Abstract
Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEG–amine) or conjugation with positively charged amino acids (PEG–lysine, PEG–arginine, and PEG–histidine) were studied to increase the selectivity of PEG–dextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEG–dextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination.
Highlights
The increasing development of molecular biotechnology and molecular therapies, such as non-viral gene therapy and DNA vaccines, is reflected in an imperative demand for large amounts of plasmid DNA with a stringent clearance of impurities [1,2].In both cases, pDNA plays a very important role as a non-viral vector
It was demonstrated that positively charged amino acids that were conjugated to poly(ethylene glycol) (PEG) chains could be used as affinity ligands for the purification of pDNA in
The commercial polymer PEG–amine was shown to have the capacity to biorecognise the molecules of pDNA
Summary
The increasing development of molecular biotechnology and molecular therapies, such as non-viral gene therapy and DNA vaccines, is reflected in an imperative demand for large amounts of plasmid DNA (pDNA) with a stringent clearance of impurities [1,2]. In both cases, pDNA plays a very important role as a non-viral vector. PDNA vectors can stimulate humoral and cellular immune responses to a specific antigen, allowing for the development of DNA vaccination [5] This was recently in the spotlight, with several. Most of them are still in clinical trials, one plasmid-based vaccine for COVID-19 was recently approved in
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
