Development, characterization, and stability assessment of a lyophilized Eculizumab formulation for use as a reference material

Objectives. The study set out to develop a stable lyophilized formulation of the monoclonal antibody Eculizumab, comprehensively characterize the resulting material, and assess its stability for qualifying it as a reference material. This involved developing a matching placebo formulation, determining the optimal lyophilization conditions, and conducting a rigorous stability study.

Methods. In the development of the formulation and lyophilization conditions for Eculizumab, we tested various buffer systems and cryoprotectants. The residual moisture content in the resulting lyophilized samples was determined by Karl Fischer titration. Peptide mapping was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) following enzymatic hydrolysis with trypsin. The structural, physicochemical, and biological properties were analyzed using various analytical methods, including RP-HPLC, high-performance liquid chromatography mass spectrometry, capillary sodium dodecyl sulfate electrophoresis, size-exclusion high-performance liquid chromatography, and enzyme-linked immunosorbent assay.

Results. A placebo solution for lyophilization of Eculizumab was selected with the following composition: 20 mM sodium phosphate, 4% trehalose, 0.2% polysorbate 80, pH 7.0. The results demonstrated a high degree of similarity between the candidate reference material and Eculizumab EU. Stability studies under storage conditions at 2–8°C demonstrated the material’s stability for one year, with control points at 3, 6, 9, and 12 months.

Conclusions. The absence of any effect of the drying process on the primary and spatial structure, post-translational modifications, content of related impurities, composition of isoforms, and specific activity was confirmed. Furthermore, stability studies demonstrated no significant changes in protein quality during storage at 2–8°C for at least 12 months, which represents the entire available data period at the time of manuscript preparation. The results indicate that the developed lyophilized material is a viable candidate for an international reference material, although its official qualification would require additional collaborative trials and long-term stability data.

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