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In-situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress



Diana Gomes, Susana C. Marujo Teixeira, Juscelino Leao, Vladimir Razinkov, Wei Qi, Miguel Rodrigues, Christopher Roberts


Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice−liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as −15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near −10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (TCD ∼ −17 °C; calculated via isothermal chemical denaturation and Gibbs−Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near −5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.
Molecular Pharmaceutics


high pressure, low temperature, protein unfolding, small angle neutron scattering, cold storage, accelerated degradation, monoclonal antibodies


Gomes, D. , Marujo Teixeira, S. , Leao, J. , Razinkov, V. , Qi, W. , Rodrigues, M. and Roberts, C. (2021), In-situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress, Molecular Pharmaceutics, [online],, (Accessed July 23, 2024)


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Created November 26, 2021, Updated November 29, 2022