Harnessing the potential of 2-methyl imidazolium dihydrogen phosphate as a protein stabilizer: Assessing its toxicity and impact on aggregation, structural integrity, and functional efficacy of monoclonal antibodies

• Published in: Journal of molecular liquids 2025-01, Vol.417, p.126587, Article 126587
• Main Authors: Pachpore, Radhika, Dugam, Shailesh, Manchekar, Triveni, Kulkarni, Bhalchandra, Jagtap, Dhanashree, Dandekar, Prajakta, Jain, Ratnesh
• Format: Article
• Language: English
• Subjects: 2-MIDHP; Aggregation; Bevacizumab; Binding activity; Stability; Structural stability; Thermal stress
• ISSN: 0167-7322
• DOI: 10.1016/j.molliq.2024.126587

•Examined 2-MIDHP as a protein stabilizer for Bevacizumab in storage and thermal stress conditions.•Established 10 % v/v 2-MIDHP as a safe concentration using MTT assay.•Assessed impact on mAb aggregation and monomer stability using SEC, DLS, FFF and AUC.•Evaluated effect on mAb structure using CD and fluorescence spectroscopy, and binding activity by SPR.•Confirmed protective effect of 2-MIDHP on mAb aggregation and its potential as a safe formulation excipient. The development of safe and effective biotherapeutics faces challenges due to protein aggregation. These aggregates, formed due to various stress factors, can lead to irreversible changes and increase the risk of immunogenicity and off-target binding. Ionic liquids (ILs) have recently gained significant attention due to their potential in stabilizing proteins and enzymes. In this investigation, we have assessed the thermal stability of bevacizumab (BmAb), in presence of 2-methyl imidazolium dihydrogen phosphate (2- MIDHP), both alone and in combination with other formulation excipients. SEC, DLS, FFF-MALS and AUC were used to study the protective effect of 2-MIDHP on BmAb aggregation. CD and fluorescence spectroscopy were used to evaluate the impact of 2-MIDHP on structural changes in BmAb, while SPR was used to assess the binding activity in the presence and absence of 2-MIDHP. Subsequently, a cell viability assay (MTT) was conducted to evaluate potential toxic effects of 2-MIDHP with BmAb. Overall, our findings suggested the potential of 2-MIDHP in enhancing the stability of BmAb without any negative impact on its structural and binding activity, and its compatibility with other formulation components, thus indicating its promising prospects for the development of improved biotherapeutics.