The bispecific mode of action of faricimab comprises a new concept in the treatment of retinal diseases related to pathological angiogenesis and vascular leakage. Given this feature and the increasing demand of anti-VEGF drugs that demographic changes will bring, data regarding the long-term stability of faricimab in ready-to-use syringes is of clinical relevance for providers of anti-VEGF therapy. While being off-label, compounding of anti-VEGF drugs in syringes by qualified pharmacies provides some advantages in comparison to drug preparation by the clinician. It can increase patient safety by introducing higher standards of hygiene, save time by executing the same task more efficiently and by a larger quantity and reduce cost by splitting vials in a safe manner. While compounding and storage has been shown to be safe for other anti-VEGF agents, those findings cannot be extrapolated to faricimab due to its different antibody properties. Jørstad et al. recently published stability data for faricimab compounded in silicone oil-free syringes and did not find changes in binding affinity or stability after 37 days.[20] However, in contrast to our study, evaluation of sterility was not included in their analysis and only silicone oil-free syringes were used, without a side-by-side comparison with other syringe types. Moreover, a different assay was employed to test the binding affinity to faricimab’s targets.
In this study, compounding and storage in silicone oil-free as well as silicone oil-containing syringes were evaluated. While the silicone oil content of syringes has been associated with an increased presence of protein aggregates and particle formation in the past [13], the protective silicone layer could also serve as a barrier for interference between certain components of the syringe and the structural integrity of the biologic.[21] Thus, both syringe types were included in this study to investigate possible differences in drug integrity due to silicone-oil coating.
As previously highlighted, a significant concern with compounded antibodies is the potential formation of aggregates and structural degradation, which can lead to reduced compound activity and intraocular inflammation.[12, 22] In the present study, neither signs of oligomerization, nor changes in the size distribution, nor the formation of undissolved particles or aggregates could be found after 28 days of storage by SEC, UV-VIS and DLS. Additionally, SEC did not show structural degradation of the andibody. The presence of a second nanoparticle population in the range between 300 to 400nm derived by oligomerization processes over time, which was reported by Paul et al. for bevacizumab in syringes [14], was not observed with faricimab. Furthermore, the detected z-average hydrodynamic diameter is in accordance with other publications about the hydrodynamic diameter of particles formed by IgG1 antibodies with a molecular weight of 150kDa.[23] Also, the tertiary protein structure of faricimab remained stable.
The stability of protein based compounds is also strictly dependent on the pH of the solution. Regarding monoclonal antibodies of the IgG1 type, Zheng et al. could show that a decreased pH provokes conformational changes and an increased degradation.[24] However, the buffer capacity of the faricimab formulation maintained a stable pH of 5.5 during the whole storage duration.
The adequate binding of the biologic to its dedicated targets after compounding is of paramount importance for intravitreal antibody drugs. In contrast to Jørstad et al., who used ELISA to investigate the binding affinity to VEGF and Ang-2 separately, we established a GCI-based method similar to the surface plasmon resonance method used by Regula et al. in their evaluation of the binding properties of faricimab.[1] In our approach, faricimab was immobilized non-covalently on the measurement chip via an anti-human IgG (Fc) antibody. This gave the possibility of testing the simultaneous binding mechanism of both VEGF and Ang-2, closely mimicking the mode of action after intravitreal administration, which otherwise would require the establishment of a dual-binding ELISA method. In comparison to ELISA, GCI shows a significantly higher sensitivity and monitors the binding mechanisms in a real-time manner. Additionally, GCI works entirely label-free and therefore avoids possible negative interference with the antibody-antigen interaction.[25, 26] For both antigens, the average signal intensity seemed to be dependent on the order in which the antigens were injected. In case of VEGF, a reduction of the average signal intensity by 0.06 was visible, when it was injected after Ang-2. With Ang-2, there was an increase by 0.13, when it was injected second. Such differences created by the injection order might occur because of steric hindrances. However, in the respective replicates there was no statistically relevant difference between faricimab samples compounded and stored in syringes and the day 0 samples.
As microbiological contamination can result in endophthalmitis after intravitreal injection, we further analyzed both the safety of the compounding process and the reliability of the primary packaging system during storage based on the sterility of the product. Directly after transfering of faricimab into syringes as well as after 28 days of storage an endotoxine concentration below the detection limit and no signs of bacterial growth proof the quality of the compounding process.
The limitations of this study are as follows. First, binding affinity was tested for VEGF as well as Ang-2 only, and not for Fcγ-receptors and the neonatal Fc-receptor as performed by Jørstad et al.[20] However, the acquired stability data in this study does not implicate any structural changes of faricimab after compounding and storage. Second, this study was not specifically designed to investigate the release of silicone oil-based coating into the formulation. Such analyses would require techniques such as flow cytometry in combination with specific labelling of protein particles and silicone oil droplets, which was not within the scope of this study.[27] Third, this was a laboratory study and the compounded faricimab was not tested clinically.
In conclusion, the establishment of a safe compounding process for anti-VEGF drugs for intravitreal injections to avoid contamination as well as any negative interference with the integrity of the compound is imperative to ensure the highest possible patient safety and treatment efficacy. This study evaluated the effect of the compounding process of faricimab into silicone oil-containing and silicone oil-free polypropylene syringes and subsequent storage for up to 28 days at 2°C to 8°C under light protection. The applied methods did not show impaired chemical or physical stability nor changes in binding affinity to VEGF and Ang-2 while maintaining sterility. These findings suggest a safe use of pharmaceutically compounded faricimab in syringes after storage for at least 28 days and provide a way to increase patient safety and cost-effectiveness for intravitreal injections in real-world settings.