Therapeutic antibody formulation: Phase behavior, opalescence, viscosity, physical and chemical stability

Therapeutic monoclonal antibodies (mAbs) are promising drug candidates and have become the fastest growing type of therapeutic in development. However, antibodies are large, complex proteins that can exhibit a wide range of solution behaviors that are not desirable for therapeutic applications. These include chemical instability of the peptide sequence, precipitation of native or non-native aggregates, opalescence and high viscosity. These behaviors present economic and safety issues throughout the development, production and storage of antibody therapeutics.;The central goal of this dissertation is to develop a fundamental understanding of detrimental solution behavior and present rational approaches for modulating the behaviors. The aim is to demonstrate that antibody solution behavior can be described by fundamental principles of thermodynamics and reaction kinetics. By presenting fundamental explanations, the results and conclusions herein will aid in more rapid and rational antibody formulation in the biopharmaceutical industry.;The phase behavior of a therapeutic antibody is investigated using a hanging drop crystallization technique in conjunction with second virial coefficient (SVC) measurements from static light scattering. The SVC is correlated with reversible, amorphous precipitation events as well as liquid-liquid phase separation.;Additionally, the fragmentation of a full-length therapeutic antibody during storage is characterized with size-exclusion chromatography (SE-HPLC), light scattering and matrix-assisted laser desorption mass-spectrometry (MALDI). The fragmentation was determined to be a hydrolysis of the hinge region that was dependent on the presence of trace transition metal ions and buffer type. The conformational stability of the mAb was characterized with thermally and chemically induced unfolding and also found to be buffer dependent. The relationship between the conformational stability and the susceptibility to fragmentation is described.;Furthermore, opalescence and viscosity phenomena are measured with nephelometric turbidity and cone/plate viscometry respectively. To understand the source of the high viscosities membrane osmometry and static light scattering were used to measure the SVC at high concentration. Zeta potential measurements via laser Doppler velocimetry revealed that electroviscous effects, including electrostatic repulsion, were the source of high viscosity for this mAb. The ionic strength dependence of both opalescence and viscosity were characterized. Opalescence was seen to increase as the protein-protein interactions became more attractive and was related to general critical opalescence phenomena observed for a wide variety of fluids and fluid mixtures. A method for simultaneously minimizing opalescence and viscosity utilizing both ionic strength and high protein concentrations is demonstrated.;Finally, the aggregation pathway of a therapeutic antibody during accelerated storage stability studies is characterized with SE-HPLC coupled to molecular weight measurements from light scattering. The reaction is revealed to be third order in the activity of the mAb. Additionally, the conformational stability of the mAb as measured by differential scanning calorimetry (DSC) is determined to be to primary factor controlling the rate of aggregation. Therefore, co-solutes that increase the interfacial tension, such as sucrose and citrate lead to an increase in the thermal stability and resistance to aggregation.