Rational development of protein formulations in solid and solution states

Abstract

Development of protein formulations in the solid and solution state involves stability studies during long-term storage (2-3 years). A long-term study of a protein under conditions leading to its rapid physical and chemical degradation often results in excessive use of resources and severe time constraints. To minimize these problems, a thorough preformulation study of protein behavior under different conditions is necessary. An in depth understanding of the properties of proteins in both the solution and solid state may subsequently result in selection of conditions leading to adequate stability during storage.

Preformulation studies of a protein in solution often involve a three step approach. In this method a protein is first characterized under a range of conditions (e.g. pH, temperature, etc.), and the data is then summarized in the form of an empirical phase diagram. This information is then used to design a high throughput screening approach to identify stabilizing compounds. This approach was employed for preformulation studies of vaccines against Clostridium difficile ( C. difficile)-associated disease. Such vaccines contain formaldehyde treated toxoids A and B in free or adjuvant bound form. Studies of C. difficile toxins and toxoids under a range of conditions revealed a stabilizing effect of formaldehyde crosslinking on the thermal stability of the toxoids. Furthermore, screening for stabilizing compounds resulted in the identification of conditions and specific compounds that lead to enhanced thermal stability of free and bound to adjuvant toxoids.

Preformulation studies of proteins in the solid state usually involve characterization of an amorphous solid in general (e.g. moisture content, crystallinity, structural relaxation, etc.) and specific protein properties (e.g. extent of protein structure preservation). Unfortunately, these characteristics of the solid and protein cannot usually predict protein stability during storage. Therefore, a more in depth understanding of amorphous matrices is needed. To understand the role of interactions between protein and expient as well as the homogeneity of protein/excipient mixtures, a study of a model system containing human Growth Hormone (hGH) and sugars (sucrose and trehalose) was performed.

This study revealed that the extent of protein/excipient interaction can be used to describe the degree of homogeneity of a lyophilized mixture which can be related to the cryo- and lyo-protecting properties of the excipients. Additionally, it was seen that the rate of structural relaxation is proportional to the rate of insoluble aggregate formation. These studies of proteins in solution and the solid state allowed for the identification of conditions for long term stability studies of C. difficile vaccines and contributed to our understanding of the role of interactions between protein and excipient in lyophilized solids.