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Transferon, a biotherapeutic agent that has been used for the past 2 decades for diseases with an inflammatory component, has been approved by regulatory authorities in Mexico (COFEPRIS) for the treatment of patients with herpes infection. The active pharmaceutical ingredient (API) of Transferon is based on polydispersion of peptides that have been extracted from lysed human leukocytes by a dialysis process and a subsequent ultrafiltration step to select molecules below 10 kDa. To physicochemically characterize the drug product, we developed chromatographic methods and an SDS-PAGE approach to analyze the composition and the overall variability of Transferon. Reversed-phase chromatographic profiles of peptide populations demonstrated batch-to-batch consistency from 10 representative batches that harbored 4 primary peaks with a relative standard deviation (RSD) of less than 7%. Aminogram profiles exhibited 17 proteinogenic amino acids and showed that glycine was the most abundant amino acid, with a relative content of approximately 18%. Further, based on their electrophoretic migration, the peptide populations exhibited a molecular mass of about 10 kDa. Finally, we determined the Transferon fingerprint using a mass spectrometry tool. Because each batch was produced from independent pooled buffy coat samples from healthy donors, supplied by a local blood bank, our results support the consistency of the production of Transferon and reveal its peptide identity with regard to its physicochemical attributes.

Etanercept is a recombinant fusion protein approved for the treatment of TNF-αmediated diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, and ankylosing spondylitis. Herein, we present an evaluation of the physicochemical and biological properties of a biosimilar etanercept and its reference product followed by a clinical study in patients diagnosed with RA intended to demonstrate comparability of their immunomodulatory activity. Identity analyses showed a total correspondence of the primary and higher-order structure between the two products. In regard to intrinsic heterogeneity, both products showed to be highly heterogenous; however the biosimilar etanercept exhibited similar charge and glycan heterogeneity intervals compared to the reference product. Apoptosis inhibition assay also showed that, despite the high degree of heterogeneity exhibited by both products, no significant differences exist in theirin vitroactivity. Finally, the clinical assessment conducted in RA-diagnosed patients did not show significant differences in the evaluated pharmacodynamic markers of both products. Collectively, the results from the comparability exercise provide convincing evidence that the evaluated biosimilar etanercept can be considered an effective alternative for the treatment of RA.

According to the World Health Organization, the incidence of malignant neoplasms and endocrine, blood, and immune disorders will increase in the upcoming decades along with the demand of affordable treatments. In response to this need, the development of biosimilar drugs is increasing worldwide. The approval of biosimilars relies on the compliance with international guidelines, starting with the demonstration of similarity in their physicochemical and functional properties against the reference product. Subsequent clinical studies are performed to demonstrate similar pharmacological behavior and to diminish the uncertainty related to their safety and efficacy. Herein we present a comparability exercise between a biosimilar trastuzumab and its reference product, by using a hierarchical strategy with an orthogonal approach, to assess the physicochemical and biological attributes with potential impact on its pharmacokinetics, pharmacodynamics, and immunogenicity. Our results showed that the high degree of similarity in the physicochemical attributes of the biosimilar trastuzumab with respect to the reference product resulted in comparable biological activity, demonstrating that a controlled process is able to provide consistently the expected product. These results also constitute the basis for the design of subsequent delimited pharmacological studies, as they diminish the uncertainty of exhibiting different profiles.

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’st-testPvalues 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 (TNFαneutralization) showed no differences. Overall, this data revealed the capabilities and drawbacks of each thiol quantification technique and their correlation with protein structure.