Development of new tools for the production of plasmid DNA biopharmaceuticals

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 96-104).DNA vaccines and gene therapies that use plasmid DNA (pDNA) as a vector have gained attention in recent years for their good safety profile, ease of manufacturing, and potential to treat a host of diseases. With this interest comes increased demand for high-yield manufacturing processes. Overall, this thesis aims to develop new, innovative tools for the production of plasmid DNA biopharmaceuticals. As one part of this thesis, we designed a 1-mL fed-batch microbioreactor with online monitoring and control of dissolved oxygen, pH, and temperature, as well as continuous monitoring of cell density. We used the microbioreactors to scale down temperature-induced production of a pUC-based DNA vaccine vector, pVAX1-GFP. Scaled-down processes can facilitate high-thoughtput, low-cost bioprocess development. We found that the microbioreactors accurately reproduced the behavior of a bench-scale bioreactor as long as key process parameters, such as dissolved oxygen, were held constant across scales. The monitoring capabilities of the microbioreactors also provided enhanced process insight and helped identify conditions that favored plasmid amplification. A second aspect of this thesis involved construction and characterization of a new DNA vaccine vector based on a runaway replication mutant of the R1 replicon. Runaway replication plasmids typically show increased amplification after a temperature upshift. However, we found that our new vector, pDMB02-GFP, gave higher yields during constant temperature culture at 30"C, reaching a maximum of 19 mg pDNA/g DCW in shake flasks. We gained mechanistic insight into this behavior by measuring RNA and protein expression levels of RepA, a plasmid-encoded protein required for initiation of replication at the R1 origin. Through these studies we found that RepA levels may limit plasmid amplification at 42*C, and relieved this limitation by increasing RepA translation efficiency via a start codon mutation. We also scaled up production of pDMB02-GFP at 300C from 50-mL shake flasks to 2-L bioreactors. Initial scale up efforts resulted in increased growth rate compared to the shake flasks, accompanied by very low plasmid yields. Decreasing the growth rate by limiting dissolved oxygen increased plasmid specific yield and emerged as a viable strategy for maintaining productivity during scale up.by Diana M. Bower.Ph.D

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Development of new plasmid DNA vaccine vectors with R1-based replicons

Background: There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results: In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions: Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.MIT-Portugal ProgramPfizer Inc

Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process

The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well. Keywords: microbioreactors; Escherichia coli; plasmid biopharmaceuticals; bioprocess developmen

Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains. Keywords: Central metabolism; Escherichia coli; Metabolic engineering; Plasmid biopharmaceuticals; Strain engineerin