Antibiotic-free plasmid production system
Supports manufacture of highly supercoiled plasmid DNA molecules and higher yield of plasmid DNA, without the use of antibiotics in manufacture
Plasmid DNA is used in various applications that involve nucleic acid gene transfer protocols in routine laboratory research; as drugs in gene therapy, in vaccines, in RNAi and all applications where the informational DNA molecule is used to direct transcription of RNA and/or of proteins/peptides derived from the RNA or when the plasmid is used as an adjuvant. Use of plasmid DNA in all of the applications is predicated on the ease and cost of manufacture. Current technologies for the isolation have certain inherent issues and limitations and therefore limits further improvements in quality, yield and safety of plasmid DNA-based products.
Problem to be solved:
- Wild-type bacterial strains and mutant strains that are routinely employed in manufacturing plasmid DNA are incapable of producing high copy number plasmids by routine methods that involve alkali treatment, thus significantly reducing the yield of total plasmid DNA, and more significantly reducing the supercoiled plasmid content. In certain applications, nicked and linear plasmid DNA molecules that result from the alkali treatment of the supercoiled form during manufacture have certain theoretical safety implications when used as drugs, due to the increased probability of chromosomal insertion (integration) of plasmid DNA sequences into host chromosomes thereby increasing the probability of oncogene activation.
- The primary reason for loss of supercoil plasmid DNA during manufacture is due to the nicking of the plasmid DNA backbone. During fermentative conditions that increase plasmid copy number, the bacterial cell is unable to keep up its production to the demands of deoxy-mononucleotides, which are the building blocks of DNA. When the pools of available deoxynucleotides become rate limiting, the DNA polymerase responsible for plasmid replication and increasing plasmid copy numbers begin to utilize ribonucleotides. This phenomenon is well documented (commonly referred to as “ribo-substitution”), and it is also well documented that ribonucleotides are unstable in alkali. Upon alkali treatment the plasmid DNA backbone is nicked due to a phosphodiester bond cleavage at positions where ribonucleotides replaced the deoxynucleotides.
- Selection and manufacture of plasmid DNA molecules requires that the plasmid possess a selectable marker to ensure that plasmid is retained in the bacterial cell during fermentation. Routinely, an antibiotic resistance gene is inserted into the plasmid, which confers resistance to the bacterium during fermentation to that specific antibiotic. In order to eliminate bacteria that have lost the plasmid during fermentative growth, manufacturers routinely add the antibiotic, thus enriching plasmid-bearing cells in the fermenter. Due to increased incidence of antibiotic resistant bacteria in the ecosystem due to wide spread indiscriminate use of antibiotics; regulators are demanding that the use of antibiotics in food and drug manufacture be eliminated in the near future.
Veritas Bio Solution:
- Veritas Bio’s technology addresses both of these problems simultaneously. The technology utilizes a complementation system that not only eliminates the use of an antibiotic resistance gene as a selectable marker on the bacterium that harbors the plasmid, but also includes a gene encoded in the plasmid that causes uncontrolled production of deoxy-mononucleotides, thus preventing ribo-substitution in plasmid DNA molecules, and thereby not only significantly increasing plasmid DNA yields by nearly ten fold but also increasing supercoil content to greater than 98%.