Orthogonal Testing

Orthogonal Testing designates experimental design approaches employing different analytical methods measuring the same parameters, providing comprehensive characterisation, detecting potential artefacts from individual techniques, and increasing confidence in conclusions through convergent evidence. Regulatory agencies increasingly expect orthogonal approaches confirming critical quality attributes rather than relying on single analytical methods susceptible to interfering substances or technical limitations.

Biologics characterisation exemplifies orthogonal testing importance, with complex therapeutic proteins requiring multiple analytical methods comprehensively assessing structure, function, and quality attributes. Primary structure analysis combines peptide mapping via liquid chromatography-mass spectrometry with amino acid sequencing and intact mass measurement. Higher order structure assessment employs circular dichroism, fluorescence spectroscopy, and hydrogen-deuterium exchange mass spectrometry. Glycosylation profiling combines lectin binding assays and mass spectrometry-based glycopeptide mapping. Potency assessment employs cell-based bioassays plus binding assays, with convergent results increasing confidence in product consistency. Biomarker validation requires orthogonal methods confirming measurements reflect true biological status, with protein biomarkers verified through immunoassays and mass spectrometry employing fundamentally different detection principles. As product complexity grows, orthogonal testing continues expanding throughout pharmaceutical development.