TY - JOUR
T1 - Development and validation of a bioassay to evaluate binding of adalimumab to cell membrane-anchored TNFα using flow cytometry detection
AU - Camacho-Sandoval, Rosa
AU - Sosa-Grande, Eréndira N.
AU - González-González, Edith
AU - Tenorio-Calvo, Alejandra
AU - López-Morales, Carlos A.
AU - Velasco-Velázquez, Marco
AU - Pavón-Romero, Lenin
AU - Pérez-Tapia, Sonia Mayra
AU - Medina-Rivero, Emilio
N1 - Publisher Copyright:
© 2018 The Authors
PY - 2018/6/5
Y1 - 2018/6/5
N2 - 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 (r2 = 0.92, slope = 1.20), precise (%CV ≤ 18.31) and specific (curve fitting, r2 = 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.
AB - 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 (r2 = 0.92, slope = 1.20), precise (%CV ≤ 18.31) and specific (curve fitting, r2 = 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.
KW - Adalimumab
KW - Binding assay
KW - Bioassay validation
KW - Flow cytometry
KW - Membrane TNFα
UR - http://www.scopus.com/inward/record.url?scp=85045050940&partnerID=8YFLogxK
U2 - 10.1016/j.jpba.2018.03.057
DO - 10.1016/j.jpba.2018.03.057
M3 - Artículo
C2 - 29653347
AN - SCOPUS:85045050940
SN - 0731-7085
VL - 155
SP - 235
EP - 240
JO - Journal of Pharmaceutical and Biomedical Analysis
JF - Journal of Pharmaceutical and Biomedical Analysis
ER -