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Team:UCL London/Manufacturing/IndustryOverview
From 2011.igem.org
Contents |
Industry Overview
Plasmid technologies such as DNA vaccines are unique in the sense that they are one of the few technologies that can be created based on rational design (creating molecules with a certain function using physical models to predict their behaviour). Using this technology enables one to bypass a great deal of the research and development stages, so they can be tested during clinical trials for safety and efficacy as soon as possible. This is extremely important during epidemics when new vaccines must be developed and mass produced within a few weeks to meet public demand. Existing technologies would require half a year to produce vaccines at this scale and would employ technology and infrastructure that is immensely expensive, which drives up the cost to the consumer and makes it available only to a small minority of countries.
The reason plasmid technology is so vital is that, according to Carnes and Williams (2007)[1] it has the “potential to be the most rapidly deployed vaccine platform for pandemic application.” Another setback about existing technologies is they won´t allow the production of the 0.5- 4 mg doses of plasmid required for DNA vaccinations. To develop a manufacturing process two criteria must be met:
- The fermentation yield as well as quality must be appropriate.
- Plasmid composition and final product purity must meet current standards set by regulatory bodies such as the EMA (European Medicines Agency) or FDA (Food and Drug Administration, USA).
Plasmid DNA (pDNA) used in the production of DNA vaccines are obtained using recombinant DNA (DNA assembled from various different sources, being expressed in a single host organism aka expression vector) produced using Escherichia coli (E.coli) as an expression vector. The three most critical unit operations required in plasmid processing are:
- Fermentation
- Cell Disruption
- Downstream Purification
Fermentation
The main aim of fermentation is to optimise the volumetric and specific yield of supercoiled plasmid. This former enables more cost effective, smaller scale fermentations while the latter allows for greater plasmid purity. This is beneficial for the downstream processing steps as it enables fewer purification steps, curbing processing time. Also, reducing the volume of the fermentation paste is preferable because large amounts of fermentation paste can´t be processed for cell lysis procedures, this side steps a major bottleneck. Fermentation plays a major role in plasmid quality. It is during this stage that we ensure proper optimisation of quality plasmid i.e. plasmids in their supercoiled form which the FDA recognises to be more effective than plasmids in nicked and/or open circular forms.
Cell Disruption
After we grow the engineered cells to have the desired target plasmids, in order to extract the plasmids from the cell we must break open the cells. There are two kinds of methods for doing this: physio-mechanical and chemical methods. These steps must be done while inflicting minimal damage on the desired pDNA product caused by shear stress to the cell. Cell disruption must also be done such that there is minimal release of contaminants from the cell which may degrade and/or contaminate the pDNA and causes problems when separating the product from the lysate (fluid containing the contents of the lysed cells) during downstream processing. Another reason existing cell disruption methods are unfavourable is it requires vast amounts of harsh chemicals which result in large waste streams that are an environmental hazard.
Downstream Purification
Downstream processing aims to separate the product from the remaining lysate and is currently the most expensive part of the production process. The reason it´s so expensive is that the desired product takes up a small percentage of the total fermentation broth and hence, the lysate. Separation of pDNA from the remaining lysate is especially difficult since desired pDNA is difficult to differentiate from nucleic acid dispersed throughout the lysate.
We´ve developed a arsenal of various different weapons that we can use to battle a lot of the bottlenecks present in pDNA manufacturing. Supercoiliology and Magneto- Sites work together to increase the yield and quality of the desired pDNA. Extractery is responsible for simplifying the downstream processing by finding a more effective route to lyse the cell without destroying the pDNA. Additionally it uses enzymes which digest the plasmids which do not meet a certain standard e.g. undesirable plasmid isoforms which may have formed during fermentation as well as damaged plasmids. Stresslight 2.0 and Supercoilometer are tools used to monitor the conditions faced by the plasmids in the fermenter ensuring that the cell isn´t undergoing stress posed by hypoxia (low levels of oxygen) or shear stress in the cell while Supercoilometer gauges whether or not an appropriate amount of supercoiling has been reached within the cell.
