Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA.

Modified messenger RNA (mRNA) represents a rapidly emerging class of therapeutic drug product. Development of robust stability indicating methods for control of product quality are therefore critical to support successful pharmaceutical development. This paper presents an ion-pair reversed-phase liquid chromatography (IP-RPLC) method to characterise modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product. The optimised method could be used for separation and analysis of large RNA, sized up to 1000 nucleotides. Column temperature, mobile phase flow rate and ion-pair selection were each studied and optimised. Baseline separations of the model RNA ladder sample were achieved using all examined ion-pairing agents. We established that the optimised method, using 100 mM Triethylamine, enabled the highest resolution separation for the largest fragments in the RNA ladder (750/1000 nucleotides), in addition to the highest overall resolution for the selected modified mRNA compound (eGFP mRNA, 996 nucleotides). The stability indicating power of the method was demonstrated by analysing the modified eGFP mRNA, upon direct exposure to heat, hydrolytic conditions and treatment with ribonucleases. Our results showed that the formed degradation products, which appeared as shorter RNA fragments in front of the main peak, could be well monitored, using the optimised method, and the relative stability of the mRNA under the various stressed conditions could be assessed.

Using phosphoglucose isomerase-deficient (pgi1Δ) Saccharomyces cerevisiae to map the impact of sugar phosphate levels on D-glucose and D-xylose sensing.

BACKGROUND: Despite decades of engineering efforts, recombinant Saccharomyces cerevisiae are still less efficient at converting D-xylose sugar to ethanol compared to the preferred sugar D-glucose. Using GFP-based biosensors reporting for the three main sugar sensing routes, we recently demonstrated that the sensing response to high concentrations of D-xylose is similar to the response seen on low concentrations of D-glucose. The formation of glycolytic intermediates was hypothesized to be a potential cause of this sensing response. In order to investigate this, glycolysis was disrupted via the deletion of the phosphoglucose isomerase gene (PGI1) while intracellular sugar phosphate levels were monitored using a targeted metabolomic approach. Furthermore, the sugar sensing of the PGI1 deletants was compared to the PGI1-wildtype strains in the presence of various types and combinations of sugars. RESULTS: Metabolomic analysis revealed systemic changes in intracellular sugar phosphate levels after deletion of PGI1, with the expected accumulation of intermediates upstream of the Pgi1p reaction on D-glucose and downstream intermediates on D-xylose. Moreover, the analysis revealed a preferential formation of D-fructose-6-phosphate from D-xylose, as opposed to the accumulation of D-fructose-1,6-bisphosphate that is normally observed when PGI1 deletants are incubated on D-fructose. This may indicate a role of PFK27 in D-xylose sensing and utilization. Overall, the sensing response was different for the PGI1 deletants, and responses to sugars that enter the glycolysis upstream of Pgi1p (D-glucose and D-galactose) were more affected than the response to those entering downstream of the reaction (D-fructose and D-xylose). Furthermore, the simultaneous exposure to sugars that entered upstream and downstream of Pgi1p (D-glucose with D-fructose, or D-glucose with D-xylose) resulted in apparent synergetic activation and deactivation of the Snf3p/Rgt2p and cAMP/PKA pathways, respectively. CONCLUSIONS: Overall, the sensing assays indicated that the previously observed D-xylose response stems from the formation of downstream metabolic intermediates. Furthermore, our results indicate that the metabolic node around Pgi1p and the level of D-fructose-6-phosphate could represent attractive engineering targets for improved D-xylose utilization.