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Scale-Down Characterisation of Post-Centrifuge Flocculation Processes and the Study of its Impact upon Downstream Processing during Mammalian Cell Antibody Production

Espuny Garcia del Real, G; (2016) Scale-Down Characterisation of Post-Centrifuge Flocculation Processes and the Study of its Impact upon Downstream Processing during Mammalian Cell Antibody Production. Doctoral thesis , UCL (University College London). Green open access

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Abstract

The high demand for biopharmaceuticals has been met thanks to improved upstream productivity. Downstream, which has not advanced at the same pace, calls for new clarification strategies to cope with the increased process-related impurities associated with upstream improvements. This thesis has focused on the implementation of polyelectrolyte flocculation between centrifugation and depth filtration during the primary recovery of a monoclonal antibody Chinese hamster ovary (CHO) cell process. A high-throughput, automated ultra scale-down (USD) flocculation methodology was developed with industrial relevance in mind for process development activities. The impact flocculation had on depth filtration and protein A chromatography performance was subsequently investigated and supported by analytical assays and techniques to assess impurity removal. Cell densities at time of harvest > 20x10^6 cells/mL were critical for flocculation to occur on centrifuged CHO cell cultures. Flocculant addition time limited the flocculation scale-up success between two vessels with different geometrical ratios and working volumes (1.5 L and 800 µL). The influence of mixing time scales (macromixing, mesomixing and micromixing) on this variable defined the USD flocculation scale-up basis, which was: predominance of micromixing in the vessels (flocculant addition time scale-up) and constant power input per unit volume (impeller speed scale-up). The implementation of constant pressure, single-layer USD filtration methods with flocculated feeds was precluded due to the low filter capacity values obtained (< 35 L/m^2). In contrast, constant flux filtration with multi-layer depth filters at laboratory scale reached manufacturing-scale filter capacity values (> 490 L/m^2). Flocculation achieved larger depth filtration capacities and lower process-related impurities before protein A chromatography when compared to the current manufacturing processing option. Post-protein A chromatography, the clear eluate obtained with flocculation contrasted with the turbid eluate of the current processing option. Mass spectrometry data confirmed the turbid eluate was mainly constituted of negatively charged CHO cell protein precipitates.

Type: Thesis (Doctoral)
Title: Scale-Down Characterisation of Post-Centrifuge Flocculation Processes and the Study of its Impact upon Downstream Processing during Mammalian Cell Antibody Production
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
UCL classification: UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
URI: http://discovery.ucl.ac.uk/id/eprint/1531984
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