Investigation of the thermal shift assay and its power to predict protein and virus stabilizing conditions

Publication date: Available online 9 August 2018Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Dora Sviben, Branimir Bertoša, Andrea Hloušek-Kasun, Dubravko Forcic, Beata Halassy, Marija BrglesAbstractProtein thermal shift assay (TSA) has been extensively used in investigation of protein stabilization (for protein biopharmaceutics stabilization, protein crystallization studies or screening of recombinant proteins) and drug discovery (screening of ligands or inhibitors). This work aimed to analyze thermal shift assay results in comparison to protein polymerization (multimerization and aggregation) propensity and test the most stabilizing formulations for their stabilization effect of enveloped viruses. Influence of protein concentration, buffer pH and molarity was tested on three proteins (immunoglobulin G, ovalbumin, and albumin) and results showed that each of these factors has an impact on determined shift in protein melting point Tm, and the impact was similar for all three proteins. In case of ovalbumin, molecular dynamics simulations were performed with the goal to understanding molecular basis of protein’s thermal stability dependence on pH. Effect of three denaturing agents in a wide concentration range on Tm showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on Tm for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing p...
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research