Rapid Process Development for High Yield Plasmid DNA Fed-batch Fermentation

How to reduce plasmid-mediated metabolic burden for higher yields.
Oct 31, 2009

ABSTRACT

To commercialize DNA medicines, industrial plasmid DNA manufacturing processes that meet the quality, economy, and scale requirements projected for future products are needed. We have developed cell bank and fermentation process unit operation innovations that reduce plasmid-mediated metabolic burden, enabling improved upstream production of optimal plasmids to 2.6 g/L. Application of these processes also facilitated production of otherwise unstable direct repeat containing vectors in standard E. coli host strain DH5α, eliminating the need for specialized stabilizing strains. Fermentation yields with low yield plasmids also were improved up to three-fold by using a simple fermentation process development method requiring only one to two fermentations.


Nature Technology Corporation
Plasmid DNA is increasingly finding its way into new, experimental non-viral gene therapeutics, including DNA vaccines,1 short hairpin RNA (shRNA) gene knockdown therapeutics, gene replacement vectors, and seed constructs for viral vector production.

Host Escherichia coli strains for plasmid production require recA and endA mutations.2 Several studies on various plasmid host strains indicate that plasmid yield and quality are significantly affected by the choice of host strain. However, these studies also demonstrate that plasmid production in shake flasks is poorly predictive of plasmid production in fermentation, and that a strain's performance can be affected by the fermentation process.3 DH5α is a good host strain because it consistently produces high-quality plasmid DNA in both shake flask and fermentation culture. Evaluation of the individual contributions of the DH5α host strain, plasmid backbone, and production process to plasmid production has demonstrated high-yield plasmid fermentation is largely determined by process and vector-intrinsic factors, not strain-intrinsic factors.4

LOW METABOLIC BURDEN FERMENTATION

Constitutive, high plasmid copy number throughout the fermentation process is not necessary or desirable. Maintaining a high copy number during biomass accumulation creates an environment in which plasmid-free cells have a significant growth advantage.

Plasmid-mediated metabolic burden can inhibit biomass growth and may lead to stability or quality problems (e.g., deletions or dimers) with many plasmids. Thus, maintaining low cell stress or metabolic burden during biomass accumulation, and inducing high plasmid copy numbers for only the final portion of the process, leads to superior volumetric plasmid yields while preserving plasmid quality.


Figure 1. Illustration of the temperature-inducible fed-batch process. The initial temperature setpoint is 30 °C to keep the plasmid copy number low during growth. Feed medium containing concentrated glycerol is added according to an exponential feeding strategy to control cellular growth at about 0.12/h.
Gene therapy or DNA vaccine plasmids typically contain the pUC (temperature-sensitive) origin of replication. This temperature sensitivity is especially useful for inducing high-yield plasmid production in fermentation using a temperature-inducible, fed-batch process (Figure 1) with an optimized semi-defined medium.5 The initial setpoint temperature of 30 °C maintains the plasmid at a low copy number. This reduces metabolic burden and cell stress during bacterial growth such that the majority of the final biomass is formed under low stress conditions.


Figure 2. Time profile of a fermentation of DH5α containing a 6.5 kb DNA vaccine plasmid, temperature induced at 29 h
After sufficient biomass accumulation, the temperature is shifted to 42 °C and growth continues for up to approximately one doubling of cell mass. This process has resulted in volumetric plasmid yields up to 2,590 mg/L, and specific plasmid DNA yields up to 51 mg/g dry cell weight (DCW), or 5% of the total DCW.4 Figure 2 shows a time profile from a fermentation that reached a plasmid yield of 2,100 mg/L.