Controlled water content for evaluation of denaturation temperature of freeze-dried enzymes

Water content and storage temperature play an important role in determining the thermal stability and activity retention of biomolecules, mainly in freeze-dried proteins; for this reason, the study of the effect of both properties on the thermal stabilization of proteins is paramount to the biopharmaceutical industry. A parameter used to evaluate thermal stability of proteins is the denaturation temperature (Td), reported extensively for a large number of proteins. Although it is known that Td values are strongly dependent on moisture, water content is not necessarily considered or deliberately controlled during denaturation measurement for solid samples. In the present work a method to evaluate Td under controlled water (ca. 6.5%) was implemented and tested for solid lysozyme to overcome the limitations encountered during the determination of Td. The implemented method can be used for quality control purposes for solid protein products since it allows an objective comparison of samples differing in provenance.