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• Active thioredoxin of 90% purity and 95% recovery was purified by HEA HyperCel chromatography. • HEA HyperCel had salt-tolerant and more selective binding than Capto adhere and Toyopearl NH2-750F mixed-mode resins. • Process optimization by high-throughput screening and scale-up performance was evaluated. • Molecular dynamics simulations provide protein-ligand interactions aiding process development. [ABSTRACT FROM AUTHOR]

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the cause of the COVID-19 pandemic that originated in China in December 2019. Although extensive research has been performed on SARS-CoV-2, the binding behavior of spike (S) protein and receptor binding domain (RBD) of SARS-CoV-2 at different environmental conditions have yet to be studied. The objective of this study is to investigate the effect of temperature, fatty acids, ions, and protein concentration on the binding behavior and rates of association and dissociation between the S protein and RBD of SARS-CoV-2 and the hydrophobic aminopropylsilane (APS) biosensors using biolayer interferometry (BLI) validated with molecular dynamics simulation. Our results suggest three conditions—high ionic concentration, presence of hydrophobic fatty acids, and low temperature—favor the attachment of S protein and RBD to hydrophobic surfaces. Increasing the temperature within an hour from 0 to 25 °C results in S protein detachment, suggesting that freezing can cause structural changes in the S protein, affecting its binding kinetics at higher temperature. At all the conditions, RBD exhibits lower dissociation capabilities than the full-length S trimer protein, indicating that the separated RBD formed stronger attachment to hydrophobic surfaces compared to when it was included in the S protein. [ABSTRACT FROM AUTHOR]