Development of new tools for the production of plasmid DNA biopharmaceuticals

摘要: DNA vaccines and gene therapies that use plasmid (pDNA) as a vector have gained attention in recent years for their good safety profile, ease of manufacturing, potential to treat host diseases. With this interest comes increased demand high-yield manufacturing processes. Overall, thesis aims develop new, innovative tools the production biopharmaceuticals. As one part thesis, we designed 1-mL fed-batch microbioreactor with online monitoring control dissolved oxygen, pH, temperature, well continuous cell density. We used microbioreactors scale down temperature-induced pUC-based vaccine vector, pVAX1-GFP. Scaled processes can facilitate high-thoughtput, low-cost bioprocess development. found accurately reproduced behavior bench-scale bioreactor long key process parameters, such were held constant across scales. The capabilities also provided enhanced insight helped identify conditions favored amplification. A second aspect involved construction characterization new based on runaway replication mutant R1 replicon. Runaway plasmids typically show amplification after temperature upshift. However, our pDMB02-GFP, gave higher yields during culture at 30°C, reaching maximum 19 mg pDNA/g DCW shake flasks. mechanistic into by measuring RNA protein expression levels RepA, plasmid-encoded required initiation origin. Through these studies RepA may limit 42°C, relieved limitation increasing translation efficiency via start codon mutation. scaled up pDMB02-GFP 30°C from 50-mL flasks 2-L bioreactors. Initial efforts resulted growth rate compared flasks, accompanied very low yields. Decreasing limiting oxygen specific yield emerged viable strategy maintaining productivity up. Thesis Supervisor: Kristala L. Jones Prather Title: Associate Professor Chemical Engineering