A Review on Bioanalytical Methods for Quantifying and Structural Determination of Therapeutic Monoclonal Antibodies

Abstract

Therapeutic monoclonal antibodies require robust and validated bioanalytical methods to support pharmacokinetic evaluation, pharmacodynamic modeling, immunogenicity characterization, biosimilar comparability, and commercial quality assurance. The macromolecular nature of these tetrameric glycoproteins characterized by a high molecular mass of approximately 150 kDa, extensive post-translational modifications, variable glycan micro-heterogeneity, and susceptibility to chemical and physical degradation presents significant analytical challenges across all stages of biopharmaceutical development. Bioanalytical workflows rely on selective sample preparation methodologies, including organic solvent and acid precipitation, solid-phase extraction, immobilized Protein A or Protein G affinity chromatography, and targeted ligand or anti-idiotypic immunocapture to extract the analyte from complex physiological fluids while minimizing matrix suppression. Quantitative assessment leverages ligand-binding assays such as enzyme-linked immunosorbent assays for high-throughput screening, whereas liquid chromatography coupled with tandem mass spectrometry offers orthogonal molecular specificity through the multiple reaction monitoring of surrogate peptides generated via site-directed enzymatic proteolysis. Advanced structural characterization incorporates ultra-high-performance liquid chromatography, size-exclusion chromatography, and native high-resolution mass spectrometry across intact, middle-down, and top-down methods to resolve size variants, charge distribution profiles, disulfide linkages, and critical quality attributes. Persistent technical challenges include non-specific matrix effects, circulating anti-drug antibody complexation, and digestion-induced modifications. Multi-attribute methods, automated microfluidics, ion mobility spectrometry, and alternative microsampling can enhance analytical throughput and regulatory concordance