Bioprocess development for plasmid-based vaccine production

Clarence Ongkudon
2017
Plasmid DNA (pDNA) vaccine is a promising vaccine technology, with better safety profile, more economical production and transport logistics, than conventional viral vaccines. Most importantly, pDNA vaccines elicit different immune responses including antibody-mediated, CD4 T-cell-mediated and CD8 T-cell-mediated immune responses, for defending against viral infections and cancer. The increasing number of preclinical and clinical trials on plasmid vaccines has triggered the need to make more in
more » ... eed to make more in less time. Recent developments in the production of plasmid therapeutics involve the establishment of innovative and cost effective methods as well as simplified operations. This dissertation reports fundamental studies essential to the development of a rapid economically-viable plasmid production system which is cGMP-compatible. Optimisation of upstream bacterial fermentation and continuous downstream purification of the plasmid vaccine fraction are the main aspects considered in the project of this dissertation. Process variables required to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in E. coli DH5α were investigated. A cGMP-compatible method offering the capacity to continuously produce homogeneous supercoiled pDNA from clarified bacterial lysate using a monolithic adsorbent was developed. The method involved optimisation of the adsorbent characteristics, ligand functionalisation and chromatographic process conditions. The feasibility of using free metal ions to preferentially precipitate endotoxins (LPS) from a clarified plasmid DNAcontaining bacterial lysate was investigated. Screening of various free metal ions for effective endotoxin removal and optimisation of process conditions, such as pH, ion concentration, temperature and incubation time, using central composite design experiments were performed. The potential and advantages of using Zn2+-induced LPS aggregation as a secondary pDNA purification method was validated by studying the interaction of Zn2+ [...]
doi:10.4225/03/5890168bd46e2 fatcat:yezj76e6ufbtdm26ufr24fcwv4