Antibody-based therapeutics are the fastest-growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. Although the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small-molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, thereby altering therapeutic effect and disposition in ways that are incompletely understood. Although excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B₂) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy is able to measure diffusive B₂-values directly in undiluted serum. Apparent second virial coefficient (B_2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the approach of isolating specific components and completing independent cross-term virial coefficient measurements may not be an effective approach to characterizing nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general and advance the development of therapeutic antibodies and other protein-based therapeutics.

基于抗体的疗法是市场上增长最快的药物类别，用于治疗侵袭性癌症、慢性自身免疫性疾病和许多其他疾病状态。尽管治疗性抗体的特异性、亲和力和多功能性可以提供优于传统小分子药物的优势，但它们的开发和优化可能更具挑战性和耗时。这部分是因为用于体外表征的理想制剂缓冲系统不能充分反映这些药物一旦施用于患者时所经历的拥挤的生物环境（血清、内体腔等）。这种环境会干扰抗体与其抗原和受体的结合，以及同源和异源聚集，从而以不完全理解的方式改变治疗效果和处置。尽管排除体积效应被认为有利于结合，但在拥挤条件下与共溶质的弱相互作用会抑制结合。第二维里系数 (B ₂ ) 参数量化了这种弱相互作用，并且可以通过多种技术在稀溶液中确定，但在复杂生物流体中的类似方法还没有很好地建立。在这里，我们证明荧光相关光谱能够直接测量未稀释血清中的扩散B _2值。表观第二维里系数 ( B _{2, app}) 对血清中抗体的测量表明，吸引和排斥相互作用之间平衡的变化会极大地影响全球的非理想性。此外，我们的研究结果表明，分离特定成分和完成独立的跨项维里系数测量的方法可能不是表征血清中非理想性的有效方法。这里介绍的方法可以丰富我们对生物环境对蛋白质一般影响的理解，并促进治疗性抗体和其他基于蛋白质的疗法的发展。