Pharmaceutical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA.
Analytical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA.
Pharm Res. 2018 Sep 25;35(11):220. doi: 10.1007/s11095-018-2500-8.
Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs.
Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [C-H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments.
Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution.
The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.
Fc 结构域是单克隆抗体（mAb）和基于 Fc 的融合蛋白的一个组成部分。在 Fc 结构域中进行工程突变是实现治疗性 mAb 所需效应功能和临床疗效的常用方法。然而，分子工程（通过改变糖基化模式或 Fc 结构域中的氨基酸突变）是否会影响 Fc 结构域的高级结构，从而导致 mAb 聚集倾向增加，这仍然存在争议。
在这里，我们使用来自具有相似糖基化模式的选定辉瑞 mAb 的 Fc 结构域的 NMR 指纹分析来解决这个问题。具体来说，我们使用 Fc 片段的高分辨率 2D [C-H] NMR 光谱，该光谱指纹甲基侧链残基，以探究高级结构与 mAb 储存稳定性的相关性。还进行了热量热法研究以评估 mAb 片段的稳定性。
与 NMR 指纹分析不同，热量热法获得的完整 mAb 和片段（Fc 和 Fab）的热融温度与 mAb 的聚集倾向没有任何相关性。尽管这些 mAb 的序列同源性>97%，但 NMR 数据表明 Fc 结构域的高级结构可能是动态的，并且可能导致溶液中存在独特的构象。
这些 mAb 的整体糖基化模式相似，这些构象可能与 Fc 结构域的固有可塑性有关，并且可能作为 mAb 整体聚集的早期瞬态。
