Proteins: Thermal Unfolding

Abstract

Thermal unfolding represents an important tool to evaluate protein stability and is extensively used in the development of protein‐based products. In order to assess the protein preference to maintain its folded (active) conformation, the protein is exposed to increasing levels of denaturing stress (temperature in the case of thermal unfolding) and protein stability is determined based on the stress level required to produce a significant fraction of unfolded protein. This chapter provides a description of theory and experimental technique, as well as multiple examples of the application of protein thermal unfolding in the development of therapeutic protein products. The first part describes several experimental techniques used for data acquisition and thermodynamic models needed to perform data analysis of thermal unfolding. The following section provides a detailed description of the factors that affect the thermal stability: protein structure, presence of ligands or excipients, and solvent conditions. Finally, examples of thermal unfolding studies used to support various stages of product development are presented, including protein molecular design, manufacture and purification process development, and stable formulation selection. Thermal unfolding in combination with results provided by other techniques can also be used in high‐throughput screening for a variety of applications and for protein characterization.

An erosion‐type hydrolysis behavior of insoluble protein fraction from Chlorella protothecoides

“…For example, protein fibrils from whey protein isolate were produced by acid treatment at pH 2 for 20 h at 90 °C . For soluble proteins, heat treatment may lead to an unfolding that makes the protein backbone more accessible to attack by acids . In a previous study, we found that an insoluble protein‐rich fraction from Chlorella protothecoides was resistant to acid hydrolysis at room temperature when subjected to a treatment with 0.05–6 mol L −1 HCl .…”