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

通过高场核磁共振波谱技术在水-空气和水-油界面原位探测单克隆抗体的高阶结构以检测病毒感染

• 批准号: 10593377
• 负责人: Hadi Mohammadigoushki
• 金额: $ 22.49万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-17 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593377
• 关键词: Adsorption; Affect; Air; Autoimmune Diseases; Binding; Biological Response Modifier Therapy; COVID-19; COVID-19 treatment; Cardiovascular Diseases; Chemicals; Communicable Diseases; Development; Diffusion; Formulation; Foundations; Frequencies; Future; Grant; Higher Order Chromatin Structure; Hydrophobicity; In Situ; Ionic Strengths; Isotope Labeling; Kinetics; Knowledge; Laboratories; Life; Location; Magnetic Resonance Imaging; Malignant Neoplasms; Maltose; Measurement; Measures; Middle East Respiratory Syndrome Coronavirus; Modeling; Molecular; Molecular Probe Techniques; Monoclonal Antibodies; NMR Spectroscopy; Nature; Neutrons; Nuclear Magnetic Resonance; Oils; Outcome Study; Patients; Pharmaceutical Preparations; Polysorbates; Predisposition; Proteins; Relaxation; Research; Resolution; Risk; Roentgen Rays; SARS coronavirus; Safety; Spectrum Analysis; Structure; Surface; Surface Tension; Syringes; System; Techniques; Temperature; Testing; Therapeutic; Time; Transportation; Viral; Virus Diseases; Water; bioprocess; cancer therapy; experience; immunogenicity; improved; indexing; interfacial; macromolecule; magnetic field; novel; programs; quantum; risk mitigation; surfactant; therapeutic protein

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

项目概要/摘要 单克隆抗体 (mAb) 是一类重要的生物治疗药物，可以 治疗 COVID-19、癌症和其他传染病。尽管它们潜力巨大，但 对患者进行单克隆抗体的处理、储存和/或给药具有挑战性，因为单克隆抗体的存在 处理和给药过程中疏水界面的影响（静脉输液袋中的空气滞留） 或注射器内部的油水界面）可能会促进单克隆抗体对此类液体的吸附 恐惧界面。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、安全性和功效将受到影响，造成免疫- 对已经易感的患者存在基因性风险。减轻这些风险的第一步是评估 mAb（无论是折叠还是未折叠）在疏水界面处的原位 HOS。确定 由于体积的限制，单克隆抗体在此类界面处的原位结构一直是一个重大挑战 基于尺度或散射的微观结构探测技术。在这个计划中，我们将超越 这种限制并结合使用基于核磁共振谱的独特分子探测技术 Troscopy 和动态表面张力测定法解析 mAb HOS 和吸附的细节 疏水界面处的动力学。特别是，通过使用高场空间和光谱重新 解决了核磁共振光谱法，该光谱法只能通过国家高磁场使用 实验室，我们将动态评估 1) 纯 mAb 在疏水界面的原位 HOS， 2）它们与界面表面活性剂缔合的性质。我们将进行张力测量 以及纯 mAb、同位素标记 mAb 和 mAb/表面活性剂的 NMR 光谱 疏水界面处的组合。我们将测量a）动态表面张力，b）spa- 1D 1H 和 2D 1H-13C NMR 谱中的局部化学位移，c) 的扩散系数 mAb，以及 d) 不同条件下 mAb 本体和界面处的 T2 弛豫 （例如，各种 mAb 和表面活性剂浓度、溶液 pH 值和离子强度）。通过比较 根据指标 (a-d) 计算批量和接口的结果，团队将确定是否 mAb 的天然 HOS 已通过吸附到疏水界面或其相关物而改变 与表面活性剂的作用。这项研究的结果将提供第一个机制理解 mAb HOS 在疏水界面上的分布。此外，从这项研究中获得的知识 对于开发减轻单克隆抗体对疏水界面吸附的框架至关重要， 随后可用于改善患者单克隆抗体的有效部署。 1