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Investigation of largescale production and quantification methodology of monoclonal antibodies (MAbs)

Fahey, Catherese (2002) Investigation of largescale production and quantification methodology of monoclonal antibodies (MAbs). Master of Science thesis, Dublin City University.

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Abstract

The unrivalled specificity with which an antibody binds to the antigen that has stimulated its production, forms the basis for the development of a multi-million euro biotech industry based on mammalian cell culture. However, the large-scale production of monoclonal antibodies, (MAb) is faced with a number of major problems. These include the poor yield of hybridised cells and the low productivity of MAb in culture. This thesis investigates aspects of the latter of these problems by attempting to design optimised culture conditions for growth and antibody production for a number of hybridoma producing cell lines namely the hybridoma producing anti-topoisomerase Ilex MAb, 4/2D (IgGi), the hybridoma producing MAb 1/11C (IgGi), the hybridoma producing MAb to a novel complex of cytokeratin 6 and 9, 5C3 (IgGi) and the hybridoma producing anti-mdr-1 MAb 6/1C (IgGi). Assay techniques such as Radio Immunodiffusion and ELISA were investigated to determine their reliability in quantifying monoclonal antibody in cell culture supernatant. The effect of commercially available serum free and protein free media on growth characteristics and productivity of the hybridoma producing cell lines were also examined. Finally, the environmental aspects of cell culture conditions (temperature and mixing) were considered. A new system for quantifying MAb in supernatant was evaluated. This system, involving nephelometry (a turbidity-based system) was compared with the commonly used methods of ELISA and RID. The main advantage of such a system is that it gives real time analysis of antibody productivity whereas the RID method takes 2-3 days and the ELISA takes approximately 4 hours complete. After evaluation on small-scale runs, a number of 10L fermentation runs were designed and executed to examine MAb production in two of the hybridoma producing cell lines 4/2D and 5C3 at large scale. As a result of varying temperature and agitation, the culture conditions of 37 C and lOOrpm were found to be the most efficient for cell growth and antibody productivity in all cases except the culture of 4/2D in PFHM II. In this case, the most efficient culture conditions in terms of MAb production were found to be 34 C and 200rpm. This result highlights the fact that each cell line has its own customised culture conditions. Using the optimised culture conditions at large scale, it was found that quantities (mg/L) of antibody similar to those observed at small scale were being produced. The main difference however was that peak antibody productivity was occurring at day 3 and then decreasing thereafter. The exception to this was the culture of the hybridoma producing MAb, 5C3 in protein free conditions. Not only were antibody quantities increasing throughout the culture but also the levels of antibody produced were 10-15 fold in excess of the other cell line in both SSM and PFM. The results are discussed in detail and recommendations are made for future work.

Item Type:Thesis (Master of Science)
Date of Award:2002
Refereed:No
Supervisor(s):Clynes, Martin and Moran, Elizabeth and O'Driscoll, Donnacha
Uncontrolled Keywords:antibodies; antigens; binding; monoclonal antibodies; MAb
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:17245
Deposited On:21 Aug 2012 16:07 by Fran Callaghan. Last Modified 21 Aug 2012 16:07

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