Development and study of a novel anti-HER2 therapeutic conjugate based on DARPin G3

Introduction. Overexpression of human epidermal growth factor receptor 2 (HER2) is a critical driver of malignancy in multiple solid tumors, underscoring the demand for innovative targeted therapeutics. Protein–drug conjugates offer selective delivery of highly potent cytotoxic payloads directly to tumor cells. Designed ankyrin repeat proteins (DARPins) represent a viable alternative to full-length monoclonal antibodies owing to their high binding affinity, thermal stability, and efficient production in prokaryotic expression systems. However, analytical characterization and standardization of DARPin-based conjugates necessitate dedicated, validated methodology.

Aim. To develop a targeted therapeutic conjugate comprising DARPin G3 and the cytotoxic agent emtansine, and to establish standardized analytical methods tailored for its quality control as an active pharmaceutical ingredient.

Materials and methods. The study evaluated the HER2-specific therapeutic conjugate DARPin G3-ABD covalently linked to emtansine (DM1) via a non-cleavable maleimide linker. Manufacturing involved site-specific protein reduction, chemical conjugation, and size-exclusion chromatographic purification. Physicochemical characterization was performed using UV spectrophotometry, the bicinchoninic acid (BCA) colorimetric assay, nano-HPLC-MS/MS, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biological activity was assessed in HER2-overexpressing human breast adenocarcinoma SKBR3 cells using the MTT cell viability assay. Method validation was conducted in accordance with pharmacopoeial guidelines.

Results and discussion. Comprehensive analytical profiling of the conjugate encompassed identity, related impurities, quantitative assay, and in vitro potency. Identity was verified by nano-HPLC-MS/MS, demonstrating 100 % amino acid sequence coverage. SDS-PAGE revealed a single major monomeric band with high electrophoretic purity and homodimeric impurities below 6 %. Quantitative determination was evaluated by direct UV spectrophotometry at 252 nm and the BCA assay. Validation demonstrated that the UV spectrophotometric assay is specific, linear (R = 0.999), free from systematic error, and highly precise (RSD ≤ 2.06 %). Conversely, the BCA assay exhibited a statistically significant systematic bias (δ = 2.76 %), confirming the superiority of direct UV spectrophotometry for active ingredient assay. The conjugate demonstrated dose-dependent cytotoxicity against HER2-positive cells with an IC₅₀ = 40 ± 10 нМ, whereas the payload-free protein control showed no antiproliferative effect.

Conclusion. A targeted conjugate based on DARPin G3-ABD and emtansine was successfully engineered and characterized, displaying high purity, confirmed primary sequence identity, and potent HER2-dependent cytotoxic activity. The established analytical panel provides a reliable basis for quality control of the conjugate as an active pharmaceutical ingredient in preclinical development.

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