Upstream developments have led to increased mAb titres above 5g/L in 14-day fed-batch cultures, however this has come with the disadvantage of cell density exceeding 20 million cells/mL. Depth filtration remains a popular choice for harvest of CHO cell culture for bioreactors up to 2000L and single use facilities. However due to the high cell density there is also increase of process related impurities, such as cell debris, host cell protein (HCP) and DNA. This can affect the capacity of the depth filters and lead to carry over of impurities to Protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence there is a need for process understanding of impurity removal mechanisms. In this work the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. The CHO cell culture was initially harvested using Millistak+ D0HC primary depth filter for both secondary filter conditions. The viability of the cell culture is also altered to examine its effect on depth filter performance. Additionally a novel method was developed to image the fouled depth filters under a confocal microscope where PicoGreen and Nile Red were used to stain fouled depth filters for DNA and debris, respectively. Flux, tested at 75, 100 and 250 LMH, w as found to affect the maximal throughput but no significant changes were seen in the HCP and DNA breakthrough. However a drop in cell culture viability, from 87% to 37%, led to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability. The X0SP filter has a 30% higher maximal throughput, which can be explained by the confocal imaging where the debris and DNA is distributed differently in the layers of the filter pods, with more of the second tighter layer used in the X0SP.

Breakthrough of process related impurities during depth filter harvest of high cell density CHO culture

ACS Fall 2021

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