Parau, Maria; (2023) Process related impurity breakthrough from depth filtration during monoclonal antibody purification. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Upstream advances have led to increased mAb titers above 5g/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. However, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. This thesis investigates the impact of high cell density (30-40 million cells/ml) feed material on traditional cellulose and DE filters compared to synthetic polyacrylate + silica depth filters. The study 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. Further investigation is carried out to understand the effects on Protein A chromatography. In a 2:1 primary: secondary depth filter scale-down model, it was found that the primary filter was the limiting step in terms of pressure and that soluble impurities were mostly removed by the secondary filters. A direct comparison of secondary synthetic and cellulose/DE filters was performed by scaling up the primary depth filter. The viability of the cell culture, and hence DNA concentration at input, was an important factor for DNA retention. HCP was not significantly removed by the depth filtration train. The confocal imaging of the secondary filter showed that cell debris and DNA foulant were distributed differently based on viability and filter type, leading to differences in the pressure profile and impurity retention. In order to gain a deeper understanding of process interaction, three different depth filtration trains were compared where the variables were filter materials and loading. The filtrate was used in a scale-down Protein A chromatography lifetime study and a low pH hold with 0.2μm filtration. Results show that a 36% loading increase in the primary synthetic filter negatively. affected DNA retention in the secondary filter. Confocal imaging of the depth filters showed that the foulant was pushed down through the filter 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. Increased solids in the filtrate were associated with the synthetic filter trains, as was 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.

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