Abstract
The understanding and characterization of protein interactions is crucial for elucidation of complicated biomolecular processes as well as for the development of new biopharmaceutical therapies. Often, protein interactions involve multiple binding, avidity, oligomerization, and are dependent on the local environment. Current analytical methodologies are unable to provide a detailed mechanistic characterization considering all these parameters, since they often rely on surface immobilization, cannot measure under biorelevant conditions, or do not feature a structurally-related readout for indicating formation of multiple bound species. In this work, we report the use of flow induced dispersion analysis (FIDA) for in-solution characterization of complex protein interactions under in vivo like conditions. FIDA is an immobilization-free ligand binding methodology employing Taylor dispersion analysis for measuring the hydrodynamic radius (size) of biomolecular complexes. Here, the FIDA technology is utilized for a size-based characterization of the interaction between TNF-α and adalimumab. We report concentration-dependent complex sizes, binding affinities (Kd), kinetics, and higher order stoichiometries, thus providing essential information on the TNF-α–adalimumab binding mechanism. Furthermore, it is shown that the avidity stabilized complexes involving formation of multiple non-covalent bonds are formed on a longer timescale than the primary complexes formed in a simple 1 to 1 binding event.
Highlights
The understanding and characterization of protein interactions is crucial for elucidation of complicated biomolecular processes as well as for the development of new biopharmaceutical therapies
The flow induced dispersion analysis (FIDA) principle relies on Taylor dispersion analysis (TDA) for measuring the apparent hydrodynamic radius of a selective ligand as it binds to the analyte of interest[13]
The hydrodynamic radius of TNF-α is measured in the absence of adalimumab, the size of unbound TNF-α is measured confirming the structural integrity of the molecule
Summary
The understanding and characterization of protein interactions is crucial for elucidation of complicated biomolecular processes as well as for the development of new biopharmaceutical therapies. FIDA is an immobilization-free ligand binding methodology employing Taylor dispersion analysis for measuring the hydrodynamic radius (size) of biomolecular complexes. We report concentration-dependent complex sizes, binding affinities (Kd), kinetics, and higher order stoichiometries, providing essential information on the TNF-α–adalimumab binding mechanism. Flow Induced Dispersion Analysis (FIDA) is a novel analytical platform for size-based characterization of biomolecules including assessment of binding affinity (Kd), complex hydrodynamic radius, analyte quantification, Scientific Reports | (2021) 11:4754. The FIDA principle relies on Taylor dispersion analysis (TDA) for measuring the apparent hydrodynamic radius (size) of a selective ligand (the indicator) as it binds to the analyte (e.g., the protein/ biomolecule) of interest[13]. While AUC remains an important methodology, throughput and sample volume requirements limits its broad applicability
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