Identification des interactions entre les protéines et les excipients afin d'élucider les propriétés de la solution et les processus de stabilisation dans les formulations biothérapeutiques à haute concentration

High-concentration monoclonal antibody (mAb) formulations are essential for subcutaneous administration, as they allow a high therapeutic dose to be administered in small volumes. However, at concentrations above 100 mg/mL, mAbs tend to reversibly self-associate into transient oligomers, leading to a sharp increase in viscosity. This complicates the manufacture, handling, and injection of the treatment and can compromise its stability and efficacy. To limit these effects, excipients, typically amino acids, sugars, or surfactants, are added. These reduce protein-protein interactions and ensure the stability of the mAb. However, their selection remains largely empirical, as their impact on self-association and viscosity remains poorly understood.During this study, an IgG evaluated in clinical trials by Sanofi was used as a model. A set of complementary NMR methods was optimized with the aim of detecting mAb:mAb and mAb:excipient interactions under standard formulation conditions. These methods included analysis of ¹H signals from the mAb in the presence of nine different excipients, pulsed field gradient (PFG-NMR) experiments, chemical shift perturbation (CSP) analysis, saturation transfer (STD-NMR) experiments, and transverse relaxation (R2) measurements. In addition, the macroscopic viscosity of the samples was measured by rheometry, and the size distribution of mAb clusters was analyzed by dynamic light scattering (DLS). This analysis was complicated by exchanges between monomers and clusters, confirming the limited ability of DLS to describe the distribution of mAb species at high concentrations. Furthermore, the interaction parameters deduced from low-concentration analyses (kD) did not allow the viscosity at high concentrations to be predicted. However, NMR analyses made it possible to link the macroscopic viscosity measured by rheometry with the molecular interactions of the mAb. The 1D ¹H NMR intensities proved to be very sensitive to reversible oligomerization of the mAb. After correction for viscosity (measured by rheometry), a significant loss of NMR signal was observed above 50 mg/mL, indicating the formation of transient oligomers (or clusters). Among the nine excipients tested, arginine and lysine significantly reduced the loss of NMR signal, and therefore the reversible oligomerization of the mAb. Most excipients reduced macroscopic viscosity, with arginine and lysine again being the most effective. R2 analysis and CSPs showed that arginine, lysine, and sucrose interact most strongly with the surface of the mAb, while glycine and surfactants show weaker interactions, mainly detected in the presence of clusters. STD-NMR proved to be particularly sensitive to the reversible oligomeric state of the mAb and detected strong interactions with lysine, while measurements in the presence of proline showed low reproducibility. Overall, this work shows that NMR is suitable for studying weak interactions in concentrated mAb solutions. The data indicate that viscosity depends not only on the degree of mAb self-association, but also on the nature of the transient interactions between different clusters. mAb-excipient interactions may contribute to the regulation of the interaction network formed by the mAb, but cannot alone determine macroscopic viscosity. The mechanistic description of mAb self-assembly and the role of excipients by NMR helps to rationalize the molecular determinants of viscosity.