高通量小角度X射线散射揭示了与高浓度抗体粘度相关的有效结构因子转变

摘要(中文译文)

高浓度单克隆抗体（mAb）制剂常因黏度升高而受到限制，这主要由蛋白质-蛋白质相互作用驱动，使生产复杂化并限制皮下给药。在药物发现阶段进行早期黏度风险评估至关重要，但传统测量方法需要大量样品且缺乏高通量能力。在此，我们开发了一种高通量小角X射线散射（SAXS）方案，可在稀释浓度下检测mAb的自缔合，从而实现对高浓度黏度的早期预测性洞察。对21种在pH 6.0组氨酸缓冲液中配制的mAb进行了同步辐射SAXS测量。对初始10种mAb在1–150 mg/mL范围内进行分析，发现在低q区域指示颗粒间相互作用的有效结构因子转变始终出现在25 mg/mL以下。随后，利用自动化液体处理和流通池在1–25 mg/mL范围内对另外11种mAb进行分析，以实现高通量筛选。高黏度mAb在浓度≤10 mg/mL时表现出可检测的低q上扬，而低黏度mAb则表现为下降。基于有效结构因子转变的分类标准在150 mg/mL时准确分类了所有高黏度和低黏度mAb，为传统方法提供了一种可扩展、样品高效的替代方案。这些结果拓展了近期关于SAXS对短程吸引力浓度依赖性敏感性的发现，证明其可在低于先前报道的浓度下出现。本研究呈现了迄今为止在单一制剂中报道的最全面、最多样的mAb SAXS数据集，为开发和验证能够更准确捕捉主导高浓度溶液行为的分子间相互作用的粗粒化模型提供了宝贵资源，从而有助于合理的抗体工程和改善可开发性。

Abstract

High-concentration monoclonal antibody (mAb) formulations are often constrained by elevated viscosity, largely driven by protein-protein interactions, which complicates manufacturing and limits subcutaneous delivery. Early viscosity risk assessment is essential during discovery, yet traditional measurements require large sample volumes, and lack high-throughput capability. Here, we develop a high-throughput small-angle X-ray scattering (SAXS) protocol to detect mAb self-association at dilute concentrations, enabling early predictive insights into high-concentration viscosity. Synchrotron SAXS measurements were conducted for 21 mAbs formulated in a histidine buffer at pH 6.0. An initial subset of 10 mAbs analyzed across 1-150 mg/mL revealed that effective structure factor transitions in the low-q region, indicative of interparticle interactions, consistently emerged below 25 mg/mL. Subsequently, 11 additional mAbs were analyzed at 1-25 mg/mL using automated liquid handling and flow cells to enable high-throughput screening. High-viscosity mAbs exhibited detectable low-q upturns at concentrations ≤10 mg/mL, whereas low-viscosity mAbs showed downturns. A classification criterion based on effective structure factor transitions accurately classified all high- and low-viscosity mAbs at 150 mg/mL, offering a scalable, sample-efficient alternative to conventional methods. These results extend recent findings on the concentration-dependent sensitivity of SAXS to short-range attractions, demonstrating that they can emerge at lower concentrations than previously reported. This study presents the most comprehensive and diverse SAXS dataset for mAbs reported to date within a single formulation, providing a valuable resource for developing and validating coarse-grained models that can more accurately capture intermolecular interactions governing high-concentration solution behavior, thereby enabling rational antibody engineering and improved developability.

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