Unidad de Desarrollo e Investigación en Bioprocesos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N Colonia, Casco de Santo Tomas Delegación Miguel Hidalgo C.P 11340, Ciudad de Mexico, Mexico.
Departamento de Farmacología y Unidad Periférica de Investigación en Biomedicina Translacional (CMN 20 de noviembre, ISSSTE), Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de Mexico, Mexico.
J Pharm Biomed Anal. 2018 Jun 5;155:235-240. doi: 10.1016/j.jpba.2018.03.057. Epub 2018 Mar 30.
Physicochemical and structural properties of proteins used as active pharmaceutical ingredients of biopharmaceuticals are determinant to carry out their biological activity. In this regard, the assays intended to evaluate functionality of biopharmaceuticals provide confirmatory evidence that they contain the appropriate physicochemical properties and structural conformation. The validation of the methodologies used for the assessment of critical quality attributes of biopharmaceuticals is a key requirement for manufacturing under GMP environments. Herein we present the development and validation of a flow cytometry-based methodology for the evaluation of adalimumab's affinity towards membrane-bound TNFα (mTNFα) on recombinant CHO cells. This in vitro methodology measures the interaction between an in-solution antibody and its target molecule onto the cell surface through a fluorescent signal. The characteristics evaluated during the validation exercise showed that this methodology is suitable for its intended purpose. The assay demonstrated to be accurate (r = 0.92, slope = 1.20), precise (%CV ≤ 18.31) and specific (curve fitting, r = 0.986-0.997) to evaluate binding of adalimumab to mTNFα. The results obtained here provide evidence that detection by flow cytometry is a viable alternative for bioassays used in the pharmaceutical industry. In addition, this methodology could be standardized for the evaluation of other biomolecules acting through the same mechanism of action.
作为生物制药的活性药物成分的蛋白质的物理化学和结构特性是发挥其生物活性的决定因素。在这方面，旨在评估生物制药功能的测定法提供了确认性证据，证明它们含有适当的物理化学特性和结构构象。在 GMP 环境下进行生产的关键要求是对生物制药关键质量属性评估方法进行验证。在此，我们提出了一种基于流式细胞术的方法，用于评估重组 CHO 细胞上膜结合 TNFα（mTNFα）的阿达木单抗亲和力。该体外方法通过荧光信号测量溶液中的抗体与其在细胞表面上的靶分子之间的相互作用。在验证过程中评估的特性表明，该方法适合其预期用途。该测定法表现出准确性（r=0.92，斜率=1.20）、精密度（%CV≤18.31）和特异性（曲线拟合，r=0.986-0.997），可用于评估阿达木单抗与 mTNFα 的结合。这里获得的结果提供了证据，表明流式细胞术检测是制药行业中使用的生物测定法的可行替代方法。此外，该方法可用于评估通过相同作用机制起作用的其他生物分子的评估。
