title: Comparability of a three-dimensional structure in biopharmaceuticals using spectroscopic methods.
abstract: Protein structure depends on weak interactions and covalent bonds, like disulfide bridges, established according to the environmental conditions. Here, we present the validation of two spectroscopic methodologies for the measurement of free and unoxidized thiols, as an attribute of structural integrity, using 5,5'-dithionitrobenzoic acid (DTNB) and DyLight Maleimide (DLM) as derivatizing agents. These methods were used to compare Rituximab and Etanercept products from different manufacturers. Physicochemical comparability was demonstrated for Rituximab products as DTNB showed no statistical differences under native, denaturing, and denaturing-reducing conditions, with Student's t-test P values of 0.6233, 0.4022, and 0.1475, respectively. While for Etanercept products no statistical differences were observed under native (P = 0.0758) and denaturing conditions (P = 0.2450), denaturing-reducing conditions revealed cysteine contents of 98% and 101%, towards the theoretical value of 58, for the evaluated products from different Etanercept manufacturers. DLM supported equality between Rituximab products under native (P = 0.7499) and denaturing conditions (P = 0.8027), but showed statistical differences among Etanercept products under native conditions (P < 0.001). DLM suggested that Infinitam has fewer exposed thiols than Enbrel, although DTNB method, circular dichroism (CD), fluorescence (TCSPC), and activity (TNF α neutralization) showed no differences. Overall, this data revealed the capabilities and drawbacks of each thiol quantification technique and their correlation with protein structure.
authors: Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V. Cruce de Carreteras Acatzingo-Zumpahuacán S/N, Tenancingo, 52400 Mexico, MEX, Mexico.
authors: Víctor
authors: 0000-0001-6088-0765
authors: V
authors: Pérez Medina Martínez
authors: Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Avenida Tecnológico y Antonio García Cubas S/N, 38010 Celaya, GTO, Mexico.
authors: Mario E
authors: ME
authors: Abad-Javier
authors: Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V. Cruce de Carreteras Acatzingo-Zumpahuacán S/N, Tenancingo, 52400 Mexico, MEX, Mexico.
authors: Alexis J
authors: AJ
authors: Romero-Díaz
authors: Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Avenida Tecnológico y Antonio García Cubas S/N, 38010 Celaya, GTO, Mexico.
authors: Francisco
authors: F
authors: Villaseñor-Ortega
authors: Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V. Cruce de Carreteras Acatzingo-Zumpahuacán S/N, Tenancingo, 52400 Mexico, MEX, Mexico.
authors: Néstor O
authors: NO
authors: Pérez
authors: Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V. Cruce de Carreteras Acatzingo-Zumpahuacán S/N, Tenancingo, 52400 Mexico, MEX, Mexico.
authors: Luis F
authors: LF
authors: Flores-Ortiz
authors: Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V. Cruce de Carreteras Acatzingo-Zumpahuacán S/N, Tenancingo, 52400 Mexico, MEX, Mexico.
authors: Emilio
authors: 0000-0001-8862-1378
authors: E
authors: Medina-Rivero