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 (K_d), 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.

蛋白质相互作用的理解和表征对于阐明复杂的生物分子过程以及开发新的生物药物疗法至关重要。通常，蛋白质相互作用涉及多重结合、亲合力、寡聚化，并且取决于局部环境。考虑到所有这些参数，当前的分析方法无法提供详细的机械表征，因为它们通常依赖于表面固定，无法在生物相关条件下进行测量，或者没有结构相关的读数来指示多个结合物种的形成。在这项工作中，我们报告了使用流动诱导分散分析 (FIDA) 在体内类似条件下对复杂蛋白质相互作用进行溶液内表征。FIDA 是一种无固定化配体结合方法，采用泰勒色散分析来测量生物分子复合物的流体动力学半径（大小）。在这里，FIDA 技术用于对 TNF-α 和阿达木单抗之间的相互作用进行基于大小的表征。我们报告了浓度依赖的复杂大小、结合亲和力（K _d )、动力学和更高阶的化学计量，从而提供有关 TNF-α-阿达木单抗结合机制的重要信息。此外，研究表明，与在简单的 1 比 1 结合事件中形成的初级复合物相比，涉及形成多个非共价键的亲合力稳定复合物的形成时间更长。