High Recovery Chromatographic Purification of mRNA at Room Temperature and Neutral pH

Abstract

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM^® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5-7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3-7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H₂O at room temperature.

Keywords: chromatographic monoliths; isoelectric point; liquid chromatography; mRNA stability; nucleic acids separation; platform purification; preparative chromatography; weak anion-exchanger.

Figure 1

Zeta potential of chromatographic monoliths as a function of pH.

Figure 2

Elution profile of 1 µg of eGFP mRNA from 0.1 mL CIMmic PrimaS disk (A) and 0.1 mL CIMmic Swiper disk (B) using Gradient 1. Solid line represents the absorbance measured at 260 nm, while dashed line represents pH trace.

Figure 3

Elution profiles showing the effect of anions on mRNA–pDNA separation using CIMmultus Swiper 1 mL column in pH gradient from 6.0 to 7.5. Gradient 7 (A) and Gradient 8 (B) were performed at approximately 6–7 mS/cm, while conductivity in Gradient 9 (C) was around 16 mS/cm. Solid line represents the absorbance measured at 260 nm, while dashed line represents pH trace.

Figure 4

Comparison of mRNA–pDNA separation in NaCl gradients at pH 6.0 ((A), Gradient 10) and pH 5.0 ((B), Gradient 11) using CIMmultus Swiper 1 mL column. Solid line represents the absorbance measured at 260 nm, while dashed line represents conductivity trace.

Figure 5

Separation of RNAs of different sizes from their respective linear DNA templates using Gradient 8 for eGFP mRNA ((A), size 995 nt), mFIX mRNA ((B), size 4000 nt) and saRNA ((C), size 10,000 nt). Solid line represents the absorbance measured at 260 nm, while dashed line represents pH trace.

Figure 6

mRNA purification from IVT reaction mixture using CIMmultus Swiper 1 mL column. Preparative chromatogram (A) and AGE analysis of the collected fractions (B). RR—RiboRuler High Range RNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA), L—load, FT—flow-through fraction, W—salt wash fraction, E—elution fraction, CIP—CIP fraction. AGE loading mass was 80 ng.

Figure 7

Stability study of eGFP mRNA purified with CIMmultus Swiper 800 mL column. AGE of the eGFP mRNA sample after purification (lane 0) and after storage at −80 °C (lane 1), −20 °C (lane 2), 4 °C (lane 3), room temperature (lane 4), and 37 °C (lane 5) for 34 days. RR—RiboRuler High Range RNA Ladder (Thermo Fisher Scientific).