Abstract
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.
Althea Technologies
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As the fields of DNA vaccines and gene therapy mature, many companies are using high cell density production methods that
have proven to be effective in the production of recombinant proteins. However, adopting a high cell density process for DNA
production has some unique challenges. This article will examine some of the considerations that should be evaluated before
adopting high cell density fermentation for DNA production. Specifically, which type of production (batch, fed-batch, or continuous
fermentation), which media and components, and which strategies for growth control for the high cell density methods will
be discussed.
The production costs associated with developing biopharmaceuticals is an increasingly important consideration for companies
that develop these products. As products approach commercialization and companies have to reconcile the high cost of production
with relatively low reimbursement rates, this consideration receives even more attention. There are many aspects of production
that can be evaluated and optimized. Specifically, production costs may be reduced through efficient vector design, strain
selection, and optimizing upstream and downstream production processes. Significant efforts have been made to increase the
productivity of recombinant proteins produced in E. coli, including the use of high cell density fermentation processes. The progression of plasmid DNA products in development pipelines
has also prompted the application of high cell density processes in the production of E. coli–derived plasmid DNA products.
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.
