Department of Biochemical Engineering, University College London, London, UK.
Research and Development, FUJIFILM Diosynth Biotechnologies (FDB), Billingham, UK.
Biotechnol Prog. 2023 May-Jun;39(3):e3329. doi: 10.1002/btpr.3329. Epub 2023 Feb 23.
Upstream advances have led to increased mAb titers above 5 g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilizing these to remove process-related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/ml). It focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. An increase in loading of the primary-synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary-synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems.
上游技术的进步使得在 14 天补料分批培养中抗体滴度超过 5g/L。这伴随着更高的细胞密度和与过程相关的杂质，如 DNA 和宿主细胞蛋白 (HCP)，这给下游操作带来了挑战。深层过滤仍然是收获 CHO 细胞培养物的流行选择，并且人们有兴趣利用这些在收获阶段去除与过程相关的杂质。收获阶段的操作也已被证明会影响 Protein A 层析步骤的性能。此外，制造商正在寻求摆脱纤维素和硅藻土 (DE) 等天然材料，以提高过滤器的一致性和供应安全性。因此，人们对深层过滤有了更多的了解和认识的需求。本研究考察了深层过滤材料和加载量对使用具有工业相关性的高细胞密度进料（4000 万细胞/ml）的 Protein A 树脂寿命的影响。它侧重于通过突破研究和一种用于原位成像污染物的新型共焦显微镜方法来保留与过程相关的杂质，如 DNA 和 HCP。初级合成过滤器的加载量增加了三分之一，导致二级过滤器中 DNA 更早突破，在 50L/m 的通量下，DNA 浓度增加了一倍以上。对深层过滤器的共焦成像表明，随着加载量的增加，污染物被推向过滤器结构的深处。初级合成过滤器中的另外两层在初级和次级过滤器中都形成了更好的压力分布，但对保留 HCP 或 DNA 没有帮助。使用合成滤器组时，最终滤液的滤液澄清度（以 OD600 测量）降低了 1.6 倍。这也与 Protein A 柱进料中的沉淀有关。在使用合成滤器组的情况下，100 个循环后树脂的共焦成像显示，DNA 在珠粒外部的积聚与合成滤器组有关，这可能导致传质问题。
