Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626660
Title: Host cell protein characterisation during the downstream processing of monoclonal antibodies from mammalian cells
Author: Tarrant, R. D. R.
ISNI:       0000 0004 5362 8424
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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Abstract:
The biopharmaceutical industry is at a pivotal moment of its short history, with ‘blockbuster’ monoclonal antibody (mAb) biosimilars emerging and the ‘next generation’ of molecules beginning to make their mark. Concomitantly, regulatory constraints on manufacturers have amplified, with processes now requiring in-depth characterisation. Scale-down mimics of process steps and supportive analytical methods are therefore becoming indispensable. The work presented in this thesis has focussed on the downstream processing of mammalian cell derived mAbs. With industrial relevance considered throughout, a scale-down primary recovery and chromatography process was implemented. This was supported by ELISA, mass spectrometry and gel-based methods to investigate host cell proteins (HCPs), which are rigorously controlled process-related impurities. The interface of upstream and downstream processing was identified as a critical aspect of bioprocessing which can dictate impurity profiles. Preliminary work demonstrated that small particle debris and cell properties, such as viability and size, all contribute to primary recovery performance. Subsequent investigations of mild hypothermic cell culture showed that cell membrane rigidity also plays an important role, as centrifugation clarification was up to 4% greater for temperature adapted cells. However, HCPs were adversely affected, increasing by ~50%. To further understand harvest material properties, a polyethylenimine flocculation process was developed. This improved centrifugation performance, by reducing shear susceptibility, and increased subsequent depth filtration capacity up to 2.1 fold. Benefits to purification were also demonstrated, with DNA and negatively charged HCPs being removed. Protein A chromatography was the final focus of work, owing to its prominence in purification. Four different resins were evaluated; non-specific adsorption of HCPs was only pronounced for the resin with a controlled pore glass back-bone, but ‘product association’ was significant for all. To help analyse this sub-set of HCPs, an immuno association disruption methodology was developed and may prove invaluable in future characterisation efforts to improve product purity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.626660  DOI: Not available
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