Fluorescence-based thermal stability screening is concentration-dependent and varies with protein size

Abstract

Proteins are used in many areas including diagnostic and therapeutical applications. Screening protein stability is an essential step for production, pharmacokinetic/pharmacodynamic studies, and determination of storage conditions. Instability of proteins can cause serious problems such as activity loss and unexpected adverse effects, so determination of sensitive and reliable methods for protein stability measurement is crucial. There are several “gold-standard” protein stability tests such as differential scanning calorimetry (DSC), but they are usually not suitable for high-throughput settings and consume large amounts of proteins. Instead, more high-throughput methods such as fluorescent-based assays can be used and validated to make stability screening process more straight-forward, easier, and lower-cost. Here, two methods were systemically compared to see whether their measurements depended on protein sizes. DSC and Sypro Orange dye-based fluorescent assay were compared for various proteins with different sizes and quaternary structures. This is the first systemic comparison of these two methods for thermal stability testing for different ranges of proteins in the literature. It was shown that protein melting temperature (Tm) measured by fluorescent assay highly depends on protein concentration and protein size. Larger proteins with multi-domain structures such as monoclonal antibodies gave more deviated and lower than expected Tms compared to small proteins. It has been concluded that fluorescent-based thermal stability assays are more suitable for smaller proteins, but protein concentrations used are still needed to be optimized in their settings for more reliable results.