Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647183
Title: A micro-scale study of primary clarification options for the processing of a Fc-fusion protein
Author: Lau, E. C.
ISNI:       0000 0004 5365 6249
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Abstract:
This thesis describes the use of ultra scale-down (USD) methods to address the primary recovery challenges posed by the processing of a novel, highly glycosylated Fc-fusion protein expressed in a Chinese Hamster Ovary (CHO) cell line. A feeding strategy coupled with an early cell culture harvesting time point delivered a broth whereby the Fc-fusion protein (~ 8.5 mg L-1)was minimally contaminated with "near-neighbour" variants of the glycosylated fusion protein. Such variants are not purified from the product during protein A recovery, making their reduced presence a critical feature of process success. However, USD shear studies demonstrated the need for low shear stress processing of the cell culture broth if the Fc-fusion protein was not to be contaminated by such variants due to intracellular release during cell removal. USD centrifugation studies showed that the processing of cell culture material using a continuous centrifuge equipped with low stress feed zones would avoid such contaminants. USD depth filtration, again a low stress operation,produced well-clarified process fluids without thecontamination of glycosylated variants. In addition, the charged filter material facilitated the reduction of host cell contaminants, such as DNA (reduced by ~ 50 %) and lipids (reduced by ~ 40 %), in the process fluidsto a greater extent than achieved by centrifugation. The predictionsfrom both centrifugation and filtrationstudies were successfully verifiedat pilot-scale. Depth filtration operated at constant flux rate was used as an alternative to constant pressure operation used in the USD studies. The preliminary analysis showed similarpredicted filter capacities provided the filtrate quality based on % solids remaining was the key design parameter. Again, the benefits of low shear stress operation were realised with no release of Fc-fusion protein contaminants. Finally, preliminary studies of packed beds of "big" beads for protein A affinity capture of the Fc-fusion protein directly from cell culture broth were carried out. Almost complete flow through of the cells was achieved when loading the cell culture into the packed beds and the majority of the Fc-fusion protein was recovered. Again, the characteristics of low shear stress operation were observed with no cell damage or release of glycosylated variants in the column flow-through fractions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.647183  DOI: Not available
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