Both conformational and colloidal stability of therapeutic proteins must be closely monitored and thoroughly characterized to assess the long-term viability of drug products. We characterized the IgG1 NISTmAb reference material in its histidine formulation buffer and report our findings on the higher order structure and interactions of NISTmAb under a range of conditions. In this paper we present the analysis of experimental small-angle scattering data with atomistic molecular simulations to characterize the monodisperse dilute solution of NISTmAb. In part II we describe the characterization of the NISTmAb at high protein concentration (Castellanos et al. 2018). The NISTmAb was found to be a flexible protein with a radius of gyration of 49.0 ± 1.2 Å in histidine formulation buffer using a variety of neutron and X-ray scattering measurements. Scattering data were then modeled using molecular simulation. After building and validating a starting NISTmAb structure from the Fc and Fab crystallographic coordinates, molecular dynamics and torsion-angle Monte Carlo simulations were performed to explore the configuration space sampled in the NISTmAb and obtain ensembles of structures with atomistic detail that are consistent with the experimental data. Our results indicate that the small-angle scattering profiles of the NISTmAb can be modeled using ensembles of flexible structures that explore a wide configuration space. The NISTmAb is flexible in solution with no single preferred orientation of Fc and Fab domains, but with some regions of configuration space that are more consistent with measured scattering profiles. Analysis of inter-domain atomistic contacts indicated that all ensembles contained configurations where residues between domains are ≤ 4 Å, although few contacts were observed for variable and C _H 3 regions.

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必须密切监测治疗性蛋白质的构象和胶体稳定性，并对其进行彻底表征，以评估药物产品的长期生存能力。我们在组氨酸制剂缓冲液中鉴定了IgG1 NISTmAb参考物质，并报告了我们在一系列条件下对NISTmAb的更高阶结构和相互作用的发现。在本文中，我们通过原子分子模拟对实验性小角度散射数据进行分析，以表征NISTmAb的单分散稀溶液。在第二部分中，我们描述了高蛋白浓度下NISTmAb的表征（Castellanos等人，2018年））。使用多种中子和X射线散射测量，NISTmAb是一种柔性蛋白质，在组氨酸制剂缓冲液中的回转半径为49.0±1.2Å。然后使用分子模拟对散射数据进行建模。在从Fc和Fab晶体坐标建立并验证了起始NISTmAb结构后，进行了分子动力学和扭转角蒙特卡洛模拟，以探索NISTmAb中采样的构型空间，并获得具有原子细节的结构体与实验相符的集合体。数据。我们的研究结果表明，NISTmAb的小角度散射轮廓可以使用探索广泛配置空间的柔性结构体来建模。NISTmAb在解决方案中非常灵活，没有Fc和Fab结构域的单个首选取向，但具有一些配置空间区域，这些区域与测得的散射曲线更加一致。对域间原子接触的分析表明，尽管在变量和C上几乎没有观察到接触，但所有团簇都包含结构域之间残基≤4Å的构型。 _H 3个地区。

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