Upstream advances have led to increased mAb titers above 5g/L in 14-day fed-batch cultures, however, this has come with the challenge 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. Due to the high cell density, there is also an 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 improved process understanding of the mechanisms impurity removal in these filters. <br/>Three depth filter trains were compared, one Millistak+ HC (cellulose and DE) and two Millistak+ SP (polyacryclic and silica) series where the throughput is also controlled. This is followed by a 100 cycles Protein A chromatography lifetime study. Preliminary analysis of the results shows that an increase of 35% throughput (possibly due to additional layers in the SP primary filter) and led to a 58% higher DNA concentration at 50L/m<sup>2 </sup>in the secondary filters. DNA breakthrough in the primary depth filters was not affected by filter type. The higher throughput is also associated with a 36% average increase in DNA concentration in the Protein A eluates.  Confocal imaging of the secondary depth filters shows similar amount of DNA and cell debris being captured overall. However when the throughput is increased, more is retained in the layer 2, while in the other conditions it is more evenly distributed between the 2 layers. <br/>Additional work will be presented on confocal data used to identify unfolded protein resulting from repeated CIP. The effect of depth filter type and operation on the low pH hold post-Protein A chromatography and subsequent 0.2µm filtration will also be described. Taken together the results show the potential for depth filters based on new materials to improve impurity removal prior to chromatography when appropriately sized in a two stage filtration process.

Analysis of depth filter performance using breakthrough studies and confocal microscopy and its interaction with protein A resin lifetime and low pH viral inactivation

ACS Spring 2022

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