Reid, E.C.T.Q.;
(2007)
A tool-kit for in-process determination and control of structural and conformational authenticity of complex biopharmaceuticals.
Doctoral thesis , University of London.
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Abstract
The work presented here focuses on a Chinese hamster ovary cell line producing monoclonal IgG. A key challenge in the production of mAbs is to produce a consistent glycoform profile between batches, since glycosylation can impact efficacy. This work examines the effect of time of harvest and means of cell removal on the molecular structure of recombinant IgG. The glycosylation status of IgG was compared at different stages of fermentation through analysis of intact mAb using liquid-chromatography-electrospray-time-of-flight-mass-spectrometry. Heterogeneity in glycosylation patterns, as well as C-terminal lysine and N- terminal glutamine residues, could be seen in all samples and an increase in the proportion of shorter glycans was observed over time of culture, indicating time of harvest could impact upon the efficacy of the product. A shear device mimicking the feed zone of a large-scale disc stack centrifuge was used to allow better prediction of the effects of early stage cell recovery on the structural authenticity of the protein. The composition of the intracellular material, in terms of H2L2 tetramers, HL dimers and H and L single chains, is clearly different to the extracellular intact mAb, however, their relatively low concentration gives little change to the overall profile when released into the product stream. Shear rate and time seem to have little effect on the molecular structure of the mAb. The effect of holding time and temperature before cell removal was also examined. While neither seems to have an effect on the glycosylation pattern of the mAb, there was an increase in "half-antibodies" with holding time, from 0 h to 24 h, when holding at either +4 C or +37 C. This work highlights the importance of looking at the interaction between stages and the bioprocessing units as a whole rather than single steps.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | A tool-kit for in-process determination and control of structural and conformational authenticity of complex biopharmaceuticals. |
| Identifier: | PQ ETD:592746 |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by Proquest |
| UCL classification: | UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1445426 |
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