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Journal of Analytical Methods in Chemistry - Volume 2014 2014, Article ID 950598, 11 pages -

Research Article

Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V. Cruce de Carreteras Acatzingo-Zumpahuacán S-N, Tenancingo, 52400 Mexico, MEX, Mexico

Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Avenida Tecnológico y Antonio García Cubas S-N, 38010 Celaya, GTO, Mexico

Received 17 February 2014; Accepted 23 April 2014; Published 22 May 2014

Academic Editor: Josep Esteve-Romero

Copyright © 2014 Víctor Pérez Medina Martínez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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 -test values of 0.6233, 0.4022, and 0.1475, respectively. While for Etanercept products no statistical differences were observed under native and denaturing conditions , 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 and denaturing conditions , but showed statistical differences among Etanercept products under native conditions . DLM suggested that Infinitam has fewer exposed thiols than Enbrel, although DTNB method, circular dichroism CD, fluorescence TCSPC, and activity TNFα neutralization showed no differences. Overall, this data revealed the capabilities and drawbacks of each thiol quantification technique and their correlation with protein structure.

Author: Víctor Pérez Medina Martínez, Mario E. Abad-Javier, Alexis J. Romero-Díaz, Francisco Villaseñor-Ortega, Néstor O. Pérez



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