The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case

The remarkable impact of mRNA vaccines on mitigating disease and improving public health has been amply demonstrated during the COVID-19 pandemic. Many new mRNA-based vaccine and therapeutic candidates are in development, yet the current reality of their stability limitations requires their frozen storage. Numerous challenges remain to improve formulated mRNA stability and enable refrigerator storage, and this review provides an update on developments to tackle this multi-faceted stability challenge. We describe the chemistry underlying mRNA degradation during storage and highlight how lipid nanoparticle (LNP) formulations are a double-edged sword: while LNPs protect mRNA against enzymatic degradation, interactions with and between LNP excipients introduce additional risks for mRNA degradation. We also discuss strategies to improve mRNA stability both as a drug substance (DS) and a drug product (DP) including the (1) design of the mRNA molecule (nucleotide selection, primary and secondary structures), (2) physical state of the mRNA-LNP complexes, (3) formulation composition and purity of the components, and (4) DS and DP manufacturing processes. Finally, we summarize analytical control strategies to monitor and assure the stability of mRNA-based candidates, and advocate for an integrated analytical and formulation development approach to further improve their storage, transport, and in-use stability profiles

Physicochemical and Biopharmaceutical Characterisation of Small Drug Molecules by Capillary Electrophoresis

Capillary Electrophoresis (CE) was explored as a means for physicochemical and biopharmaceutical characterisation of small drug molecules. Special attention was paid to the characterisation of acid-base and lipophilic properties of drug compounds by analysing their migration behaviour in different CE systems. The thesis comprises an overview of the field together with separate studies on the different topics. The utility of CE for the determination of pKa of labile drug compounds was investigated. A general methodology was developed comprising key steps such as the use of a stabilising sample diluent, electromigration injection, and analyte characterisation by UV-Vis spectroscopy. The methodology was successfully applied for two sets of drug compounds, labile at low and high pH, respectively. CE was also evaluated for experimental modelling of passive intestinal membrane permeability by studying analyte migration in liposomal, microemulsion and micellar electrolytes. Good correlation is reported between CE migration and Caco-2 cell absorption estimates and for in vitro inhibition of thrombin. Interestingly, a slightly better correlation was obtained for liposomal electrolytes. The utility of liposomes in CE was further extended by developing a novel procedure for immobilising liposomes inside fused silica capillaries. This approach enabled direct on-line coupling of liposome CE to high sensitivity mass spectrometry. The utility of liposome-coated capillaries is demonstrated for estimating drug passive intestinal membrane permeability. Its use in biopharmaceutical drug profiling is discussed. Utilising advanced molecular descriptors, commonly applied to in silico prediction of passive intestinal membrane permeability, migration of analytes in micellar CE systems could be well predicted. The novel approach was based on hierarchical multivariate analytics and use of molecular descriptors for both analytes and micellar media surfactants. Demonstrated results propose that the CE format could be useful to validate how representative molecular descriptors are for describing molecular behaviour in complex liquid media, e.g. physiological systems

Physicochemical and Biopharmaceutical Characterisation of Small Drug Molecules by Capillary Electrophoresis

Capillary Electrophoresis (CE) was explored as a means for physicochemical and biopharmaceutical characterisation of small drug molecules. Special attention was paid to the characterisation of acid-base and lipophilic properties of drug compounds by analysing their migration behaviour in different CE systems. The thesis comprises an overview of the field together with separate studies on the different topics. The utility of CE for the determination of pKa of labile drug compounds was investigated. A general methodology was developed comprising key steps such as the use of a stabilising sample diluent, electromigration injection, and analyte characterisation by UV-Vis spectroscopy. The methodology was successfully applied for two sets of drug compounds, labile at low and high pH, respectively. CE was also evaluated for experimental modelling of passive intestinal membrane permeability by studying analyte migration in liposomal, microemulsion and micellar electrolytes. Good correlation is reported between CE migration and Caco-2 cell absorption estimates and for in vitro inhibition of thrombin. Interestingly, a slightly better correlation was obtained for liposomal electrolytes. The utility of liposomes in CE was further extended by developing a novel procedure for immobilising liposomes inside fused silica capillaries. This approach enabled direct on-line coupling of liposome CE to high sensitivity mass spectrometry. The utility of liposome-coated capillaries is demonstrated for estimating drug passive intestinal membrane permeability. Its use in biopharmaceutical drug profiling is discussed. Utilising advanced molecular descriptors, commonly applied to in silico prediction of passive intestinal membrane permeability, migration of analytes in micellar CE systems could be well predicted. The novel approach was based on hierarchical multivariate analytics and use of molecular descriptors for both analytes and micellar media surfactants. Demonstrated results propose that the CE format could be useful to validate how representative molecular descriptors are for describing molecular behaviour in complex liquid media, e.g. physiological systems

Physicochemical and Biopharmaceutical Characterisation of Small Drug Molecules by Capillary Electrophoresis

Capillary Electrophoresis (CE) was explored as a means for physicochemical and biopharmaceutical characterisation of small drug molecules. Special attention was paid to the characterisation of acid-base and lipophilic properties of drug compounds by analysing their migration behaviour in different CE systems. The thesis comprises an overview of the field together with separate studies on the different topics. The utility of CE for the determination of pKa of labile drug compounds was investigated. A general methodology was developed comprising key steps such as the use of a stabilising sample diluent, electromigration injection, and analyte characterisation by UV-Vis spectroscopy. The methodology was successfully applied for two sets of drug compounds, labile at low and high pH, respectively. CE was also evaluated for experimental modelling of passive intestinal membrane permeability by studying analyte migration in liposomal, microemulsion and micellar electrolytes. Good correlation is reported between CE migration and Caco-2 cell absorption estimates and for in vitro inhibition of thrombin. Interestingly, a slightly better correlation was obtained for liposomal electrolytes. The utility of liposomes in CE was further extended by developing a novel procedure for immobilising liposomes inside fused silica capillaries. This approach enabled direct on-line coupling of liposome CE to high sensitivity mass spectrometry. The utility of liposome-coated capillaries is demonstrated for estimating drug passive intestinal membrane permeability. Its use in biopharmaceutical drug profiling is discussed. Utilising advanced molecular descriptors, commonly applied to in silico prediction of passive intestinal membrane permeability, migration of analytes in micellar CE systems could be well predicted. The novel approach was based on hierarchical multivariate analytics and use of molecular descriptors for both analytes and micellar media surfactants. Demonstrated results propose that the CE format could be useful to validate how representative molecular descriptors are for describing molecular behaviour in complex liquid media, e.g. physiological systems

Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides

This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation