4.1.1 Viral vectors used as gene therapies and in the production of gene-modified cell therapies are manufactured from plasmids — small, typically circular, double-stranded DNA units found in many bacterial species.
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4.2.1 The origin of replication is a DNA sequence on the plasmid that regulates the number of plasmid copies per cell. The pUC plasmids are a derivative of the plasmid pBR322, which contains a natural origin of replication and is controlled to about 25 copies per cell. A deletion in the rop gene on pBR322 removes the restriction of replication and increases the overall copy number.[68] Although pBR322 is consistently maintained at about 25 copies per cell, the synthetic plasmids can achieve over 150 copies per cell. Thegene, however, also ensures the 25 copies of pBR322 are equally distributed to daughter cells during cell division, whereas high-copy plasmids are distributed randomly to daughter cells.
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4.3.1 The manufacture of plasmids is performed using competent Escherichia coli cells, of which there are many host strains available. DH1, DH5, DH5a, DH10B, JM109, and HB101 are the most commonly employed strains for plasmid production.[73] However, E. coli strains with unique genotypes for cloning unstable inserts (commercially known as Stbl competent cells) are often used for transgene plasmids (plasmids used for viral vector manufacturing that contain the gene of interest, or GOI) flanked by ITR sequences.
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4.4.1 After a production strain is created, the cell is cultured in a fermenter to very high cell densities, which can readily achieve >25% solids in well-optimized processes. The key to achieving high cell densities is to optimize the feed schedule so that acetate does not accumulate in the medium. Glucose is the most common carbon source used in industrial production, but glycerol feeds are also employed because E. coli produces acetate from glycerol at a lower rate than it produces acetate from glucose. Feed schedule optimization is often a critical part of E. coli upstream process development, and there are multiple approaches. Feed schedule algorithms are often proprietary among big pharma and CDMOs but a few academic groups have published extensively on feed schedule optimization, and their work is familiar to many industrial researches.[75][76][77]
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4.5.1 Once cell paste has been harvested by centrifugation, the cells must be lysed and the plasmid DNA purified. The basic chemistry of purification is the same at bench scale and large scale: alkaline lysis, neutralization with a chaotrope, precipitation of the large (chromosomal) DNA, and chromatography.
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4.6.1 The quality of the plasmids used to produce viral vectors directly impacts the quality and safety of gene and gene-modified cell therapies based on those vectors. The FDA advises against the use of research-grade plasmids in clinical trials that were produced in nonsegregated research laboratories without proper cleaning and segregation procedures.[78]
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4.7.1 Different types of cell banks are used to produce plasmids of different quality grades. A research cell bank (RCB) is typically generated in a nonsegregated research laboratory and used to produce research-grade plasmids intended for use in laboratory studies only. Non-GMP cell banks are used for the production of non-GMP plasmid DNA of higher quality than research-grade materials, generated in a segregated laboratory, and intended for use in preclinical and early-stage clinical trials. GMP master cell banks (MCBs) are used to produce GMP plasmids for use in early- or late-stage clinical trials and commercial products.
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4.8.1 Fill/finish operations must meet rigorous sterility requirements in accordance with aseptic manufacturing procedures and today are often performed using fully disposable systems. The FDA only requires GMP filling of plasmid DNA in the viral vector manufacturing process for phase III clinical trial material or production of a commercial product. Plasmid DNA used in the manufacture of viral vectors for phase I and II clinical trials does not have to be filled under GMP quality standards
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