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10 BioPharm International ® Emerging Therapies eBook September 2023 www.biopharminternational.com Cell and Gene Therapies methods rely on viruses, notably AAV and LV tech- nologies, but other non-viral methods are also used, including nanoparticles, cationic lipids, polymers, exosomes, and even naked DNA. There are alternative technologies that exist for de- livering genes t hat do not involve pDNA but use pDNA as starting material. Minicircles, for example, do not contain any microbial sequence, and their smaller size contributes to higher efficiency of downstream applications, such as the generation of viral particles. However, processes to produce good manufacturing practice (GMP) material would need significant fur- ther development to make them a competitive option compared to pDNA manufacturing. Messenger RNA is another example. There are some advantages that mRNA has over pDNA, as it is directly translated into a protein, with no requirement to reach the nucleus to accomplish transcription. In addition, mRNA is considered to be safer, as in contrast to pDNA, it cannot integrate into the genome, if one disregards retroposition. However, mRNA does have the disad- vantage of being less stable than pDNA, and re-dosing is likely to be required to achieve the desired results. Other therapeutic applications of pDNA include as a starting material for RNA-derived therapies such as self-amplifying RNA (saRNA) based products. Plas- mids even have potential as direct-acting drugs, with the naked pDNA molecule applying directly into the target tissue. The main dissimilarities between different pDNAs across various applications relate to their sequence and size. If the pDNA is being used to generate viral particles, then it will contain repeated sequences, al- lowing for the integration of the gene of interest into the viral capsid. In contrast, for mRNA, it will contain a long polyA sequence to increase the stability of the transcribed mRNA molecule. And with saRNA, the size of the plasmid is relatively larger than it would be for other applications. Making plasmids The manufacture of pDNA is normally performed using bacterial fermentation in Escherichia coli (E. coli.) The first step is the development of a fully char- acterized E. coli cell bank, and these cells will then be used to replicate the pDNA molecule. High titers of pDNA can be achieved via a fed-batch fermentation process, and once this is complete, the cells are lysed in a controlled manner using an alkaline treatment, and the cell debris then removed. Next, the clarified cell lysate is concentrated, and ultrafiltration/diafiltration (UF/DF) is used to purify it further, ahead of a chromatographic step to remove any residual material from the E. coli. A further UF/ DF step, with filtration down to 0.2 microns, com- pletes the process. There are challenges involved in the manufacture of pDNA, with the main ones including scalability, effi- ciency, and processing times, tied to the impact of dif- ferent raw materials in the process. There are also the inherent limits of E. coli. But with a sufficiently flexible platform process, the critical process parameters can be optimized for each individual product. Quality of product is also a big consideration in any manufacturing process, and pDNA has a number of requirements that need to be met. Firstly, there is the plasmid's identity to confirm, and ensuring that there are no mutations in its sequence, which would typically be performed using a method such as Sanger sequencing. Safety is another important factor, in- cluding the presence of any E. coli residuals, such as genomic DNA, residual RNA or host cell proteins, and particular care should be taken to ensure there are no endotoxins, because of their immunogenicity. No other microorganisms should be present, either, including mycoplasma. There are various orthogonal methods that can be used for this to ensure accuracy, precision, and reliability, and these would be common to those used to analyze other biologic materials. Finally, there is the pDNA's content and purity to as- certain. Its activity is linked to different isoforms, with the supercoiled form generally considered to be the active one. There is, therefore, a quality requirement for the final product to contain at least 80–85% of the active supercoiled isoform. Depending on the analysis requirements, orthologous methods such as agarose gel electrophoresis based densitometry or capillary gel electrophoresis can be utilized. Regulatory requirements When plasmids are used directly as therapies, it is clear that current GMP (CGMP) principles apply, as As these products are still in their relative infancy, the regulatory landscape continues to evolve in parallel to the science and technology that underpins their development.