Protein stabilization in spray drying and solid-state storage by using a ‘molecular lock’ - exploiting bacterial adaptations for industrial applications

Small, stable biomedicines, like peptides and hormones, are already available on the market as spray dried formulations, however large biomolecules like antibodies and therapeutic enzymes continue to pose stability issues during the process. Stresses during solid-state formation are a barrier to formulation of large biotherapeutics as dry powders. Here, we explore an alternative avenue to protein stabilisation during the spray drying process, moving away from the use of excipients. In thermophilic proteins, the presence of C-termini extensions can add to their stability by increasing molecular rigidity. Hence, we explored a unique thermostable amino acid extension in the C-terminal of an aldehyde dehydrogenase tetramer originating from Thermus thermophilus HB27 (ALDHTt), and its ability to stabilise the large enzyme against drying stresses. The presence of the C-terminal extension was found to act like a ‘molecular lock’ of the oligomeric state of the ALDH tetramer upon spray drying. Removal of the extension, mimicking the structure of mesophilic ALDHs, promoted the formation of aggregates and dissociative states. The ALDH protein with the ‘molecular lock’ retained ∼24% more activity after spray drying and retained up to 16% more activity during solid state storage than its mutant. We proposed a mechanism for the protection of oligomeric proteins by the distinct C-terminal extension under stresses involved in solid formation. Additionally, the process of spray drying an excipient-free ALDH is achieved using a design of experiments approach, increasing its breadth of application in the biocatalysis of aldehydes.