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

Area of Science:

• Biophysical Chemistry
• Protein Science
• Pharmaceutical Formulation

Background:

• Biopharmaceutical freezing can cause protein damage via conformational changes and adsorption to ice interfaces.
• Directly measuring protein structure in sub-0 °C environments is challenging due to water freezing at ambient pressure.
• Existing analytical techniques often require extrapolation from higher temperatures, limiting cold-regime accuracy.

Purpose of the Study:

• To develop and apply in situ analytical methods for assessing protein conformational stability at sub-zero temperatures.
• To investigate the effects of low temperature and high pressure on protein structure and self-association.
• To provide a more direct method for characterizing biopharmaceutical behavior under cold stress.

Main Methods:

• Utilized small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) for in situ analysis.
• Employed a high-pressure (HP) environment (up to 3 kbar) to prevent water freezing at sub-zero temperatures (-15 °C to -5 °C).
• Analyzed protein tertiary structure, folding, and oligomerization under varied pressure, temperature, and pH (pD) conditions.

Main Results:

• α-chymotrypsinogen A (aCgn) maintained its structure under acidic conditions across tested pressures and temperatures.
• Ovalbumin exhibited reversible structural changes and oligomer formation near -10 °C under neutral conditions, linked to cold denaturation.
• Anti-streptavidin IgG1 (AS-IgG1) showed significant structural changes near -5 °C and 2 kbar, suggesting unfolding intermediates (e.g., molten globules).

Conclusions:

• The in situ high-pressure approach enables direct characterization of protein conformational stability under cold stress.
• This method assesses structural alterations, self-association, and reversibility, offering an alternative to indirect extrapolation methods.
• Findings are crucial for understanding and preventing biopharmaceutical degradation during cold storage and processing.