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Physical instability of aqueous drug solutions, such as precipitation upon storage, has so far been difficult to predict or model. Understanding the molecular basis of such phenomena can help mitigate by influencing the product composition and by providing a mechanistic basis of experimental and in silico investigations. In this study, inconsistent precipitation of a model drug, GNE-01 in aqueous solutions was investigated. Chromatographic analyses of the drug solution that showed precipitation upon storage versus the one that did not indicate lack of covalent modification or degradation of the drug, suggesting that the precipitation was a physical phenomenon. Molecular level investigations were conducted using surface tension measurement and nuclear magnetic resonance (NMR) spectroscopy. The studies revealed self-association of the weakly basic drug in solution at slightly acidic pH values which was strengthened by the presence of polyionic excipients. The role of polyionic excipients in facilitating drug precipitation on storage was indicative of shifting solution equilibria in favor of a lower solubility drug-excipient complex. This study highlighted the importance of molecular understanding in mitigating difficult to predict physical instability of self-associating drugs in solution., Competing Interests: Declaration of Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Monoclonal antibody (mAb) therapies are rapidly growing for the treatment of various diseases like cancer and autoimmune disorders. Many mAb drug products are sold as prefilled syringes and vials with liquid formulations. Typically, the walls of prefilled syringes are coated with silicone oil to lubricate the surfaces during use. MAbs are surface-active and adsorb to these silicone oil-solution interfaces, which is a potential source of aggregation. We studied formulations containing two different antibodies, mAb1 and mAb2, where mAb1 aggregated more when agitated in the presence of an oil-water interface. This directly correlated with differences in surface activity of the mAbs, studied with interfacial tension, surface mass adsorption, and interfacial rheology. The difference in interfacial properties between the mAbs was further reinforced in the coalescence behavior of oil droplets laden with mAbs. We also looked at the efficacy of surfactants, typically added to stabilize mAb formulations, in lowering adsorption and aggregation of mAbs at oil-water interfaces. We showed the differences between poloxamer-188 and polysorbate-20 in competing with mAbs for adsorption to interfaces and in lowering particulate and overall aggregation. Our results establish a direct correspondence between the adsorption of mAbs at oil-water interfaces and aggregation and the effect of surfactants in lowering aggregation by competitively adsorbing to these interfaces.

Proteins are surface-active molecules that have a propensity to adsorb to hydrophobic interfaces, such as the air-liquid interface. Surface flow can increase aggregation of adsorbed proteins, which may be an undesirable consequence depending on the application. As changes in protein conformation upon adsorption are thought to induce aggregation, the ability to measure the folded state of proteins at interfaces is of particular interest. However, few techniques currently exist to measure protein conformation at interfaces. Here we describe a technique capable of measuring the hydrophobicity, and therefore the conformation and folded state, of proteins at air-liquid interfaces by exploiting the environmentally sensitive fluorophore Nile red. Two monoclonal antibodies (mAbs) with high (mAb1) and low (mAb2) surface activity were used to highlight the technique. Both mAbs showed low background fluorescence of Nile red in the liquid subphase and at a glass-liquid interface. In contrast, at the air-liquid interface Nile red fluorescence for mAb1 increased immediately after protein adsorption, whereas the Nile red fluorescence of the mAb2 film evolved more slowly in time even though the adsorbed quantity of protein remained constant. The results demonstrate that hydrophobicity upon mAb adsorption to the air-liquid interface evolves in a time-dependent manner. Interfacial hydrophobicity may be indicative of protein conformation or folded state, where rapid unfolding of mAb1 upon adsorption would be consistent with increased protein aggregation compared to mAb2. The ability to measure protein hydrophobicity at interfaces using Nile red, combined with small sample requirements and minimal sample preparation, fills a gap in existing interfacial techniques.

Adsorption of antibody therapeutics to air-liquid interfaces can enhance aggregation, particularly when the solution does not contain protective surfactant or when the surfactant is diluted as occurs during preparation of intravenous infusion bags. The ability to predict an antibody's propensity for interfacially mediated aggregation is particularly useful during product development to ensure the quality, potency, and safety of the therapeutic. To develop a predictive tool, we investigated the surface pressure and surface excess of a panel of 16 antibodies as well as determined their aggregation propensity at the air-liquid interface in an agitation stress model. Our data demonstrated that the initial rate of surface pressure increase upon antibody adsorption to the air-liquid interface strongly predicted the extent of agitation-induced aggregation. Other factors, including the hydrophobicity, equilibrium surface pressure, and interfacial concentration of an antibody, were not adequate predictors of its susceptibility to aggregation. In addition to developing a predictive tool, we extended the interfacial characterization to better understand the mechanisms of antibody aggregation at an air-liquid interface during agitation stress. We believe that the kinetics of antibody rearrangement and conformational change after adsorbing to the interface, leading to the development of attractive antibody-antibody interactions, dictated the extent of aggregation. Overall, our results demonstrate how surface pressure measurements can be implemented as a rapid screening tool for the identification of antibodies with a high propensity to aggregate upon adsorption to an air-liquid interface while also furthering our understanding of interfacially mediated protein aggregation.

Contrast in confocal microscopy of phase-separated monolayers at the air-water interface can be generated by the selective adsorption of water-soluble fluorescent dyes to disordered monolayer phases. Optical sectioning minimizes the fluorescence signal from the subphase, whereas convolution of the measured point spread function with a simple box model of the interface provides quantitative assessment of the excess dye concentration associated with the monolayer. Coexisting liquid-expanded, liquid-condensed, and gas phases could be visualized due to differential dye adsorption in the liquid-expanded and gas phases. Dye preferentially adsorbed to the liquid-disordered phase during immiscible liquid-liquid phase coexistence, and the contrast persisted through the critical point as shown by characteristic circle-to-stripe shape transitions. The measured dye concentration in the disordered phase depended on the phase composition and surface pressure, and the dye was expelled from the film at the end of coexistence. The excess concentration of a cationic dye within the double layer adjacent to an anionic phospholipid monolayer was quantified as a function of subphase ionic strength, and the changes in measured excess agreed with those predicted by the mean-field Gouy-Chapman equations. This provided a rapid and noninvasive optical method of measuring the fractional dissociation of lipid headgroups and the monolayer surface potential.