abstract
The large-scale production of therapeutically targeted-deoxyribonucleic acid (DNA) has passed through several challenges, postponing the tangible implementation of an effective, economic and sustainable manufacturing system. Such challenges comprise the need to develop an integrative downstream process able to extract, purify and long-term preserve DNA, whilst reducing the risk of degradation by endonucleases that would compromise their effectiveness as therapeutic products. In this work, three-phase partitioning (TPP) systems formed by the application of aqueous biphasic systems (ABS) composed of several biocompatible cholinium-based ionic liquids (ILs), are proposed for the separation of double stranded DNA (dsDNA) from the endonuclease deoxyribonuclease I (DNase I). By taking advantage of the tailor-made properties of ILs, dsDNA can be completely extracted to the IL-rich phase, whereas DNase I is precipitated at the ABS interphase. The ABS/TPP formed by IL cholinium glycolate ([N111(2OH)][Gly]) fulfills the aim of this work, i.e. at ensuring the technical viability of IL-based ABS/ TPP for the "one-pot" extraction, purification and long-term preservation of dsDNA. The results reveal the po-tential of this system to be applied in the bioprocessing of DNA, particularly relevant when envisioning DNA-based therapeutic products.
keywords
3-PHASE PARTITIONING ILTPP; AQUEOUS BIPHASIC SYSTEMS; PLASMID DNA; POLY(ETHYLENE GLYCOL); POLYETHYLENE-GLYCOL; CHEMICAL-STABILITY; 2-PHASE SYSTEMS; TEMPERATURE; RECOVERY; RNA
subject category
Engineering
authors
Dinis, TBV; Valente, AI; Tavares, APM; Sousa, F; Freire, MG
our authors
Ana Paula Mora Tavares
Assistant Researcher
Mara Guadalupe Freire Martins
Coordinating Researcher
Teresa B. V. Dinis
PhD StudentProjects
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
CICECO - Aveiro Institute of Materials (UIDP/50011/2020)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
acknowledgements
The authors would like to acknowledge the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MEC (PIDDAC) and CICS-UBI projects UIDB/00709/2020 & UIDP/00709/2020 financed by national funds through the FCT/MCTES. Ana P.M. Tavares acknowledges the FCT for the research contract CEECIND/2020/01867 and Ana I. Valente acknowledges the FCT PhD grant (SFRH/BD/08352/2021) .