Cell Harvest

Centrifugation and microfiltration or ultrafiltration are the main harvesting options. In this case centrifugation is used as an example.

Cell Lysis (Autolysis)

A number of issues must be addressed before any existing process can be utilised for cost effective commercial plasmid manufacture. While yields from current high yield fermentation processes are acceptable, cell lysis methodologies based on alkaline and heat lysis are bottlenecks. Autolysis is a new technology for cell disruption; it has a great potential to eliminate the critical process bottlenecks in industrial plasmid DNA manufacturing.

In this novel process, host strain has been synthetically modified to express λR phage endolysin specifically during the 42°C induction phase of the inducible plasmid fermentation process. The expressed endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall. Plasmid DNA can be selectively extracted from genomic DNA and cell debris with a low salt, slightly acidic buffer, an outer membrane permeabilizing agent and a cytoplasmic membrane permeabilizing agent. According to Carnes, A.E.^[16], the purity of plasmid DNA can achieve 98.3% after acidic plasmid extraction.

Comparison between Heat-Inducible Autolysis with Alkaline Lysis and Heat Lysis

To date, the most commonly used methods for plasmid DNA recovery are alkaline lysis and heat lysis ^[7].

A critical issue with alkaline lysis methods has been the scale-up of mixing. That is because as soon as the SDS-alkali penetrates the cell there is a potential for pH damage to supercoiled plasmid DNA. If batch mixing intensity is increased, the precipitated chromosomal E. coli DNA is susceptible to shear damage ^[8].

The heat-induced lysis requires the use of lysozyme for efficient recovery of plasmid DNA. The limitation for this method is the potentially high levels of gDNA in the clarified lysate ^[7]. Besides, Lysozyme is the major expense in heat induced lysis processes, and its availability in a pandemic situation would be an issue ^[16].

Since the use of alkaline and heat lysis in large scale is highly restricted due to the difficulties involved in dealing with mixing, shear stress, and flocculation issues, new technologies such as autolysis described above could be an alternative which breaks the critical processing bottleneck ^[7].

In contrast to heat-inducible lysis and alkaline, autolysis applies synthetic biology, with an endolysin enzyme that is localized to the cytoplasm, permitting controlled cell wall disruption and plasmid release, without releasing the majority of genomic DNA and cell debris. These autolytic strains can be used without any required process modification, since heat induced expression of the endolysin and plasmid amplification can be coordinated.