Role of surfactants in the stabilization of protein formulations

Abstract

Surfactants are widely used in protein formulations to mitigate interfacial stresses. In aqueous solutions, the stabilization effect of surfactants is brought out by competitively replacing proteins from surface adsorption, and by interacting with proteins to reduce their aggregation propensity. There are three surfactants used in protein products – polysorbate 20 and 80 (PS20 and PS80) as well as poloxamer 188 (P188). The first objective of this thesis was to compare the stabilization effect of these surfactants on a model protein, lactate dehydrogenase (LDH). The dynamic surface tension analysis results showed that polysorbates were more effective than P188 in preventing LDH surface adsorption and effectively stabilized LDH against mechanical stress during shaking. However, based on near UV circular dichroism results, polysorbates perturbed LDH higher order structure through hydrophobic interaction and accelerated protein destabilization during quiescent incubation, while P188 did not have such an adverse effect. In frozen and freeze-dried protein formulations, surfactants are used to prevent ice surface-induced protein destabilization. Thus, the second objective was to investigate the stabilization effect of surfactants in frozen protein solutions, and to study the impact of surfactant phase transformation (i.e., crystallization) on protein stability. The phase behavior of surfactants was studied using synchrotron X-ray diffractometry and differential scanning calorimetry. Similar to polysorbates, P188 was effective in preventing ice surface-induced LDH destabilization. However, P188 crystallization undermined its stabilization function when used at a low concentration. The addition of noncrystallizing solutes, such as protein and sugar, by inhibiting surfactant crystallization, improved its function as a stabilizer in frozen solutions. Overall, P188 can be an alternative surfactant to polysorbates in preventing ice surface-induced protein destabilization. In addition, the high crystallization propensity of P188 renders it an additional functionality as a bulking agent in freeze-dried formulations, ensuring elegant lyophiles. The third objective aims to explore the dual functionality of P188 – a stabilizer in frozen solutions and a bulking agent in lyophiles. During lyophilization, the surfactant substantially crystallized upon drying, providing elegant freeze-dried cakes. However, P188 alone did not function as a lyoprotectant and had to be used in combination with a sugar. Overall, the thesis highlights the importance of surfactants in stabilizing protein against interfacial stress, and the multifunctionality of P188, an alternative candidate to polysorbates, in frozen and freeze-dried formulations.