Multi-step conformational transitions in heat-treated protein therapeutics can be monitored in real time with temperature-controlled electrospray ionization mass spectrometry

• Published in: Analyst (London) Vol. 143; no. 3; pp. 670 - 677
• Main Authors: Wang, Guanbo, Bondarenko, Pavel V, Kaltashov, Igor A
• Format: Journal Article
• Language: English
• Published: England Royal Society of Chemistry 07.02.2018
• Subjects: Agglomeration; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - therapeutic use; Antithrombins - chemistry; Antithrombins - therapeutic use; Biopolymers; Electrospraying; Free energy; Heat; Heat stress; Heat treatment; Hot Temperature; Human behavior; Humans; Immunoglobulin G - chemistry; Immunoglobulin G - therapeutic use; Ionization; Ions; Mass spectrometry; Monoclonal antibodies; Oligomerization; Protein Conformation; Protein Denaturation; Protein Stability; Proteins; Proteins - chemistry; Proteins - therapeutic use; Real time; Scientific imaging; Spectrometry, Mass, Electrospray Ionization; Spectroscopy; Thermal denaturation; Thermal stability
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c7an01655g

Heat-induced conformational transitions are frequently used to probe the free energy landscapes of proteins. However, the extraction of information from thermal denaturation profiles pertaining to non-native protein conformations remains challenging due to their transient nature and significant conformational heterogeneity. Previously we developed a temperature-controlled electrospray ionization (ESI) source that allowed unfolding and association of biopolymers to be monitored by mass spectrometry (MS) in real time as a function of temperature. The scope of this technique is now extended to systems that undergo multi-step denaturation upon heat stress, as well as relatively small-scale conformational changes that are precursors to protein aggregation. The behavior of two therapeutic proteins (human antithrombin and an IgG1 monoclonal antibody) under heat-stress conditions is monitored in real time, providing evidence that relatively small-scale conformational changes in each system lead to protein oligomerization, followed by aggregation. Temperature-controlled ESI MS is particularly useful for the studies of heat-stressed multi-domain proteins such as IgG, where it allows distinct transitions to be observed. The ability of native temperature-controlled ESI MS to monitor both the conformational changes and oligomerization/degradation with high selectivity complements the classic calorimetric methods, lending itself as a powerful experimental tool for the thermostability studies of protein therapeutics.