Storing proteins under low temperature (T) conditions is recommended as it favors the protein native state and slows aggregation events during months or even years (e.g. under refrigerated conditions). Accelerated stability testing is a standard approach to estimate the time required for protein degradation and is generally performed under “hot” T (HT) stress. For instance, the time required for protein monomer loss (Δm) within initial rate conditions, t80 (Δm ≤ 20% loss), is the most relevant for a commercial shelf-life prediction. Quantifying aggregation rates under sub-freezing T using an isochoric method (liquid state) has shown promising results as cold T (CT) and/or pressure (P) stress can also shift the thermodynamic equilibrium and increase the fractional unfolded population. Nevertheless, the combined effect of P vs CT conditions on protein denaturation have been poorly characterized. A model protein alpha-Chymotripsinogen A (aCgn) at neutral pH was chosen as a means to interpret simple folding transition systems under CT and high P and bridge the effectiveness of isochoric methods to protein stability predictions. In situ SANS measurements under CT and high P environments show that high-P effects can be reasonably disregarded for the present system. Similar findings were also observed for aCgn at lower pH conditions and Ovalbumin (Ova) at neutral pH. The partial conformational changes observed for Ova seem to be mainly due to the proximity to the cold denaturation T rather than a P effect. The results show that for aCgn at neutral pH the isochoric method can provide more realistic shelf life predictions with less extreme T extrapolations and negligible pressure induced denaturation.

Improving aggregation rate predictions under low temperature storage conditions using an isochoric method

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.