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 (TNFα neutralization) showed no differences. Overall, this data revealed the capabilities and drawbacks of each thiol quantification technique and their correlation with protein structure.

New Alternatives for Autoimmune Disease Treatments: Physicochemical and Clinical Comparability of Biosimilar Etanercept

“…These results are supported by a previous report of the three-dimensional structure comparability between Infinitam and its reference product. Particularly, CD and TCSPC responses under native, denaturing, and denaturing-reduced conditions revealed similar structural features in both molecules, including their ordered and disordered regions that determine specific steric hindrances, as evidenced by the accessibility of free-thiols [ 11 ]. Accordingly, comparable target recognition is expected as long as charge heterogeneity ranges for both molecules overlap.…”

“…Glycan microheterogeneity was studied using hydrophilic interaction ultra-performance-liquid-chromatography (HILI-UPLC). Sample treatment and analysis conditions were performed as previously described by Flores-Ortiz et al, 2014 [ 10 ] (MS, RP-UPLC-MS/MS, CD, and CEX-UPLC); Pérez Medina-Martínez et al, 2014 [ 11 ] (TCSPC); Espinosa-de la Garza et al [ 12 ] (cIEF); and Miranda-Hernández et al, 2015 [ 13 ] (HILI-UPLC).…”

Physicochemical and Biological Characterization of a Biosimilar Trastuzumab

“…Purity was determined by capillary gel electrophoresis under reducing (CGE-R) and nonreducing (CGE-NR) conditions and size exclusion ultra-performance-liquid-chromatography (SE-UPLC). Sample treatment and analysis conditions were performed as previously described for RP-UPLC-MS/MS, DSC, CD, CEX, CGE-R, and CGE-NR by Flores-Ortiz et al, 2014 [ 23 ]; TCSPC by Pérez Medina Martínez et al, 2014 [ 24 ]; cIEF by Espinosa-de la Garza et al [ 25 ].…”