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.

Figure 1

Far- and near-UV CD spectra of (a) Rituximab and (b) Etanercept products under (1) native, (2) denaturing, and (3) denaturing-reducing conditions. Dotted lines represent Mabthera (Rituximab) and Enbrel (Etanercept), while continuous lines represent Kikuzubam (Rituximab) and Infinitam (Etanercept) representative batches. Main plot shows near-UV CD spectra (240–350 nm) and graphic insert shows far-UV CD spectra (190–300 nm). Near- and Far-UV characteristic spectra of Rituximab are shown in (a) and insert shows antiparallel β-sheets coming from CH₁, CH₂, and CH₃ and V_L and V_H domains. Etanercept spectra (b) show an irregular structure directed probably by complex and diverse glycan substituents. Each sample was run in triplicate; average is represented as single spectrum (details are described in Section 2.3.6).