Chmielowski Rebecca A, Mathiasson Linda, Blom Hans, Go Daniel, Ehring Hanno, Khan Heera, Li Hong, Cutler Collette, Lacki Karol, Tugcu Nihal, Roush David
Merck & Co., Inc., Biologics and Vaccines, Downstream Process Development & Engineering, 2015 Galloping Hill Road, Kenilworth, NJ, 07033, USA.
GE Healthcare, R&D BioProcess, Life Sciences, Björkgatan 30, 751 84, Uppsala, Sweden.
J Chromatogr A. 2017 Dec 1;1526:58-69. doi: 10.1016/j.chroma.2017.10.030. Epub 2017 Oct 13.
Advances in cell culture technology have enabled the production of antibody titers upwards of 30g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC). Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31g/L. Initial experimentation showed a challenge with determining a difference in change in UV280nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beer's Law given a known absorbance of the media background and impurities or experimentally determined using various UV280nm cell pathlengths. Based on these results, a 0.35mm pathlength at UV280nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations.
细胞培养技术的进步使得抗体滴度能够达到30g/L以上。这些高产细胞培养系统可能会因柱负载较低而导致下游纯化过程中的生产力瓶颈，尤其是在初级捕获色谱步骤中。有助于解决这一瓶颈的替代色谱解决方案包括使用连续处理系统，如周期性逆流色谱（PCC）。最近的研究提供了优化和改进PCC设计的方法，以处理高达约3g/L的细胞培养滴度。本文定义了一种PCC的连续上样策略，该策略与细胞培养背景无关，适用于高达约31g/L的细胞培养滴度。初步实验表明，确定细胞培养进料与单克隆抗体（mAb）浓度之间的UV280nm信号变化差异（即ΔUV）具有挑战性。进一步研究发现，不含抗体的细胞培养原料的UV280nm吸光度超出了给定细胞光程的线性检测范围。额外的实验表明，对于各种细胞培养进料，ΔUV的差异可以根据培养基背景和杂质的已知吸光度通过比尔定律进行理论预测，也可以使用各种UV280nm细胞光程进行实验测定。基于这些结果，选择UV280nm处0.35mm的光程进行动态控制，以克服背景信号。孔扩散模型与实验前沿分析数据显示出良好的一致性，这导致在3C-PCC实验中定义了20%至70%的ΔUV设定点范围。在各种细胞培养进料和高达约41g/L的滴度之间，洗脱池的产品质量是可接受的。结果表明，为实现稳健的动态控制并维持3C-PCC产量，应采用以下ΔUV设定点：对于滴度大于10g/L的情况，根据高细胞浓度进料（HCCF）的UV吸光度，设定点约为20%至45%；对于滴度高达10g/L的情况，设定点约为45%至70%，与HCCF的UV吸光度无关。本文提出的策略和结果表明，连续色谱步骤中的柱负载可以独立于细胞培养原料和滴度进行动态控制，并允许在下游连续操作中增强过程控制。
