phase provide increased surface area and improved accessibility to larger RNA molecule, allowing for improved chromatographic resolution. Additionally, the small column dimensions of 2.1 x 30 mm make it an ideal choice for high-throughput analysis.
Oligonucleotides Analysis by HILIC in the Absence of Ion-Pair Reagents
Oligonucleotides (ONs) have captured significant attention as promising therapeutics agents over the past decades, driving the need for effective analytical techniques. Liquid Chromatography-Mass Spectrometry (LC-MS) is the commonly used method for ONs analysis due to its high sensitivity and resolution. Ion-pair reversed-phase (IP-RP) HPLC is prevalently used for the separation of ONs. However, the use of ion-pairing reagents in IP-RP HPLC present several drawbacks, including the requirement for dedicated systems and increased complexity, as well as potential interference with MS analysis. Presented here is the use of Hydrophilic Interaction Liquid Chromatography (HILIC) as an alternative for ONs analysis, overcoming these drawbacks.
Application Data
COSMOSIL RNA-RP1 Application Data (12.15.2021)Product specifications
| Packing Material | RNA-RP1 | RNA-SEC-1000 | RNA-SEC-2000 |
|---|---|---|---|
| Separation mode | Reversed-phase | Size-exclusion | |
| Silica gel | Fully-porous high-purity spherical silica gel | ||
| Average particle size (µm) | 5 | ||
| Average pore size (nm, approx.) | — | 100 (1,000 Å) | 200 (2,000 Å) |
| Bonded phase | Octadecyl group | Hydrophilic group | |
| Suitable pH range | 2 – 7.5 | ||
| Maximum pressure (MPa) | 15 | ||
Reference:
| Paper: | Separation and purification of short-, medium-, and long-stranded RNAs by RP-HPLC using different mobile phases and C18 columns with various pore sizes |
| Author: | Makoto Ozaki, Tomomi Kuwayama, Motoshi Shimotsuma, Tsunehisa Hirose |
| Article: | Analytical Methods. 2024;16:1948-1956. DOI:https://doi.org/10.1039/D4AY00114A |
| Overview: |
Nucleic acids, which have been employed in medicines for various diseases, are attracting attention as a new pharmaceutical model. Depending on the target substances, nucleic acid medicines with various nucleic acid chain lengths (several tens of nucleotides [nt] to several thousands of nt) exist. The purification of synthesized nucleic acids is crucial as various impurities remain in the crude product after synthesis. Presently, reversed-phase high-performance liquid chromatography (RP-HPLC) represents an effective purification method for nucleic acids. However, the information regarding the HPLC conditions for separating and purifying nucleic acids of various chain lengths is insufficient. Thus, this technical note describes the separation and purification of short-, medium-, and long-stranded nucleic acids (several tens of nt to thousands of nt) by RP-HPLC with various mobile phases and octadecyl-based columns with various pore sizes, such as normal (9–12 nm), wide (30 nm), and super wide (>30 nm) pores.  |
| Paper: | Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores |
| Author: | Tomomi Kuwayama, Makoto Ozaki, Motoshi Shimotsuma & Tsunehisa Hirose |
| Article: | Analytical Sciences volume 39, 417-425 (2023) https://doi.org/10.1007/s44211-022-00253-w |
| Overview: | 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.
|
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Ordering Information
COSMOCORE 2.6C18
| Product | Cat.No. | PKG Size | Price | |
|---|---|---|---|---|
| COSMOCORE 2.6C18 packed column (2.1 mm I.D. x 100 mm) | 12614-71 | 1 pkg | 814.00 | Buy |
COSMOSIL RNA-RP1
| Product | Cat.No. | PKG Size | Price | |
|---|---|---|---|---|
| COSMOSIL RNA-RP1 Packed Column (2.0 mm I.D. x 30 mm) | 22524-41 | 1 pkg | 1,426.00 | Buy |
| COSMOSIL RNA-RP1 Packed Column (2.0 mm I.D. x 100 mm) | 21078-31 | 1 pkg | 1,584.00 | Buy |
| COSMOSIL RNA-RP1 Packed Column (4.6 mm I.D. x 100 mm) | 21079-21 | 1 pkg | 1,584.00 | Buy |
| COSMOSIL RNA-RP1 Packed Column (10.0 mm I.D. x 100 mm) | 21080-81 | 1 pkg | 3,724.00 | Buy |
COSMOSIL RNA-SEC-1000
| Product | Cat.No. | PKG Size | Price | |
|---|---|---|---|---|
| COSMOSIL RNA-SEC-1000 Packed Column (4.6 mm I.D. x 250 mm) | 21088-01 | 1 pkg | 2,119.00 | Buy |
| COSMOSIL RNA-SEC-1000 Packed Column (7.5 mm I.D. x 300 mm) | 19380-21 | 1 pkg | 2,996.00 | Buy |
| COSMOSIL RNA-SEC-1000 Guard Column (7.5 mm I.D. x 50 mm) | 20785-91 | 1 pkg | 1,017.00 | Buy |
COSMOSIL RNA-SEC-2000
| Product | Cat.No. | PKG Size | Price | |
|---|---|---|---|---|
| COSMOSIL RNA-SEC-2000 Packed Column (4.6 mm I.D. x 250 mm) | 21095-01 | 1 pkg | 2,119.00 | Buy |
| COSMOSIL RNA-SEC-2000 Packed Column (7.5 mm I.D. x 300 mm) | 19381-11 | 1 pkg | 2,996.00 | Buy |
| COSMOSIL RNA-SEC-2000 Guard Column (7.5 mm I.D. x 50 mm) | 21096-91 | 1 pkg | 1,017.00 | Buy |
SunShell HILIC Diol MTF
| Product | Cat.No. | PKG Size | Price | |
|---|---|---|---|---|
| SunShell HILIC Diol MTF (2.1 x 100 mm) | LL696MTF | 1 pkg | 777.00 | Buy |