DEVELOPMENT OF A SOLVATOCHROMIC TOOL TO CLASSIFY PHARMACEUTICAL EXCIPIENTS BY POLARITY: INVESTIGATING SOLVENT POLARITY ON PROTEIN LIQUID FORMULATIONS VIA SOLVATOCHROMISM

Therapeutic proteins have emerged as a major class of pharmacotherapies involved in the mitigation of an increasing number of maladies. However, protein formulations pose challenges in terms of their physicochemical stability and the need for complex, time consuming methods for characterization. For instance, it is well known that the stability of a protein at a specified pH can vary as a function of the buffer utilized. This rather common phenomenon, which extends to other excipients besides buffers, is not well understood. Current efforts in protein formulation development predominantly rely on extensive vehicle screening. In this thesis, it is hypothesized that excipients alter the aqueous environment surrounding proteins through electrostatic and polarity effects, subsequently impacting protein functionality. One critical question is whether it is possible to experimentally detect differences in polarity exerted by buffers and excipients under typical conditions, in which the solvent consists of 99+% water and very low concentrations of all combined additives. To address this question, a solvatochromic-based approach was developed in this investigation and found to be able to detect electrostatic differences between similar salts, such as NaCl and KCl, in equimolar concentrations.

Further investigation explored whether the solvatochromic method could detect differences produced by solution chemistry effects that are much weaker than electrostatic effects, specifically, polarity effects on the solvent exerted by non-electrolytes. It was found that the solvatochromic method can differentiate between closely related non-electrolytes such as trehalose and maltose, both glucose disaccharides with different linkages for the glucose subunits (Chapter 2). To understand the implications of these polarity effects on protein functionality, the enzyme activity was assessed using various enzymes as model compounds in the presence of different excipient systems. Changes in enzyme activity were observed in response to the excipients present in solution. Multivariate analysis, integrating electrostatic/polarity effects from excipients alongside enzyme activity data, revealed that specific polarity regions correlate with optimal activity for a given protein (Chapter 3). These findings suggest that protein formulation screening can be efficiently streamlined, potentially leading to a more rational development of stable protein therapeutic formulations.

Finally, solvatochromism was used as an initial polarity assessment tool for non-polar compounds, revealing differences in the microenvironment of structurally similar hydrocarbons, such as cis- and trans- decalin. Despite their comparable molecular structures, these isomers exhibited distinct solvatochromic responses, indicating variations in their effective polarity. These findings are particularly significant for further exploration in the aviation fuel industry, where subtle differences in hydrocarbon composition can impact fuel performance, including energy density and combustion efficiency (Chapter 4). These results highlight the potential of solvatochromism as a highly sensitive analytical technique, showing its potential for detecting subtle variations in molecular environments.