Bioanalytical assays for clinical similarity assessment of biotherapeutics: evolving platforms and regulatory...

The rapid expansion of biosimilars following the patent expiry of biologic therapies has increased the need for robust bioanalytical strategies capable of supporting biosimilarity assessment and regulatory decision-making. Within the totality-of-evidence framework, bioanalytical assays play a pivotal role in generating comparative pharmacokinetic (PK), pharmacodynamic (PD), and immunogenicity data that reduce residual uncertainty and demonstrate comparability between a biosimilar candidate and its reference product. The selection of appropriate analytical methodologies is therefore critical to ensuring reliable evaluation of clinical performance, immunogenicity risk, and therapeutic equivalence.This review critically evaluates the application of major bioanalytical platforms used in biosimilar development, including ligand binding assays (LBA), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and hybrid immunoaffinity-LC-MS/MS approaches. Their respective strengths, limitations, and regulatory relevance are discussed in the context of PK quantification, PD biomarker assessment, anti-drug antibody (ADA) detection, and neutralizing antibody (NAb) evaluation. Comparative analysis focuses on key assay performance characteristics such as sensitivity, specificity, selectivity, dynamic range, multiplexing capability, susceptibility to matrix and drug interference, and operational complexity. The contribution of each platform to addressing residual uncertainty during biosimilarity assessment is also examined.Furthermore, the review highlights recent technological advances that have improved assay performance and expanded the analytical toolbox available for complex biologics and emerging biosimilar modalities. A practical decision-making framework is proposed to guide assay selection according to study objectives, biomarker availability, molecular complexity, immunogenicity risk, and the degree of residual uncertainty remaining after analytical similarity assessment. Literature was identified through systematic searches of PubMed and Google Scholar covering publications from 2005 to 2026, with emphasis on recent developments in bioanalytical technologies and evolving regulatory expectations. This review provides a biosimilar-focused perspective on the strategic integration of bioanalytical approaches to support efficient development programs, strengthen the totality of evidence, and facilitate evidence-based regulatory submissions.