Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341889
Title: Control of recombinant protein productivity and quality in mammalian cell culture
Author: Rendall, Mark Harcus
ISNI:       0000 0001 3513 8470
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2000
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Abstract:
Various bioprocess parameters such as choice of host cell, or changes in the cell culture environment including: elevated ammonia or presence of serum, can influence the N-glycosylation of recombinant proteins with possible alterations in stability, antigenicity and clearance rate in vivo. The combination of capillary isoelectric focusing, HPLC and MALDI-mass spectrometry enabled elucidation of the composition and putative structure of the N-glycosylation of a model recombinant protein, TIMP-1. Additionally, a novel serial dual column assay was demonstrated for the rapid (< 45 mins) and routine quantification of TIMP-1 sialic acid isoforms. Cell line specific changes in the N-glycosylation of TIMP-1, produced by glutamine synthetase amplified Chinese hamster ovary (GS-CHO) and GS-NSO cell lines indicated that NSO TIMP-1 associated N-glycans exhibited reduced sialylation with significantly elevated levels of potentially immunogenic N-glycolyl neuraminic acid residues as opposed to the predominant n-acetyl neuraminic acid form. NSO, but not CHO, TEMP-1 N-glycans also had termini ending in immunogenic a1-3 linked galactose residues. A comparison of CHO recombinant protein production and N-glycosylation in 7 % serum-supplemented and serum-free media suggested that spinner-flask cultures in the latter had reduced overall cell growth (as measured by cumulative cell hours) but a higher overall cellular TIMP-1 productivity. Few changes were observed in sialylation due to variable serum content. Increases in the CHO intracellular sugar nucleotide pool of UDP-HexNAc (UDP-N- acetylglucosamine + UDP-N-acetylgalactosamine) through artificially elevated 1 Introduction ammonia concentrations coincided with reduced TIMP-1 N-glycan sialylation but predominantly unchanged antennarity. Culture supplementation with 20 mM N-acetyl-mannosamine (ManNAc), a precursor for sialic acid synthesis resulted in raised intracellular CMP-sialic acid levels and increased TIMP-1 sialylation. Elevated ammonia induced decreases in TIMP-1 sialylation were largely reversed with artificially increased intracellular CMP-NeuAc levels through ManNAc coaddition. High intracellular CMP-NeuAc overcoming UDP-GlcNAc mediated inhibition of CMP-NeuAc transport into the Golgi is a likely mechanism for this. A non-specific, pH determined reduction in sialylation is also likely.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.341889  DOI: Not available
Keywords: Biotherapeutic
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