PROCESS DEVELOPMENT CASE STUDIES
Some plasmids are low yielding in the 30–42 °C inducible fermentation process because of cell lysis after plasmid induction.
We hypothesize that these plasmids are toxic to the production cell at moderate copy number, such that lysis occurs at a threshold
copy number reached shortly after plasmid induction. Consistent with this, fermentation yields (and cell bank viability) from
low yielding plasmid-containing cell lines were much higher when seed stocks were created at 30 °C, rather than at 37 °C.4
We have determined that fermentation yield with such plasmids can be improved through simple process modifications to: 1)
Increase biomass before induction by performing temperature induction at a higher OD600; or 2) Induction for a shorter duration to limit cell lysis; or 3) Induction at a lower temperature (37 °C) to reduce copy
The following case studies using seed stocks manufactured at 30 °C in strain DH5α illustrate rapid process improvement using
combinations of these modifications. In all cases, culture lysis and poor plasmid yields after the first pilot fermentation
using standard induction conditions were indicative that the plasmid was toxic to the host at moverate copy number. Volumetric
yield (mg/L plasmid) improvements ranged from 60–170% (Table 2: one process development run) to 194% (Table 3: three-fold
yield increase with two process development runs).
Table 2. Improved plasmid yield after fermentation process modification
Case Study 1
In this optimization with a proprietary 9-kb plasmid, fermentation run 1 induction was at 42 °C at 50 OD600 for 10.5 h. After the observed extensive lysis at harvest, the second fermentation was induced at 55 OD600 for 10 h (shorter induction at higher OD600). This resulted in improved harvest biomass and specific yield, resulting in a 60% improvement in volumetric yield (Table
Case Study 2
In this optimization with a proprietary 12-kb plasmid, induction at 42 °C was at 50 OD600 for 10.5 h in fermentation run 1. After the observed extensive lysis and very poor yield at harvest, the second fermentation
was induced at 37 °C at 60 OD600 for 9 h (shorter, reduced temperature induction at higher OD600). This resulted in improved harvest biomass and specific yield, resulting in a 170% improvement in volumetric yield (Table
Case Study 3
In this optimization with a proprietary 3.5-kb eukaryotic GFP expression plasmid, fermentation run 1 induction was at 42 °C
at 50 OD600 for 9 h. After the observed extensive lysis and poor yield at harvest, the second fermentation was induced at 55 OD600 for 10 h (induction at higher OD600). This resulted in improved harvest biomass and specific yield, resulting in a 72% improvement in volumetric yield (Table
Case Study 4
This was a proprietary 6-kb DNA vaccine plasmid containing a toxic influenza serotype H1 hemagglutinin gene. In the first
fermentation (run 1), culture lysis was visible after 4 h induction at 42 °C (starting OD600 was 60). Only modest plasmid specific yield improvement (1.2 to 3.2 mg/L/OD600) was observed by 30–42 °C induction resulting in a final volumetric yield of 260 mg/L (Table 3).
Table 3. Improved plasmid yield after fermentation process modification
For the first process development run (run 2), a strategy to more modestly induce copy number (using 37 °C rather than 42
°C induction) was attempted. The 37 °C induction was performed at 53 OD600 for 6 h. However, specific yield during the 37 °C stage increased from 1.0 to only 2.3 mg/L/OD600. Therefore the 37 °C stage was followed by a shift to 42 °C at 83 OD600 for 4 h. Specific yields further increased to 4.6 mg/L/OD600 (44% improvement compared to run 1) after the 42 °C stage. This improved overall volumetric yield compared to run 1 by 68%
to 436 mg/L (Table 3).
For the second process development run (run 3) the fermentation was induced for 9 h at 42 °C (starting OD600 was 50). Despite culture lysis during the last several hours of induction resulting in overall lower biomass yields, three-fold
improved overall plasmid yields were obtained (Table 3: 194% improvement in volumetric yield, and 253% improvement in specific
yield compared to run 1). This demonstrates that, although a small subpopulation of cells is sensitive to plasmid accumulation
and lysis early, the bulk of the cells are high producers with this plasmid. Optimization of the process to favor accumulation
of the high producing cells led to dramatic overall process yield improvement.
Thus, consistent with the hypothesis that these plasmids are toxic to the production cell at moderate copy number, simple
process modifications that increased biomass and delayed high copy plasmid induction dramatically improved yield.
Expression from cryptic promoters in plasmid sequences could drive copy number-dependent expression of toxic peptides (from
the sense or antisense strand) in E. coli. Alternatively, RNA sequences or structures may be involved. Surprisingly, two classes of toxic plasmids were identified
that are either 1) universally toxic or 2) conditionally toxic to a subset of cells (e.g., influenza serotype H1 hemagglutinin
plasmid, case study 4). Interestingly, an influenza serotype H3 hemagglutinin plasmid that was toxic at moderate copy number
in freshly transformed cells, but not fermentation cells, has also been identified.4 Although universal toxicity may be attributed to the production of cryptic proteins, peptides, or RNA structures that disrupt
core metabolic functions or membrane integrity, the cellular targets for conditional plasmid toxicity are unknown.