Recombinant protein and plasmid DNA production using microbial expression systems is the cornerstone of many biologics manufacturing processes. High cell density (HCD) methods are commonly used for these processes because of the advantages they provide, including high cell productivity, high concentration levels, and lower setup costs. Additionally, the increased yield in product translates to a more cost-effective and shorter overall project length.
High cell density (HCD) processes have many inherent advantages. Specifically, HCD reduces the time required in a fermenter in either a contract manufacturing facility or in captive space. Secondly, plasmid processes can produce yields as high as 1–2 g/L, which is a marked improvement over standard fermentation methods, and this results in fewer required fermentation runs. Various HCD process parameters have been evaluated in the development of a large-scale process for the production of clinical-and commercial-grade plasmid DNA.
Vector Design and Strain Selection
The first consideration in designing an efficient process should be the vector design and host cell line selection. Plasmid size is a critical criteria in vector design. All nonessential sequences should be removed so that the plasmids are as small as possible. In addition to the potential regulatory and therapeutic challenges, many larger plasmids also create manufacturing hurdles by placing a metabolic burden on the host cell line by reducing resources required for plasmid replication. This, in turn, results in reduced yields.
Formulation is also a consideration that must be made at the stage of vector design. Although naked DNA has inherent advantages over more complex formulations, including safety and simplicity, transfection efficiencies are generally low. There are strategies that may be used during vector design, including translational engineering, that can remove secondary structure and add translational pause sites, which may increase expression.
The use of ampicillin and other B-lactams is discouraged because of hypersensitivity reactions in some patients.1 If they are used, the FDA often requires a justification and details of the precautions that should be taken to prevent these reactions.1 Kanamycin is the most commonly used means of selection. Cell line selection is one of the most critical criteria in the design of HCD processes. Bacterial cell lines offer an advantage over mammalian cell lines in that many cell lines may be evaluated simultaneously and very quickly for either protein or plasmid production. In addition to evaluating yields, the purity and quality of the plasmid in various cell lines will impact manufacturing decisions. In our experiments, some cell lines, such as DH10β, and to a lesser extent, DH5α, have been found to be consistently higher producing in HCD processes.