Novartis Global Drug Development/Technical Research & Development, Technical Development Biosimilars, Lek Pharmaceuticals d. d., Kolodvorska 27, Mengeš, Slovenia.
Jožef Stefan Institute, Jamova 39, 1000, Ljubljana, Slovenia.
Pharm Res. 2020 Jan 6;37(2):27. doi: 10.1007/s11095-019-2733-1.
The ability to predict an antibody's propensity for aggregation is particularly important during product development to ensure the quality and safety of therapeutic antibodies. We demonstrate the role of container surfaces on the aggregation process of three mAbs under elevated temperature and long-term storage conditions in the absence of mechanical stress.
A systematic study of aggregation is performed for different proteins, vial material, storage temperature, and presence of surfactant. We use size exclusion chromatography and micro-flow imaging to determine the bulk concentration of aggregates, which we combine with optical and atomic force microscopy of vial surfaces to determine the effect of solid-liquid interfaces on the bulk aggregate concentration under different conditions.
We show that protein particles under elevated temperature conditions adhere to the vial surfaces, causing a substantial underestimation of aggregation propensity as determined by common methods used in development of biologics. Under actual long-term storage conditions at 5°C, aggregate particles do not adhere to the surface, causing an increase in bulk concentration of particles, which cannot be predicted from elevated temperature screening tests by common methods alone. We also identify specific protein - surface interactions which promote oligomer formation in the nanometre range.
Special care should be taken when interpreting size exclusion and particle count data from stability studies if different temperatures and vial types are involved. We propose a novel combination of methods to characterise vial surfaces and bulk solution for a full understanding of protein aggregation processes in a sample.
在产品开发过程中，预测抗体聚集倾向的能力尤为重要，以确保治疗性抗体的质量和安全性。我们在没有机械应力的情况下，证明了容器表面在高温和长期储存条件下对三种单抗聚集过程的作用。
我们对不同的蛋白质、小瓶材料、储存温度和表面活性剂的存在进行了系统的聚集研究。我们使用尺寸排阻色谱和微流动成像来确定聚集体的体相浓度，将其与小瓶表面的光学和原子力显微镜结合使用，以确定在不同条件下固-液界面对体相聚集体浓度的影响。
我们表明，在高温条件下，蛋白质颗粒会附着在小瓶表面，导致常用的生物制品开发方法对聚集倾向的测定大大低估。在 5°C 的实际长期储存条件下，聚集体颗粒不会附着在表面，导致颗粒的体相浓度增加，这不能仅通过常用的高温筛选试验来预测。我们还确定了特定的蛋白质-表面相互作用，这些相互作用促进了纳米范围内的低聚物形成。
如果涉及不同的温度和小瓶类型，则在解释稳定性研究中的尺寸排阻和颗粒计数数据时应特别小心。我们提出了一种新的方法组合，用于对小瓶表面和体相溶液进行特征化，以全面了解样品中的蛋白质聚集过程。
