ABSTRACT
INTRODUCTION: SB15 is a proposed biosimilar product of reference aflibercept (Eylea®), an approved biological drug product for retinal diseases including neovascular age-related macular degeneration (nAMD). This study aimed to assess the analytical similarity between SB15 and its commercially available reference product (RP) sourced from the United States (US-aflibercept) and European Union (EU-aflibercept) in terms of structural, physicochemical, and biological properties. METHODS: A panel of state-of-the-art analytical methods was used for the comprehensive characterization of SB15 and US/EU-aflibercept. In terms of the structural and physicochemical properties, primary structure; post-translational modifications (PTM); higher-order structure; purity and impurities; charge variants; and glycosylation were compared. In addition, biological characterization including mechanism of action (MoA)-related and Fc-related biological activities was conducted. RESULTS: Analytical similarity between SB15 and US/EU-aflibercept was demonstrated. The primary and higher-order structure of SB15 was confirmed to be comparable to that of US/EU-aflibercept. In addition, there were no meaningful differences in the physicochemical properties in terms of size and charge heterogeneity between SB15 and its RP. SB15 and RP were similar in biological activities including MoA-related binding activities, potencies, and Fc-related biological functions. Consequently, SB15 was confirmed to be highly similar to US/EU-aflibercept. CONCLUSIONS: Based on a comprehensive analytical similarity assessment of structural, physicochemical, and biological properties, SB15 was demonstrated to be highly similar to US/EU-aflibercept RP, supporting safe and effective use of SB15.
ABSTRACT
Extracellular vesicles (EVs) are becoming increasingly important in liquid biopsy for cancer because they contain multiple biomarkers, including proteins and RNAs, and circulate throughout the body. Cancer cell-derived EVs are highly heterogeneous, and multiplexed biomarker detection techniques are required to improve the accuracy of diagnosis. In addition, in situ EV biomarker detection increases the efficiency of the detection process because EVs are difficult to handle. In this study, in situ simultaneous detection of EV surface proteins, programmed cell death-ligand 1 (PD-L1), and internal miRNA-21 (miR-21) analyzed by conventional flow cytometry was developed for a breast cancer liquid biopsy. However, the majority of EVs were not recognized by flow cytometry for biomarker detection because the size of EVs was below the detectable size range of the flow cytometer. To solve this problem, the formation of EV clusters was induced by 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol-DSPE during biomarker detection. Consequently, both PD-L1 and miR-21 detection signals from cancer cell-derived EVs were drastically increased, making them distinguishable from normal cell-derived EVs. The in situ simultaneous cancer biomarker detection from EV clusters analyzed by flow cytometry contributes to an increase in the sensitivity and accuracy of the EV-based liquid biopsy for cancer.
