Impact of formulation pH on physicochemical protein characteristics at the liquid-air interface

Both, formulation parameters and the presence of liquid-air interfaces are known to affect the aggregation of protein drugs. In this study, the impact of pH on the liquid-air interfacial behavior of three proteins, a polyclonal and two monoclonal antibodies (IgG, mAB₁ and mAB₂) was investigated using different surface sensitive methods. Equilibrium surface pressure values revealed only a minor impact of pH. Infrared Reflectance Absorbance Spectroscopy (IRRAS) proved not only the presence of the proteins at the interface but also showed that the secondary structure was not considerably affected by the adsorption to the interface independent of pH between pH 3 and 9. Additionally, the physical resistance of the film as determined by the interfacial compressibility in a Langmuir trough was not affected by pH. Compression of the interfacial film caused the formation of telescoped areas which were no longer present after decompression at all pH values as investigated by underwater Atomic Force Microscopy (AFM). Brewster Angle Microscopy (BAM) showed some slight changes in the film reflectivity depending on pH, indicating changes in the interfacial film thickness. IRRAS experiments at different angles of incidence as well as section analysis of AFM images proved not only that the film thickness increased upon compression, but also that the interfacial film is thinner at pH 4 than at pH 9. Continuous compression and decompression of the protein film resulted in particle formation with increasing numbers of particles at higher pH value as detected by Light Obscuration (LO) and Micro-Flow Imaging (MFI). The use of different surface sensitive methods provides expedient information on how liquid-air interfacial events are affected by formulation pH. These findings enable a better understanding of not only the events and processes happening at the interface but can also be directly linked to the interface-related formation of particles.