A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.

Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.

Stabilization of Ostwald ripening in low molecular weight amino lipid nanoparticles for systemic delivery of siRNA therapeutics.

Lipid nanoparticles (LNPs) represent the most clinically advanced technology for the systemic delivery of therapeutic siRNA in vivo. Toward this end, a novel class of LNPs comprising low molecular weight (MW) ionizable amino lipids having asymmetric architecture was recently reported.1 LNPs of these amino lipids, termed asymmetric LNPs, were shown to be highly efficacious and well tolerated in vivo; advances were enabled by improved endosomal escape, coupled with enhanced amino lipid metabolism and clearance. In this work, we show that, in contrast to their desirable pharmacological performance, asymmetric LNPs present a significant pharmaceutical developability challenge, namely physical instability limiting extended shelf life. Using orthogonal characterization methods, we identify the mechanism of LNP instability as Ostwald ripening and establish it to be driven predominantly by the asymmetric amino lipid component. Through rational optimization of LNP physical and macromolecular properties, we are able to significantly attenuate or entirely eliminate the Ostwald ripening instability. Modulation of LNP size, for example, effectively halts particle growth. Similarly, optimization of LNP macromolecular packing through deliberate selection of structurally matched colipids significantly diminishes the rate of ripening. This later experimental observation is substantiated by molecular dynamics simulations of LNP self-assembly, which establish a quantitative dependence of LNP macromolecular order on colipid structure. In totality, the experimental and molecular dynamics outcomes of this work support the rational design of LNP physical and chemical properties leading to effective Ostwald ripening stabilization and enable the advance of asymmetric LNPs as a clinic-ready platform for siRNA therapeutics.

Investigation of two-dimensional high performance liquid chromatography approaches for reversed phase resolution of warfarin and hydroxywarfarin isomers.

Several offline and online 2D HPLC methods were investigated for the reversed phase resolution of a complex mixture of closely related warfarin and hydroxywarfarin isomers. By combining reversed phase achiral/chiral HPLC separation with UV-triggered fraction collection and subsequent chiral/achiral reversed phase HPLC analysis of collected fractions, complete resolution of all 12 components of the mixture was possible. In addition, a faster method was developed from online 2D HPLC analysis where multicomponent fractions from the first dimension are simultaneously chromatographed in the second dimension.

Stability and compatibility of reconstituted ertapenem with commonly used i.v. infusion and coinfusion solutions.

PURPOSE: The stability of ertapenem sodium in various commonly used i.v. infusion solutions and its compatibility with coinfusion solutions was studied. METHODS: Ertapenem was reconstituted with sterile water for injection and then diluted with various commercial i.v. infusion solutions to concentrations of 10 and 20 mg/mL. The solutions were stored in flexible polyvinyl chloride containers at 4 and 25 degrees C and in sterile glass vials at -20 degrees C. The drug's stability at 4 degrees C was monitored daily for up to 10 days, at 25 degrees C at appropriate hourly intervals for up to 30 hours, and at -20 degrees C. The daily for up to 14 days. Compatibility with the coinfusion solutions was monitored for up to eight hours at room temperature. Stability assays were conducted until the ertapenem concentration decreased by 10% or the corresponding degradation products exceeded the approved specifications. Ertapenem concentrations were determined by a stability-indicating high-performance liquid chromatography assay. RESULTS: Ertapenem was more stable in solutions stored at 4 degrees C versus 25 degrees C. Samples frozen at -20 degrees C showed extreme variability. Ertapenem 10 mg/mL was stable for a longer time than at the 20-mg/mL concentration. Ertapenem demonstrated the greatest stability in 0.9% and 0.225% sodium chloride solutions. CONCLUSION: Ertapenem sodium injection 10 and 20 mg/mL are relatively stable in sodium chloride injections and Ringer's solution when stored at 25 and 4 degrees C, but are unstable in mannitol and dextrose solutions. The drug can be coinfused with hetastarch, heparin sodium, and potassium chloride over several hours.