Following intravitreal (IVT) injection of protein-loaded long acting delivery (LAD) system, therapeutic protein remains in vitreous humor (VH) and gets exposed at physiological pH and temperature for significantly longer duration. Therefore, it is of prime interest to study the stability of protein in VH at physiological conditions. However, so far, this has been proven to be very challenging. In previous work, we have shown that isolated VH exhibits unique behavior of pH alkaline shift upon incubation and formation of smaller MW degradation products. Hence, we have successfully developed a customized dual-chamber ex-vivo intravitreal model (Ex-Vit) effective in maintaining pH and osmolality of VH constant throughout study period, thus enabling long-term stability investigations of protein in the isolated VH. Stability of a model bi-specific mAb (mAb) was evaluated in the isolated porcine VH at 37°C for 3 months. Physical stability of mAb was studied by particle analysis, DSC, SEC, CE-SDS, and microscopically, whereas chemical stability was examined by IEC and LC-MS/MS. The potency (binding affinity) of the protein in the stability samples was estimated by SPR-Biacore. MAb exhibited significantly less fragmentation in VH relative to PBS. Similarly, the loss of binding affinity of mAb was noticeably higher in PBS (~50%) at 3 month time point when compared to VH (~40%). Results indicate that studied mAb exhibited better stability in VH compared to PBS, suggesting that for long-term IVT applications, it would be beneficial to investigate stability in VH instead of PBS. In summary, newly developed Ex-Vit model is suitable to predict long-term stability of proteins and other molecules following IVT injection. Another perspective of application is to estimate release characteristics from controlled release IVT drug delivery systems.
•Currently, PBS is considered as a gold-standard to evaluate protein stability or release behavior of therapeutics following intravitreal injection. It is mainly due to the lack of advanced and more predictive in-vitro/ex-vivo models. •The ExVit model discussed in this talk can be used to predict long-term stability of therapeutic protein in the isolated VH. Hence, the results obtained using ExVit model would be more realistic to the in-vivo situation than the PBS.
•It can be a simple and cost effective experimental tool which can be easily set-up in any lab.
•Accounted to these facts, it can offer tremendous advantage during conventional protein formulation development and also for the development of LAD intended for intraocular delivery.