Increasing plasmid production efficiency

The research published in an early view article in the journal Biotechnology Progress details the integration of bacterial capture using swelling bentonite clay and a polycation followed by in situ lysis step that is followed by elution of the main genomic impurities before specific elution of the partially purified plasmid.

The increasing interest in genetic medicines such as gene therapy and DNA vaccines has led to increased needs for more efficient extraction processes from the host bacteria, often E. coli (Escherichia coli).

"From the viewpoint of designing an efficient plasmid DNA production process, integration of bacteria via filtration with in situ cell lysis would be of interest, especially if the elution of the plasmid DNA from the filter cake could be combined with partial purification," write the authors.

Plasmid DNA intended for human use needs to be free of the hosts genomic DNA, RNA and endotoxins while staying in its natural 'topoisomeric' form rather than the more linear denatured forms that can form on lysis of the bacteria, leading to the researchers using as mild conditions as possible.

After culturing the bacteria the bacterial suspension was incubated with the poly cationic ployethyleneimine (PEI) and sodium-activated bentonite for 1 hour.

The mixture was then diluted and celite (a chalk-based filter agent) added before filtration through a candle filter to create a filtration 'cake'.

The researchers then tried a series of various lysing experiments and found that using the Triton lysis method which involves heating the 'cake' to 37°C in the presence of buffers and surfactants allowed efficient rupturing of the bacterial cell walls.

However, they noticed that the plasmid was eluted in conjunction with large amounts of RNA and genomic DNA and devised a method to bind the plasmid to the cake during the lysis step.

The researchers then added guanidinium hydrochloride to the lysis buffer solution which caused the plasmid DNA to stick to the cake.

By careful selection of the elution buffer, the plasmid DNA could be efficiently and selectively released from the filter cake, with only low amounts of RNA being eluted at the same time.

The authors note that: "for an application in gene therapy, further purification is necessary, for example by column chromatography or alternative means."