Investigation of Mechanically-induced aggregation of the concentrated and diluted Zytux® (rituximab biosimilar) by size exclusion Chromatography

Abstract

Introduction: Mechanical stress is one of the common methods to evaluate the stability of therapeutic protein formulations including monoclonal antibodies. In this method, proteins are faced with various stresses such as the liquid-air interface, contact with container surfaces, the formation of vacuum bubbles by agitation, and localized temperature elevation. Mechanical stress increases the contact of protein molecules with hydropHobic surfaces or air/water surfaces, which leads to antibody aggregation and the formation of aggregate products, which can reduce therapeutic effects and even increase their immunogenicity.Aim: The objective of this research is to examine the pHenomenon of aggregation induced by mechanical stress( shaking, and stirring) in various concentrations of Zytux monoclonal antibody (rituximab biosimilar) by the use of size exclusion Chromatography.Method: Samples of Zytux monoclonal antibodies were prepared at various concentrations and exposed to mechanical stress through shaking and stirring at a constant speed of 200 rpm. The samples were periodically sampled and analyzed throughout the experiment.Results: The data obtained from the experiment demonstrated that the application of mechanical stress through shaking led to an increase in aggregation. This increase can be attributed to the higher amounts of mechanical stress generated during shaking, as compared to stirring. Moreover, the level of aggregation has demonstrated an upward trend in correlation with elevated concentration levels, mostly attributed to the intrinsic propensity of Zytux to aggregate in conditions of high concentration.Conclusion: The research findings highlight the significance of comprehending the impact of mechanical stress on the stability and propensity for aggregation of Zytux. This understanding is particularly crucial in the context of formulation and storage conditions, as mechanical stress can be introduced during various stages such as production, transportation, and product handling.