Characterization of protein-protein interactions for optimizing formulation and physical stability of high protein concentration solutions

Current trends in market for high dose therapeutic proteins require concentrated liquid formulations for patient convenience, in home subcutaneous administration, to cut manufacturing costs and to improve product marketability. Protein-protein interactions in these solutions need to be characterized to prepare these solutions with desired viscosity and physical stability during storage. The nature and consequences of protein-protein interactions in concentrated protein solutions is reviewed. An ultrasonic shear rheometer based on impedance analysis of piezoelectric quartz crystals was developed for rheological analysis and viscosity measurement of liquids at small sample volumes. Solution viscosities of aqueous solutions of sucrose, urea, PEG-400, glucose, and ethylene glycol were measured at 25°C. The measured viscosities were reproducible and consistent with the literature values. Characterization of viscoelastic fluids was conducted and storage modulus (G\u27) and loss modulus (G ) were measured. Bovine serum albumin solutions were analyzed in order to establish the utility of the developed ultrasonic rheometer for studying subtle differences in protein solution rheology as a function of solution conditions. Results of high-frequency rheology analysis were consistent with the structural information reported for the protein in the literature. Rheological analysis and biophysical characterization conducted on a model monoclonal antibody, IgG2, between pH 4.0 to 9.0 and ionic strengths between 4 mM and 300 mM demonstrated the significant role of protein-protein interactions in governing the solution behavior of protein in concentrated solutions. Results from these studies indicated that solution G\u27 could serve as a parameter for assessing protein-protein interactions in high protein concentration solutions. Its validity for this purpose was confirmed by static and dynamic light scattering measurements under relatively dilute solution conditions. The measured second virial coefficient (B 22) and interaction parameter (kD) were found to be consistent with the solution G\u27 measurements. Extent of aggregate formation after storing the IgG2 solutions at 25°C and 37°C for three months was higher for the solution conditions exhibiting sharper increase in solution G\u27 with protein concentration and for which B22 and kD were lower. The results demonstrated the utility of ultrasonic G\u27 measurements for characterizing protein-protein interactions and for predicting favorable solution conditions for formulating high protein concentration solution formulations.