Characterization of Aggregated Antibody-Silicone Oil Complexs; From Perspectives of Morphology, 3D Image, and Fcγ Receptor Activation.

Kiyoshi, Masato; Tada, Minoru; Shibata, Hiroko; Aoyama, Michihiko; Ishii-Watabe, Akiko

Pre-filled syringes (PFS) have been in widespread use as an administration device for therapeutic antibodies in recent decades. Generally, the inner barrel and syringe of PFS are coated with silicone oil (SO) for lubrication. Multiple studies have focused on the fact that the SO adsorbs denatured antibody molecules, and induces antibody aggregation. Aggregated antibodies are recognized as a potential risk for evoking immunogenic responses in patients. The characteristics of the aggregated antibody-SO complexes, including their concentration, population, shape, three-dimensional (3D) image, and Fcγ Receptors (FcγRs) activation have been obscurely acknowledged so far. In the present work, we prepared aggregated antibody-SO complexes by agitation and analyzed using multifaceted techniques such as flow imaging, confocal fluorescence microscopy, and cell-based assays for FcγRs activation. The results emphasized that the SO accelerates the increase in sub-visible particles and antibody aggregation. The confocal fluorescence microscopy analysis revealed the high-resolution 3D images of aggregated antibody-SO complexes. The FcγRs reporter cell assay clarified that the pre-mixed and agitated Ab + SO have higher FcγRs activation capability compared to the agitated Ab. Overall, this study advances the view that SO has an effect to increase the risk of agitation-induced aggregated antibody particles.