Molecular interactions and viscosity regulation mechanism of IgG4 antibody in high-concentration solutions.

A major bottleneck in developing high-concentration (≥ 100 mg/mL) monoclonal antibody (mAb) formulations for subcutaneous injection is the exponential increase in viscosity, often exceeding the 20 mPa·s clinical threshold value. Using an IgG4 antibody (pI ≈ 6.9) as the model in a pH 5.5 sodium citrate buffer, we innovatively used amino acids and salts (AAs/salts) as "molecular probes" to investigate their viscosity-reducing efficacy, mechanisms, and effects on the formulation stability. Compared with the control group without viscosity-reducing agents, L-Arginine (Arg) achieved a maximum viscosity reduction of 65% at a low concentration (100 mM). Their core mechanism differs from the nonspecific ionic shielding of NaCl and other salts. The guanidine group of arginine interacts with negatively charged microregions on the antibody surface to alleviate intermolecular electrostatic attraction, the main cause of elevated viscosity, whereas conventional inorganic salts exert limited charge shielding effects. Stability tests at 40 °C confirmed that Arg and ArgHCl effectively maintained antibody colloidal and conformational stability, whereas sodium succinate markedly increased the risk of protein fragmentation and aggregation. Furthermore, ITC measurements exhibit non-sigmoidal thermal curves with no obvious saturation behavior, and reliable thermodynamic binding parameters cannot be accurately fitted. Combined with the rheological characteristics and universal effects of ionic excipients, non-specific electrostatic interactions are inferred to dominate viscosity regulation, and the unique molecular structure of arginine accounts for its superior viscosity-reducing capacity. This study proposes a feasible excipient design strategy to achieve synergistic viscosity reduction and stability enhancement, providing mechanistic support for the development of high-concentration subcutaneous antibody formulations.