(464d) Correlating Surface Activity with Interface-Induced Aggregation in a High Concentration Mab Solution

Therapeutic proteins encounter multiple types of stress during manufacturing, storage, and administration. The presence of interfaces constitutes a known source of stress, which causes protein unfolding and ultimately leads to aggregation. Therefore, surfactants are often added to therapeutic drugs to armor interfaces, avoiding protein adsorption. Usually, best practices involve the addition of surfactants above their critical micelle concentration (CMC), meaning that the surfactant concentration is high enough to cover the air-solution interface and to produce micelles in the bulk. Among the commonly used surfactants are polysorbates such as PS20. Current industrial trends involve the formulation and use of high-concentration protein drugs, that present different physicochemical properties than dilute solutions such as much higher viscosity. Aggregation kinetics may involve multiple steps of unfolding, (re)folding, oligo and polymerization, and while previous studies with dilute protein solutions have shown that particle formation in the bulk of quiescent samples is dependent on protein concentration, studies involving high-concentration protein solutions are still incipient, particularly when evaluating interface-induced protein aggregation. Therefore, this work is focused on evaluating if high-concentration protein solutions pose an additional challenge in terms of protein stability. Initially, surface pressure measurements of mAb solutions at a wide range of concentrations were carried out using a tensiometer technique. Following this, samples were collected either from the bulk or aspirated from the interface to evaluate protein particle formation via microfluid imaging (MFI). Our results showed that after saturation of the interface, the maximum surface pressure of neat mAb solutions did not change, irrespective of bulk concentration. Further, particle counts increased linearly with bulk concentration even for a mAb solution at 170 mg/mL. Subsequent studies involved the addition of PS20 below and above CMC (25 and 200 ppm respectively, where CMC of PS20 is about 60 ppm) to mAb solutions. For those studies, we used two representative mAb concentrations: “low-concentration” solutions at 10 mg/mL, and “high-concentration” solutions at 170 mg/mL. We notice that for low-concentration solutions the addition of PS20 resulted in the predominance of surfactant molecules at the interface and that the particle counts significantly decreased by the addition of surfactant, reaching a 10-fold decrease above CMC. Conversely, high-concentration protein solutions resulted in the coadsorption of protein and surfactant molecules to the interface, especially when added by surfactant below CMC. However, for high-concentration protein formulation, particle counts were not lowered by PS20 presence, even at surfactant concentrations as high as 200 ppm (about three times CMC). For the high-concentration samples, the addition of a large excess of surfactant (2,000 ppm) was necessary to produce a reduction in protein particle formation, similar to the 200 ppm in low-concentration mAb samples. Our results contribute to the understanding that a minimum surfactant-to-protein ratio is necessary for surface protection by competitive adsorption, and the addition of surfactant solely based on its CMC may not be adequate when dealing with protein therapeutics at high concentrations.