Wide Pore Polymeric Reversed-Phase PLRP-S Columns for mRNA Analysis

Since the discovery of effective methods of delivery, messenger RNAs (mRNAs) have become increasingly popular in healthcare. These large, complex molecules are designed to allow cells to produce critical proteins and enzymes that can help fight disease. The synthesis of mRNA occurs in several stages: initiation, elongation, editing (processing), and termination. This procedure is very complex, and for this reason it is essential to carry out extensive quality control to ensure that the correct molecule has been created, or to detect the presence of impurities. Impurities might also be the results of product degradation during storage. Liquid chromatography is a well-established separation technique in this regard, but getting an effective analytical separation can prove challenging.

Analytical methods play a pivotal role in evaluating the integrity of RNA molecules. One common degradation pathway involves the fragmentation of large RNA molecules into smaller fragments. Techniques such as anion exchange (AEX), IP-RP, and size exclusion chromatography (SEC) are routinely employed to identify these impurities. Among these techniques, IP-RPLC is frequently favored, offering the highest resolution. IP-RP can offer the ability to characterize a wide range of RNA compounds through modification of chromatographic conditions, including choice of ion-pairing agents, column temperature, and mobile phase composition. Traditional alkyl-bonded silica stationary phases are limited to relatively small pore sizes, rendering them unsuitable for large mRNA molecules due to their size. In contrast, polymeric reversed-phase sorbents, such as the polystyrene/ divinylbenzene-based PLRP-S, are more advantageous and can be manufactured in significantly larger pore sizes, including 1,000 and 4,000 Å. In this application note, we investigate the difference in performance of 1,000 and 4,000 Å PLRP-S columns with two model mRNA compounds, Cas9 mRNA and EPO mRNA. Cas9 mRNA encodes the CRISPR-associated protein 9 and is 4521 nucleotides in length; EPO mRNA is only 855 nucleotides long.

In this application note, we explore some of the key parameters when developing an ion-pair reversed-phase (IP-RP) separation of mRNA. An IP-RP method was developed for purity determination of mRNA samples. Good elution and separation for mRNA samples of various lengths were obtained in a short run time and with limited carryover. The PLRP-S polymeric materials were an ideal choice, providing the best pore size for the mRNA under investigation. More specifically, using the Agilent PLRP-S 4000 Å column, high resolution and peak separation for both native and heated sample were easily obtained.