Protein aggregation is a common problem during the manufacture and storage of proteins. To ensure maintenance of their biological activity and to avoid degradation, rigorous conditions for biopharmaceutical storage are usually necessary. As part of the drug development process, regulators require stability studies that attest the product’s continuous safety and efficacy through its proposed shelf-life.
Stability of biopharmaceutical formulations is generally evaluated by accelerated stability studies. The most common approach to accelerate the degradation process consists in increasing temperature (20 ºC to 40ºC the molecule’s maximum stability temperature) and then modeling degradation kinetics at high temperature (by approaching Tm) and extrapolating the mechanisms to calculate rates at typical storage temperatures (much lower). However, these actual accelerated methods have major limitations, such as its restriction to temperatures far above the freezing point, which raises many concerns to establish correspondences to stability under low temperature storage and particularly frozen storage.
At SmartFreeZ we developed the Cold Stability Acceleration (CSA) method that complements the generalized approach for accelerated studies and helps to understand the impact of cold stresses on proteins. Several factors, such as the interaction with ice–liquid interfaces and/or freeze concentration of the protein and solutes as well as crystallization of solutes, are associated with the aggregation of proteins during freezing and frozen storage. To better understand the contribution of temperature, solutes concentration and ice-liquid interface, these variables must be controlled. Therefore, the ideal would be able to lower the temperature without freezing, i.e. lower the temperature without the presence of ice and all its implications (ice-liquid interface, freeze concentration, etc). The CSA method avoids freezing of the solution, enabling to measure degradation kinetics below the normal freezing temperature down to -25°C. Protein unfolding due to cold denaturation accelerates aggregation kinetics enabling to evaluate in a few days the impact of temperature and cryopreservatives on formulations. Moreover, the stability measurements obtained with the CSA method shed light on protein behavior over a broad temperature range (including frozen storage temperatures), enabling consistent models and avoiding the uncertainty associated to extrapolations.
SmartFreeZ also uses the CSA method to perform stability studies of biopharmaceutical formulations to its customers using a small volume, helping them to anticipate aggregation during processing and storage, improve stability and extend shelf life of protein therapeutics.