April 3, 2023 PAO-04-23-NI-01
small circular DNA molecules found in bacteria and some other microbes. Engineered plasmids were i
100–1,000 g of plasmid DNA could be required per year to meet the demand formore than 1 kg of pDNA
Owing to the specialized nature and unique set of skills required for pDNA manufacturing, most production takes place at contract development and manufacturing organizations (CDMOs).5 In fact, between 80% and 90% of plasmids used for biopharmaceutical applications are produced by CDMOs.3 Despite the expansion and/or addition of capacity by leading service providers, insufficient production space remains an issue. Sponsors seeking pDNA production support can wait longer than 12 months to gain access to GMP plasmid production capacity.6 This issue is only expected to increase, as developers are more and more frequently using GMP-grade plasmids for the production of therapeutic products not just for late-phase clinical trials and commercial production, but for early-phase studies.7
Escherichia coli bacteria. As understanding of the mechanisms involved in plasmid production have increased, the design of fed-batch processes using carefully engineered E. coli strains that provide increased yields of pDNA has been possible.8 Advances in strategies around cell lysis— which is required to free the generated plasmid DNA from the E. coli cells — have also contributed to higher plasmid recoveries. Processes, as a result, are becoming increasingly sophisticated.
agitation, pH, temperature, dissolved carbon dioxide, pressure, and foam formulation — is essential to ensure the highest possible yields.9
less than 3% pDNA, which must be separated from a wide range of impurities, including RNA, genomic DNA, host-cell proteins, endotoxin, and unwanted plasmid isoforms.10 Finally, while the 2007 FDA guidance for DNA vaccines11 requires that the supercoiled DNA content be at least 80%, the actual expectation today is for the percentage of supercoiled isoforms to be greater than 90%.2
While plasmid production is complex, the numerous CPPs that can influence plasmid titer and quality provide opportunities for improving performance. Optimizing the sequences of the inserted gene of interest (GOI) and the plasmid itself are essential.5 Culture media and cell line selection, control of the bacterial growth rate, reaction temperature, pH and dissolved oxygen content, reducing cell stress, and avoiding DNase contamination are examples of means for impacting both yield and quality.5,8 Appropriate fed-batch profiles that lower metabolic burden also contribute to higher pDNA production. Optimization of the lysis step, meanwhile, can boost plasmid recoveries. Continuously processing E. coli lysate has been shown to provide more control than traditional batch processing, resulting in reproducible production of high-quality pDNA across scales.1
More extensive use of process analytical technologies (PAT) provides a means for achieving these goals. Automation is another important strategy for realizing process control.9
E. coli strains and plasmids have on performance. The best strategy for establishing such a platform is to use a design-of-experiment (DoE) approach to fully characterize and identify all of the important CPPs for each step.
size and sequence affect process performance E. coli strains during process development in order to identify optimal solutions for different pDNA products.1 Deployment of single-use technologies from research to GMP production is also important for achieving the necessary level of flexibility and scalability needed to meet growing pDNA demand.8
Dr. Challener is an established industry editor and technical writing expert in the areas of chemistry and pharmaceuticals. She writes for various corporations and associations, as well as marketing agencies and research organizations, including That’s Nice and Nice Insight.