Abstract：
Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.
摘要：
治疗性单克隆抗体(mAb)对于治疗多种疾病至关重要。免疫球蛋白G（IgG）是mAb的最主要形式，但在生产过程中易于聚集。IgG聚集体的检测和去除是耗时且费力的。色谱法通常是纯化生物药物的中心,并且是mAb生产中必不可少的。蛋白质纯化系统通常配备有用于监测pH值的检测器，UV吸光度,和导电性，便于净化过程的优化和控制。然而,目标产品和污染物种的特定在线检测，如骨料，目前不可能使用对流技术。在这里，我们展示了一种新颖的光纤在线传感器，基于局域表面等离子体共振（LSPR），用于在亲和层析期间特异性检测IgG和IgG聚集体。将具有蛋白A传感器芯片的流动池连接到亲和柱的出口,所述亲和柱连接到以实验室规模至中试规模操作的三个不同色谱系统。在使用pH梯度和等度洗脱的蛋白A柱上的纯化期间,在线分析含有各种IgG浓度和聚集体含量的样品。由于亲和力的影响，IgG聚集体在与传感器芯片结合后显示出比单体更慢的解离动力学。证明了在样品之间检测聚集体浓度低于1％和聚集体含量差异小于0.3％的可能性。骨料的在线检测可以避免耗时的离线分析，并促进自动化和过程强化。
