Elucidating the role of 2-methyl imidazolium dihydrogen phosphate in preventing aggregation of Bevacizumab: A biophysical investigation

Abstract

Evaluating the stability and biological activity of monoclonal antibodies is an essential step during the development process in order to ensure their optimum performance. Monoclonal antibodies (mAbs) are stable against aggregation over a narrow pH range and rapidly aggregate in solutions with pH outside these ranges. The pH of solutions can be controlled by using pH buffers, which determine the surface charge on mAbs and thereby affect their electrostatic interactions. Lower pH may lead to protein cleavage and isomerization, while higher pH may cause deamidation and oxidation reactions, increasing the aggregation potential. Surfactants generally prevent aggregation by reducing the tension at the air–water interface, which reduces the possibility of interactions of mAbs with this interface. Apart from surfactants, salts and sugars are also conventionally added to mAb formulation buffers to prevent aggregation. In this work, we have investigated the role of a biocompatible ionic liquid, 2-methyl imidazolium dihydrogen phosphate (2-MIDHP), to prevent aggregation of Bevacizumab. We employed a pH stress model to study the aggregation behaviour of Bevacizumab. The aggregation behaviour of Bevacizumab was analysed using size exclusion-High performance liquid chromatography (SE-HPLC) dynamic light scattering (DLS), differential scanning calorimetry (DSC), and field flow fractionation (FFF). The aggregation and monomer content were monitored over the pH range of 2 to 6.2 using SE-HPLC. Results indicated that 2-MIDHP was able to control aggregation of Bevacizumab at pH 2, for upto 4 days, as compared to the control sample, which showed an increase in aggregate content for upto 4 days, followed by degradation. These results were further orthogonally confirmed using techniques like DLS, DSC, and FFF. 2-MIDHP was thus found to be a potent, acid-resistant, mAb aggregation controlling agent.