Fermentation

Comparison between Batch Fermentation and Fed-Batch Fermentation (for DNA vaccine production)

Fed-batch fermentation is widely applied in industrial production of DNA vaccine production due to the controllable growth rate by adjusting the addition of limiting nutrient ^[1]. Studies found that lower growth rates lead to increased plasmid copy numbers, higher growth rates in fermentation are associated with lower percentages of supercoiled plasmid and plasmid instability ^[2][3]. Also, the conversion of substrate to biomass is very efficient in fed-batch fermentation.

In contrast, control of growth rate is difficult in batch fermentation as the specific growth rate will be the maximum growth rate during exponential phase. As discussed above, high growth rate is undesirable for plasmid production.

Gyrase

Some limitations of current manufacturing process:

• The unique physical properties of plasmid DNA have raised problems related to industrial manufacture. The relatively higher molecular weight and low compactability make them more susceptible to shear and flow stress ^[4].
• Besides, the FDA recognizes that open-circular, linear, and nicked forms may be less effective therapeutically than supercoiled DNA ^[5]. Those other forms can be very difficult to separate from the supercoiled plasmid during purification.

When Supercoiliogy is applied:

• A study done by Catanese Jr, D.J. et al,(2011) ^[6], has shown that negative supercoiled plasmid affords higher shear-resistance than either the relaxed, open circular or nicked forms of the plasmid. For instance, the DNA vectors with a size of 999bp persisted shear force generated by sonication 58% longer than its nicked counter parts. As part of the cell factory, the introduction of gyrase expression cassettes allows over-expression of gyrase, which not only increases the proportion of supercoiled plasmid, but also improves the quality of supercoiling. Plasmid ‘self-protection’ can be automatically actualized within the cell. And the integrality and functionality of the product can be safeguarded.
• The shear force resistance has been found to be inversely correlated with DNA size. Plasmid DNA above certain size (3000bp) gets degraded rapidly. Supercoiled DNA molecules have a much higher possibility to survive shearing than other topoisomers due to its reduced size. This facilitates the DNA vaccine manufacturers as more genetic information can be inserted into the plasmid vectors.
• Due to the fact that the undesired forms of plasmid DNA such as open-circular, linear, and nicked forms can be very difficult to separate from the supercoiled plasmid during purification. optimizing yield of supercoiled plasmid by over-expression of gyrase improves plasmid purity in downstream processing ^[7]. In addition, if the specific yield of supercoiled plasmid per mass of cells is very high, the total dry weight of cells per liter need not be exceptional and this reduces the downstream solids loading ^[8], which can have a significant effect on reducing processing time and investment cost.
• By over-expression of gyrase, the transfection efficiency of plasmid DNA into the patient cells can be significantly improved. This will be discussed in details in the medical session.

Magneto-Sites

Significance 1: Specificity

The general purpose of constructing a host strain with strong Gyrase Binding Site (GBS) inserted into the plasmid of interest is to raise the specificity of supercoiling by gyrase. Apart from plasmids that carry the product gene, plasmids with stress detectors are also transformed into the host cell for monitoring purpose as part of our project design. GBS has shown to exhibit a strong enhancement of gyrase-catalysed supercoiling ^[9]. This has also been proved by our experimental result. Based on the chloroquine gel electrophoresis, plasmids with a strong GBS from Mu bacteriaphage was 47.4% more supercoiled than plasmids without any GBS involved. Thus it helps keeping the proportion of non-product related plasmids in supercoiled plasmids as low as possible, which could ease the downstream purification steps considerably. Significance 1: Consistency

Also, our characterizing experiment result also demonstrates that GBS helps in improving the uniformity of supercoiled target plasmids. According to the chloroquine gel electrophoresis result, the consistency of plasmids with Mu GBS was 66.4% higher than control plasmids. Therefore, the repeatability of the bioprocess and the consistency of the product can be greatly improved, and the batch to batch variation will be less of an issue.

Stresslights 2.0

Some challenges faced by the current DNA vaccine industry:

The process development of manufacturing plasmid DNA generally starts with the selection and optimization of the fermentation conditions ^[10]. Information has been reported showing that the growth of the plasmid DNA-containing host cells on a very large scale could be a great challenge ^[8].

The extent of dissolved oxygen is one of the factors that have critical effects on any fermentation processes in general. For industrial DNA vaccine production in particular, reduced culture aeration has been proved to result in elevated DNA supercoiling ^[11,12]. Also, oxygen has been shown to play a role in plasmid stability ^[13], and the formation of nicked, relaxed, linear plasmids or multimers can also be affected by oxygenation ^[14]. Apart from the oxygen content, other change of growth conditions such as shear stress generated along the process of fermentation could also play an important role on the yield of biomass, plasmid copy number and cell specific growth rate.

Stresslights 2.0 can make a difference:

As to monitor the fermentation conditions, the traditional approach is to take samples regularly and followed by analysis, which is relatively costly, time consuming and there may be a certain chance of human error. To bypass all the mentioned disadvantages, stresslights offer a detection of the alteration of the fermentation condition and report this immediately in terms of change in fluorescence. Our project, in particular, mNark and Spy promoters are used for detection of hypoxia and shear stress respectively. Apart from producing and supercoiling plasmids, the cell acts as a sensor which has a great implication in finding an optimal condition in fermentation in order to improve both quality and quantity of supercoiled plasmid DNA. Based on UCL 2009 iGEM project, we’ve improved the sensitivity of the stresslights by adding an LVA tag to the reporter protein so that they can be degraded faster and provide more accurate stress readings.

Supercoilometer

Some limitations of current manufacturing process:

The US Food and Drug Administration (FDA) recommends that vaccines contain at least 80% supercoiled plasmid. This requirement is based on an understanding that supercoiled content is a key indicator of plasmid quality supercoiled plasmid DNA is often considered to be more effective at transferring gene expression than open circular and linear variants^[15]. However, the current conventional method of measuring the extent of supercoiling depends on chloroquine gel electrophoresis which takes at least 20 hours for a single run and samples need to be taken from the bioreactor for process monitoring. In such case, the process is time-consuming and the chance of introducing experimental error is high.

Stresslights 2.0 can make a difference:

The supercoilometer is a useful tool for the measurement and monitoring of the quality of the supercoiled plasmid being produced in vivo during fermentation. This will make the traditional protocol for mini-prepping the cell samples and running time-consuming chloroquine gels, involving ethidium bromide unnecessary. It will therefore facilitate the manufacturing process by making the superhelical-density measurement of the target plasmids much quicker, safer and automated.