Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.

中文翻译：

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静电相互作用调节IgG1和IgG4P抗体的差异聚集倾向，并通知带电荷的残基取代，以改善显影性。

天然状态聚集是治疗性抗体开发中的重要问题。对mAb天然状态聚集机制的增强了解将允许基于序列的选择和设计具有改进的可开发性的治疗性mAb。我们研究了静电相互作用如何影响七个人IgG1和IgG4P mAb同种型对的自然状态聚集，每对均具有相同的可变域，对于每个IgG1和IgG4P构建体而言，可变域都不同。在代表生理条件的pH 7.4和代表常用储存条件的pH 5.0下测定相对聚集倾向。我们的工作表明，相对于恒定域的净电荷状态，可变域的净电荷状态主要负责IgG1和IgG4P mAb的不同自然状态聚集行为。该观察结果表明，多结构域蛋白的全球净电荷不是聚集倾向的可靠预测指标。此外，我们在可变域的框架内展示了一种设计策略，以减少被鉴定为易于聚集的mAb的自然状态聚集倾向。重要的是，用替代的人类种系残基替代特定鉴定的残基以优化Fv电荷，导致pH 5.0和7.4时聚集潜力降低，从而提高了可开发性。该观察结果表明，多结构域蛋白的全球净电荷不是聚集倾向的可靠预测指标。此外，我们在可变域的框架内展示了一种设计策略，以减少被鉴定为易于聚集的mAb的自然状态聚集倾向。重要的是，用替代的人类种系残基替代特定识别的残基以优化Fv电荷，导致pH 5.0和7.4时聚集潜力降低，从而提高了可开发性。该观察结果表明，多结构域蛋白的全球净电荷不是聚集倾向的可靠预测指标。此外，我们在可变域的框架内展示了一种设计策略，以减少被鉴定为易于聚集的mAb的自然状态聚集倾向。重要的是，用替代的人类种系残基替代特定识别的残基以优化Fv电荷，导致pH 5.0和7.4时聚集潜力降低，从而提高了可开发性。