Willis Leon F, Toprani Vishal, Wijetunge Sashini, Sievers Annette, Lin Laura, Williams Jeanine, Crowley Tom J, Radford Sheena E, Kapur Nikil, Brockwell David J
Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds UK LS2 9JT.
Pharmaceutical Research and Development, Pfizer Inc. 1 Burtt Road, Andover, Massachusetts, USA, 01810.
J Pharm Sci. 2023 Oct 28. doi: 10.1016/j.xphs.2023.10.031.
The development time of therapeutic monoclonal antibodies (mAbs) has been shortened by formulation platforms and the assessment of 'protein stability' using 'developability' assays. A range of assays are used to measure stability to a variety of stresses, including forces induced by hydrodynamic flow. We have previously developed a low-volume Extensional Flow Device (EFD) which subjects proteins to defined fluid flow fields in the presence of glass interfaces and used it to identify robust candidate sequences. Here, we study the aggregation of mAbs and Fc-fusion proteins using the EFD and orbital shaking under different formulations, investigating the relationship between these assays and evaluating their potential in formulation optimisation. EFD experiments identified the least aggregation-prone molecule using a fraction of the material and time involved in traditional screening. We also show that the EFD can differentiate between different formulations and that protective formulations containing polysorbate 80 stabilised poorly developable Fc-fusion proteins against EFD-induced aggregation up to two-fold. Our work highlights common platform formulation additives that affect the extent of aggregation under EFD-stress, as well as identifying factors that modulate the underlying aggregation mechanism. Together, our data could aid the choice of platform formulations early in development for next-generation therapeutics including fusion proteins.
治疗性单克隆抗体（mAb）的研发时间已通过制剂平台以及使用“可开发性”分析方法对“蛋白质稳定性”的评估而缩短。一系列分析方法被用于测量蛋白质在各种应激条件下的稳定性，包括流体动力流所产生的作用力。我们之前开发了一种小体积拉伸流装置（EFD），该装置能使蛋白质在玻璃界面存在的情况下经受特定的流体流场，并利用它来识别稳健的候选序列。在此，我们使用EFD和在不同制剂条件下的振荡培养来研究单克隆抗体和Fc融合蛋白的聚集情况，探究这些分析方法之间的关系，并评估它们在制剂优化中的潜力。EFD实验使用了传统筛选所需材料和时间的一小部分，就鉴定出了最不易聚集的分子。我们还表明，EFD能够区分不同的制剂，并且含有聚山梨酯80的保护性制剂可使难以开发的Fc融合蛋白在抵抗EFD诱导的聚集方面稳定程度提高两倍。我们的工作突出了在EFD应激条件下影响聚集程度的常见平台制剂添加剂，同时也确定了调节潜在聚集机制的因素。总之，我们的数据有助于在开发早期为包括融合蛋白在内的下一代治疗药物选择平台制剂。
