Abstract
Differential scanning fluorimetry (DSF) is successfully used as a high-throughput screening method for the analysis of the protein melting temperature (T(m)) in the development of therapeutic monoclonal antibody (MAb) formulations. Typically, surfactants are utilized in MAb formulations as a stabilizer, but the commonly applied polarity-sensitive dye SYPRO® Orange shows bright fluorescence in the presence of micelles, concealing the signal of protein unfolding. Studying various MAb formulations containing polysorbate 20, polysorbate 80, or poloxamer 188 (PX 188), the molecular rotor probe 4-(dicyanovinyl)julolidine (DCVJ) was investigated. Although limited to higher MAb concentrations, DCVJ enabled the determination of T(m) in many formulations where SYPRO® Orange failed. It is important to note that careful background correction of placebo formulations is essential for the precise determination of T(m) and especially T(m onset). Thermal shifts of T(m1) (lowest observed thermal transition) indicating stabilizing or destabilizing effects of pH or excipient were in good agreement across all tested formulations and correlated well with differential scanning calorimetry measurements. Additionally, the micellization temperature of PX 188 was confirmed, which leads to a nonproteinous transition. With this new method, it is possible to apply DSF during the development of therapeutic proteins in surfactant-containing formulations.
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