Orthogonal biosimilarity testing of etanercept: A green and white analytical approach

Biosimilars offer safe and effective treatment options for patients with chronic diseases at potentially lower cost while maintaining equivalent safety, efficacy, and quality relative to innovator products. Biologics are structurally complex and sensitive molecules that require extensive physicochemical, structural, and functional characterization to support biosimilarity development and assess interchangeability. This could be done by ensuring biosimilars exhibit structural and functional equivalence to their reference products. In this study, a head-to-head comparative analytical biosimilarity orthogonal protocol was used for the evaluation of critical quality attributes (CQA) that define the performance of etanercept (a fusion protein) biosimilar in comparison to its innovator, with a novel integration of green and white analytical chemistry assessment, to evaluate the sustainability of the used orthogonal biosimilarity protocol. The analytical assessment methods used were based on peptide mapping-reverse phase HPLC, size exclusion HPLC, dynamic light scattering (DLS), reducing and non-reducing Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and reverse phase (RP-HPLC), direct UV spectrophotometry, and Enzyme-Linked Immunosorbent Assay (ELISA) technique were used to determine the content and protein binding activity of both products, respectively. The results obtained from the biosimilarity assessment showed similar profile for both products under normal conditions in terms of peak areas, retention times, apparent molecular weight bands, and binding affinity. The environmental sustainability of the studied protocol was assessed using Green Certificate-modified eco-scale evaluation, Green Analytical Procedure Index (GAPI), and Analytical Greenness metric (AGREE), in addition to the recently developed red, green, and blue 12-mode that was used to evaluate the whiteness features. Ensuring that biosimilarity evaluations meet both regulatory and environmental international standards.