Probing in situ higher order structures of monoclonal antibodies at water-air and water-oil interfaces via high-field nuclear magnetic resonance spectroscopy for viral infections

项目总结/摘要 单克隆抗体（mAb）代表一类重要的生物治疗剂，其可以 治疗COVID-19、癌症和其他传染病。尽管他们有很大的潜力，亲- 将mAb切取、储存和/或施用到患者体内是具有挑战性的，因为 处理和给药过程中疏水界面的变化（IV袋中的空气滞留 或在注射器内部的油-水界面）可促进mAb吸附到这样的水-水界面上。 疏水性界面如果mAb改变其天然（折叠）高阶结构（HOS）， 吸附到这些界面上，其质量、安全性和有效性将受到影响， 对已经易感的患者的遗传性风险。减轻这些风险的第一步是评估 单克隆抗体（折叠或未折叠）在疏水界面的原位HOS。确定 由于体积限制，在这种界面上原位构建mAb是一个主要挑战 尺度或基于散射的微结构探测技术。在这个节目中，我们将超越 这样的限制，并使用基于NMR光谱的独特分子探测技术的组合， troscopy和动态表面张力测定法来解析mAb HOS和吸附的细节 疏水界面的动力学特别是，通过使用高场空间和光谱重新， 解决了NMR光谱，是唯一可用于通过国家高磁场 实验室，我们将动态评估1）纯单克隆抗体在疏水界面的原位HOS， 和2）它们在界面处与表面活性剂缔合的性质。我们将进行张力测量 沿着纯mAb、同位素标记mAb和mAb/表面活性剂的NMR光谱 在疏水界面处的组合。我们将测量a）动态表面张力，B）spa- 在1D 1H和2D 1H-13 C NMR谱中的局部化学位移，c） mAb，和d）在不同条件下本体中和界面处mAb的T2弛豫 (e.g.,各种mAb和表面活性剂浓度、溶液pH和离子强度）。相比之下， 根据度量（a-d）分析批量和接口的结果，团队将确定 mAb的天然HOS已通过吸附到疏水界面或其缔合物而改变， 与表面活性剂。这项研究的结果将提供第一个机械的理解 疏水界面上的单克隆抗体HOS。此外，从这项研究中获得的知识 在开发减轻mAb吸附到疏水界面的框架中是必不可少的， 其随后可用于改善患者的有效mAb部署。 1

PROJECT SUMMARY/ABSTRACT Monoclonal antibodies (mAbs) represent an important class of biologic therapeutics that can treat COVID-19, cancer and other infectious diseases. Despite their promising potential, pro- cessing, storage and/or administration of mAbs into patients is challenging because the presence of hydrophobic interfaces during processing and administration (air entrapment in the IV bags or the oil-water interface at the interior of syringes) may promote mAb adsorption to such hy- drophobic interfaces. If mAbs change their native (folded) higher order structures (HOS) upon adsorption to these interfaces, their quality, safety and efficacy will be affected, posing immuno- genicity risks to already susceptible patients. The first step in mitigating these risks is to evaluate the in situ HOS of mAbs (whether folded or unfolded) at hydrophobic interfaces. Determining the in situ structure of mAbs at such interfaces has been a major challenge due to limitations of bulk scale or scattering-based microstructural probing techniques. In this program, we will go beyond such limits and use a combination of a unique molecular probing technique based on NMR spec- troscopy and dynamic surface tensiometry to resolve the details of mAbs HOS and adsorption kinetics at hydrophobic interfaces. In particular, by using high-field spatially and spectrally re- solved NMR spectroscopy that is uniquely available to use through National High Magnetic Field Laboratory, we will assess dynamically 1) the in situ HOS of pure mAbs at hydrophobic interfaces, and 2) nature of their associations with surfactants at interfaces. We will perform tensiometry along with NMR spectroscopy on pure mAbs, isotopically labeled mAbs and mAbs/surfactant combinations at hydrophobic interfaces. We will measure a) dynamic surface tension, b) spa- tially localized chemical shifts in 1D 1H and 2D 1H-13C NMR spectra, c) diffusion coefficients of the mAbs, and d) T2 relaxation of mAbs in the bulk and at the interface under different conditions (e.g., various mAbs and surfactant concentrations, solution pH and ionic strengths). By compar- ing the results of the bulk and interface in terms of metrics (a-d), the team will determine if the native HOS of mAbs has been altered by adsorption to hydrophobic interfaces or their associa- tions with surfactants. The outcome of this study will provide the first mechanistic understanding of mAbs HOS at hydrophobic interfaces. Additionally, the knowledge gained from this research is essential in developing a framework to mitigate mAbs adsorption to hydrophobic interfaces, which can be subsequently utilized to improve efficacious mAb deployment for patients. 1