Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
J Chromatogr A. 2011 Dec 16;1218(50):9010-8. doi: 10.1016/j.chroma.2011.09.086. Epub 2011 Oct 25.
In process development and during commercial production of monoclonal antibodies (mAb) the monitoring of aggregate levels is obligatory. The standard assay for mAb aggregate quantification is based on size exclusion chromatography (SEC) performed on a HPLC system. Advantages hereof are high precision and simplicity, however, standard SEC methodology is very time consuming. With an average throughput of usually two samples per hour, it neither fits to high throughput process development (HTPD), nor is it applicable for purification process monitoring. We present a comparison of three different SEC columns for mAb-aggregate quantification addressing throughput, resolution, and reproducibility. A short column (150 mm) with sub-two micron particles was shown to generate high resolution (~1.5) and precision (coefficient of variation (cv)<1) with an assay time below 6 min. This column type was then used to combine interlaced sample injections with parallelization of two columns aiming for an absolute minimal assay time. By doing so, both lag times before and after the peaks of interest were successfully eliminated resulting in an assay time below 2 min. It was demonstrated that determined aggregate levels and precision of the throughput optimized SEC assay were equal to those of a single injection based assay. Hence, the presented methodology of parallel interlaced SEC (PI-SEC) represents a valuable tool addressing HTPD and process monitoring.
在单克隆抗体 (mAb) 的工艺开发和商业化生产过程中，监测聚集体水平是强制性的。mAb 聚集体定量的标准检测方法基于高效液相色谱 (HPLC) 系统上的尺寸排阻色谱 (SEC)。其优点是具有高精度和简单性，但是，标准 SEC 方法非常耗时。由于平均每小时的吞吐量通常为两个样品，因此它既不适合高通量工艺开发 (HTPD)，也不适用于纯化工艺监测。我们展示了三种不同的 SEC 柱在 mAb-聚集体定量方面的比较，包括通量、分辨率和重现性。结果表明，具有亚二微米颗粒的短柱（150mm）可生成高分辨率（~1.5）和高精度（变异系数 (cv) < 1），检测时间低于 6 分钟。然后，该柱类型用于将交错样品注入与两列平行化相结合，以实现最短的绝对检测时间。通过这样做，成功消除了感兴趣峰前后的滞后时间，检测时间低于 2 分钟。结果表明，经优化的通量 SEC 检测法所确定的聚集体水平和精密度与基于单次进样的检测法相当。因此，所提出的平行交错 SEC（PI-SEC）方法是一种用于 HTPD 和工艺监测的有价值的工具。
