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

How to reduce plasmid-mediated metabolic burden for higher yields.
Nov 01, 2009
Volume 22, Issue 11


Figure 3. Agarose gel electrophoresis of samples from DH5α/pVLTrap fermentations from Table 1. Lane 1 is plasmid prepared directly from the seed stock, and lane 2 is plasmid from the 30–42 °C fermentation harvest (70 OD600). Lanes 3–8 are from the 37 °C throughout process, at 16 (14 OD600), 20, 24, 37, 38, and 40 (113 OD600 harvest sample) h post induction, respectively. Note the deletion product (arrow) is detected in all samples from the 37 °C fermentation.
Critically, use of the low metabolic burden process was essential to produce otherwise unstable retroviral plasmids. For example, pVLTrap, a 6.7 kilobase (kb) retrotransposon plasmid vector containing two long terminal repeats (LTRs, known to cause stability problems), was produced successfully in DH5α at 785 mg/L using the aforementioned low metabolic burden fermentation process (Table 1). However, when grown under identical conditions but at 37 °C, the region between the LTRs was entirely deleted by the end of the fermentation (Figure 3), and the truncated plasmid yield only reached 214 mg/L (Table 1).


For production of the various DNA medicines currently under investigation, it is essential that the fermentation process functions with a variety of different vector backbones and gene inserts. However, many plasmid vectors are unpredictably toxic or otherwise low yielding in standard fermentation processes. Numerous inserts also can confer low-yield plasmid production on an otherwise high-yielding plasmid backbone.

Importantly, with toxic plasmids, maintaining a low metabolic burden should begin as early as the transformation process. Propagation of cultures at 30 °C rather than 37 °C during seed stock manufacture can dramatically improve downstream fermentation yields when producing plasmids containing toxic inserts,4 presumably by reducing the copy number to limit plasmid-mediated metabolic burden during this critical step.

Table 1. Comparison of plasmid quality and yield from 37 °C throughout and 30–42 °C inducible fermentations
Use of the low metabolic burden process with 30 °C manufactured seed stocks further improved yield. A two-fold yield improvement was observed with the low metabolic burden at 30–42 °C inducible fermentation process (compared to fermentation at 37 °C throughout) with low-yielding short-hairpin RNA (shRNA) plasmids containing multiple short palindromic repeats (Table 1).

Modest yield improvement was observed with the 30–42°C inducible fermentation process compared to 37 °C throughout with a vector containing a 43 basepair (bp) palindromic repeat. Specific yields of this vector were reduced three-fold compared to a control vector without the repeat (Table 1). Reduced yields are caused by an inherent difficulty of DNA polymerases in replicating self-associating hairpin structures.

Palindromes may be deleted by intramolecular or intermolecular recombination events. The observed sequence stability of the palindromes (in both processes; Table 1) establishes that shRNA plasmids are inherently stable in DH5α fermentation cells.

Collectively, these results demonstrate plasmid production processes that use 37 °C continuously do not perform well with many unstable or suboptimal plasmids because of detrimental metabolic burden from plasmid maintenance at constitutively high levels. Using the low metabolic burden cell bank and fermentation process facilitated production of unstable plasmids such as inverted or direct repeat containing vectors in standard strains such as DH5α, eliminating the need to use specialized stabilizing host strains.

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