In this study, we conducted a comprehensive evaluation of the stability of XSB-001 when stored in syringes under diverse conditions. Utilizing validated analytical methods, we meticulously assessed the physical, chemical and biological integrity of the compound.

These results show that XSB-001 can be stored in syringes for up to 30 days, maintaining its stability in terms of physicochemical and biological properties, even under light exposure and temperature changes. It was also demonstrated when stored in syringes for up to 30 days at 5 ± 3 °C, with some samples exposed for an additional 48 h to ambient temperature and humidity, either protected from light or exposed to indoor lighting. Across all tested conditions and syringe types, XSB-001 remained stable, suggesting its suitability for prepared syringes in workflows such as internal transport and short-term transfers from department to clinical areas.

Several validated analytical methods were employed to assess physical, chemical and functional stability. Visual assessment is a fundamental quality control measure to detect macroscopic changes such as precipitation, color alteration or the presence of visible particulates. Here, where tested, all samples, under all test conditions, remained liquid, clear and/or colorless, with no visible particulates observed. This consistent outcome demonstrates that XSB-001 maintained its physical integrity across the tested conditions. Such physical stability is critical in therapeutic proteins as physical alterations can indicate aggregation or degradation processes, potentially affecting efficacy and safety [23].

Using SE-HPLC, the product’s aggregation profile was evaluated. Aggregation of XSB-001 monomers into dimers, trimers or higher-order structures is undesirable as it can reduce product efficacy and increase immunogenicity [24, 25]. The results showed low aggregate levels and suggested minimal to no degradation across all storage conditions and all three sample types, indicating that container type did not affect stability [23].

RP-HPLC separates protein variants based on hydrophobic interactions, providing insights into purity and detecting subtle conformational changes or degradation. It is particularly useful for analyzing post-translational modifications that may affect protein function [26]. Our results suggest a stable protein profile with minimal conformational alterations. The small differences may be due to minor surface interactions or mild stress induced by ambient conditions but are within the variance of the analytical method [26, 27].

Sub-visible particles (2–50 µm) are a concern due to their potential to induce immunogenic responses. The study observed variability in particle counts even though all samples, under all test conditions, were within pharmaceutically acceptable thresholds for sub-visible particulate contamination. According to USP < 789 > Particulate Matter in Ophthalmic Solutions, products pass with an upper limit of 2 particles/ml ≥ 50 μm, which can be evaluated within measurement uncertainty, guaranteeing compliance with the acceptance criteria [28]. For vials, sub-visible particle analysis indicated minimal particle formation, with low particle counts at ≥ 2 µm (135–143 particles/ml). Larger particle counts (≥ 10 µm, ≥ 25 µm, ≥ 50 µm) were similarly within acceptable limits. However, in JJ Zero Residual syringes, an increase in sub-visible particulate formation was observed, significantly higher compared to the vials or BD Luer-Lok syringes, which also showed high sub-visible particle counts in the ≥ 2 µm and ≥ 5 µm range. Although these remained within the threshold for concern, the probable explanation is that repackaging into syringes increases sub-visible particles, potentially because of factors inherent to the syringe design or materials. These increases could be attributed to the syringe itself and not to protein aggregation as no corresponding increase was observed in the SE-HPLC results [29].

To assess biological activity, the VEGF165 Reporter Gene Assay demonstrated that potency was preserved across all conditions, with values between 91 and 102%—all within the acceptable range of 85–125%. The VEGF165 RGA is a bioassay for evaluating the biological activity of anti-VEGF-A therapies, particularly the VEGF165 isoform—the most biologically active variant involved in angiogenesis and endothelial cell function [30]. It employs genetically engineered reporter cells that express VEGF receptors and a reporter gene under the control of a VEGF-responsive promoter. Upon stimulation with VEGF165, these cells activate intracellular signaling pathways that lead to reporter gene expression, typically measured by luminescence. Anti-VEGF agents, such as XSB-001, inhibit this response, allowing the assay to quantify their potency and functional activity. Therefore, with potency values ranging from 91 to 102%, within the acceptable range of 85–125%, we demonstrated that potency was maintained for all conditions and all samples. Consistent potency is crucial for therapeutic efficacy, and these results show that XSB-001's functional integrity was preserved.

The study also supports the use of specific syringe types, under defined storage conditions, reflecting real-world use of equipment, namely JJ Zero Residual and BD Luer-Lok syringes.

This study has limitations, as it did not assess microbial sterility, although procedures were aseptic. In healthcare facilities with validated aseptic compounding, the risk of microbial contamination is minimized. The results apply to settings following validated sterile techniques. XBS-001 should be administered by label guidelines in each approved country to maintain sterility during storage.