Insights into the colloidal and structural stability of Atezolizumab with nonpolar amino acid-based ionic liquids under multiple stresses: phase two.

Monoclonal antibodies (mAbs) are susceptible to physical and chemical instabilities, which compromise their therapeutic efficacy and shelf life. This study investigates the potential of renewable choline-amino acid-based ionic liquids (Ch-AA ILs), specifically choline valinate (CV) and choline glycinate (CG), to enhance the stability of the aglycosylated IgG1 antibody, Atezolizumab (Amab). Amab was formulated in varying concentrations of the ionic liquids (ILs) and subjected to ambient, thermal (40-70 °C), and chemical (urea) stress conditions. Stability was assessed using Dynamic Light Scattering (DLS) to measure the hydrodynamic diameter (D r), UV-visible spectroscopy to determine the Aggregation Index (AI), and Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) to analyse the secondary structure. The results demonstrated that CV, particularly at a 30% w/w concentration, confers superior colloidal stability to Amab compared to both CG and a conventional Tris-HCl buffer. Under significant thermal stress (70 °C for 24 hours), the 30% CV formulation maintained minimal aggregation (AI ≤ 17), whereas Amab in Tris buffer underwent extensive aggregation (AI > 160). However, an MTT assay employed to assess the biological activity of the stabilised antibody yielded inconclusive results. The ILs themselves caused a pronounced, nonspecific increase in apparent cellular metabolic activity, indicating that the assay is unsuitable for evaluating these formulations and may overestimate cell viability. This work highlights the significant promise of the green and renewable solvent CV as a stabilising excipient, enhancing the colloidal and structural stability of mAbs.