Identification of the Root Cause of Visible Particle Formation in a Therapeutic Monoclonal Antibody by LC-MS-Based Approaches

Abstract

Visible particles were observed for a therapeutic monoclonal antibody at pH 5.5. This pH was considered optimal for this antibody during preclinical development. To minimize the particle formation, the pH of the final drug substance was increased to 6.5. There were no visible particles at pH 6.5 initially, but visible particles started to form after 2 months. To minimize impact to the clinical program, a lyophilized formulation presentation was chosen. Subsequently, due to the desire for patient administration through subcutaneous injection for this antibody, a stable, high concentration solution formulation was needed. Thus, the pH was further increased to 7.2 to mitigate the visible particle formation. LC-MS-based approaches, including reduced LC−MS analysis and cathepsin D-targeted LC−MS Lys-C peptide map analysis, were developed to identify the root cause of the visible particle formation of this antibody at pH 5.5 to ensure the successful development of a stable solution formulation at pH 7.2. Results from studies supported by LC-MS-based analyses indicated that visible particles observed in the antibody protein A capture main pool sample at pH 5.0 were associated with cathepsin D clipping of the antibody. In contrast, visible particles observed in the purified antibody drug substance samples at pH 5.5 and pH 6.5 and in the antibody in-process samples (from the low pH viral inactivation, anion exchange chromatography, hydrophobic interaction chromatography, and tangential flow filtration unit operations) were associated with succinimide intermediate formation at an Asp residue with an Asp−Ser sequence motif in one of the complementarity-determining regions of the light chain. Understanding the root cause of the particle formation of this antibody at pH 5.5 enabled us to perform targeted experiments to rapidly develop a stable high concentration solution formulation for this antibody and meet project timelines.