Navigating atypical equilibrium dynamics of protein aggregates for biopharmaceutical release and stability monitoring.

Abstract

High molecular weight species (HMW) is a critical quality attribute in biopharmaceuticals requiring comprehensive characterization and accurate quantification throughout drug development and manufacturing to ensure product efficacy and patient safety. In this study, the primary goal was to develop a robust size exclusion chromatography (SEC) method to quantify HMW varying in relative proportion to its parent molecule, an Fc-fusion protein therapeutic (FC1) for quality control release and stability testing. However, a unique "partially reversible" HMW kinetics behavior was observed, with dissociation towards equilibrium occurring slowly, up to several days, with rates dependent on time, temperature, concentration, and formulation components. This kinetic behavior posed challenges to process optimization and quality control testing, as the "true" quantitative value of HMW was highly variable based on these conditions, varying by up to an order of magnitude in relative proportion. To address this, mathematical models were built to describe HMW equilibrium kinetics which supported the development of a robust SEC test method that accurately quantifies this attribute under relevant conditions. The developed method was validated and implemented in quality control laboratory for drug release and stability testing. This work also provides new insights into the need to control protein aggregation dynamics as it relates to process optimization and drug product release requirements.