0.0017% (w/v)) compared to polysorbate 20 (ca. IgG formulations. == Electronic supplementary material == The online version of this article (doi:10.1007/s11095-009-0020-2) contains supplementary material, which is available to authorized users. KEY WORDS:aggregation, fluorescence, fluorescent molecular rotors, monoclonal antibodies, size-exclusion chromatography == INTRODUCTION == Surfactants like polysorbate 20 and 80, also known as Tween 20 or 80, are commonly used excipients in formulations of therapeutic proteins. The main function of the amphiphilic polysorbates is to prevent protein adsorption at liquid-liquid, liquid-solid or liquid-air interfaces, which can lead to surface-induced denaturation and aggregation (1). A protective effect of LY 345899 polysorbates on protein stability has been shown during freeze-thawing (2), freeze-drying (3), mechanical stress (e.g. agitation, shaking or stirring (2,4,5)), and reconstitution of dried protein preparations (6), as well as for formulations containing silicone oil droplets (5). However, polysorbates can also negatively affect stability, e.g. at quiescent conditions during long-term stability (7). Furthermore, polysorbates can undergo various degradation reactions, which can lead to a loss of its stabilizing properties and chemical modifications of proteins, such as oxidation (8). PIK3CD Almost 70% of the marketed monoclonal antibody formulations contain polysorbate 20 or polysorbate 80 as stabilizing excipients (9,10). Within those commercial preparations, the polysorbate concentrations range between 0.001% (w/v) polysorbate 80 (Reopro) (11) and 0.16% (w/v) polysorbate 20 (Raptiva) (12), with most formulations containing about 0.005 to 0.02% polysorbate 20 or 80. One difference between the polysorbates is the lower critical micelle concentration of polysorbate 80 (ca. 0.0017% (w/v)) compared to polysorbate 20 (ca. 0.007% (w/v)) (8). Polarity-responsive extrinsic fluorescent dyes, like Bis-ANS, ANS or Nile Red, can enable the sensitive detection of aggregated and structurally changed protein molecules (13), which can be particularly useful during formulation screening (14). Aggregates composed of structurally perturbed monomers are more prone to interact with those dyes, than aggregates composed of native-like monomers (15). The underlying aggregation mechanism and aggregate properties, like size, can play a role as well (16), as they are often related to changes in polarity. In previous papers, we demonstrated that fluorescent dyes, above all Bis-ANS, are highly sensitive to detect heat-induced aggregation in IgG formulations, both in steady-state fluorescence spectroscopy and as dye detection in size-exclusion chromatography (HP-SEC) (15,17). From an analytical perspective, LY 345899 it needs to be considered that polysorbates will most likely interfere with this approach, as the fluorescent properties of dyes like (Bis-)ANS or Nile Red are highly sensitive to the polarity of the environment (13). Therefore, the use of these polarity-sensitive dyes can be rendered impractical in the presence of polysorbates, as dye-surfactant interactions will lead to high background fluorescence. In fact, the interaction of polarity-sensitive fluorescent dyes with surfactants is utilized to determine surfactant content and critical micelle concentration (1821). Unlike Bis-ANS, ANS and Nile Red, the fluorescent properties of fluorescent molecular rotors, such as DCVJ and CCVJ, are mainly sensitive to changes in the viscosity of the environment and less to polarity (2224). Based on this, molecular rotors have been used for non-mechanical determination of fluid viscosity (25). Publications on LY 345899 applications of fluorescent molecular rotors in the field of protein aggregate characterization are rare compared to the polarity-sensitive dyes (Bis-)ANS and Nile Red, and are so far limited to polysorbate-free formulations (15,16,23). The aim of this study was to evaluate the ability of the fluorescent molecular rotors DCVJ and CCVJ to detect aggregation in polysorbate-containing IgG formulations, in comparison with the polarity-sensitive dyes ANS, Bis-ANS and Nile Red. We demonstrate that DCVJ and CCVJ are well-suited to identify protein aggregates in thermally stressed polysorbate-containing IgG formulations by.