Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns

Abstract

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl₂) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Keywords: aggregates; fragments; messenger RNA; size exclusion chromatography; ultra-wide pore.

Figure 1

Evaluation of various packing materials with pore sizes ranging from 450 to 2500 Å for the analysis of RNA-based samples. Experiments were performed with the RNA ladder, using a mobile phase made of Tris and KCl at pH 7.5. The flow rate was 0.1 mL/min and 260 nm UV detection.

Figure 2

Evaluation of MgCl₂ addition (2 and 10 mM MgCl₂) to the mobile phase for three different samples, namely, RNA ladder, EGFP mRNA (996 nt), and Cas9 mRNA (4521 nt).

Figure 3

Analysis of Cas9 mRNA sample using a mobile phase consisting of 50 mM Tris, 200 mM KCl, and 10 mM MgCl₂ at pH 7.5. In this case, different column lengths were tested: a single column of 30 cm (A,C) or two 30 cm columns coupled in series (B) and different flow rates: 0.1 mL/min (A,B) or 0.05 mL/min (C).

Figure 4

Peak parking experiments performed with Cas9 mRNA (overlay of the 30 min elution window for each chromatogram) in the absence (blue traces) and in the presence of MgCl₂ (green traces). The values 0′, 10′, 20′, 40′, and 60′ correspond to 0, 10, 20, 40, and 60 min of flow set at 0 mL/min during the analysis (flow was stopped 10 min after the injection).

Figure 5

Analysis of 12 different mRNA samples on a 30 cm column with 50 mM Tris, 200 mM KCl, at a flow rate of 0.05 mL/min under two conditions differing by the presence of additive (blue traces, no additive; green traces, +10 mM MgCl₂).

Figure 6

Relative amount of LMWS and HMWS (expressed in %) in several mRNA samples in presence and absence of MgCl₂ in the mobile phase.

Figure 7

Comparison of unstressed vs. heat stressed at 80 °C for 10 min, or at 40 °C for 4 h for EGFP mRNA and Cas9 mRNA samples. The samples were analyzed on a 30 cm SEC column with 50 mM Tris, 200 mM KCl, and 10 mM MgCl₂ at a flow rate of 0.05 mL/min.

Figure 8

Analysis of EGFP and cas9 mRNA samples obtained from two different providers (Providers A and B). The samples were analyzed on a 30 cm SEC column with 50 mM Tris, 200 mM KCl, and 10 mM MgCl₂ at a flow rate of 0.05 mL/min.

Figure 9

Normalized chromatograms for mRNA samples of different sizes showing fast SEC separations using 4.6 × 150 mm column and 0.25 mL/min flow rate performed at ambient temperature (A, green traces) and 50 °C (B, red traces). The mobile phase was composed of 50 mM Tris, 200 mM KCl, and 10 mM MgCl₂.