Therapeutic grade RNA purification by ultrafiltration
Open Access
- Author:
- Manzano Garcia, Jesus
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 06, 2020
- Committee Members:
- Andrew Zydney, Dissertation Advisor/Co-Advisor
Andrew Zydney, Committee Chair/Co-Chair
Themis Matsoukas, Committee Member
Hee Jeung Oh, Committee Member
William O Hancock, Outside Member
Phillip E Savage, Program Head/Chair - Keywords:
- Nucleic Acids
Purification
Ultrafiltration - Abstract:
- Recent advances in the use of nucleic acids (NA) for therapeutic applications, including vaccines, gene therapies, and more recently gene editing, have created a need for the development of appropriate separation processes for the large-scale isolation and purification of these biomolecules. One attractive potential method for NA purification is ultrafiltration using semipermeable membranes both for removal of large molecular weight species (with the RNA collected in the permeate) and for RNA concentration or buffer exchange (with the RNA collected in the retentate). The objective of this dissertation was to evaluate the factors controlling the transport of NA through semipermeable ultrafiltration membranes and to explore the development of membrane-based processes for the purification of therapeutic grade DNA and RNA-based therapeutics. Experimental data were obtained for RNA transmission through a series of composite regenerated cellulose and polyethersulfone ultrafiltration membranes with different molecular weight cutoffs. Initial studies were performed using dilute solutions of Torula yeast RNA with a range of RNA size and conformation. The data showed significant RNA adsorption to the composite regenerated cellulose membrane; this effect was not seen with the polyethersulfone membranes due to electrostatic repulsion between the negatively-charged RNA and the negatively-charged membrane. Subsequent studies examined the effects of RNA size and tertiary structure using RNA with nucleotide sequences that give rise to specific structural motifs. The hairpin RNA had much higher transmission than the corresponding linear RNA with no Watson-Crick pairing. RNA transmission increased at high NaCl concentrations and in the presence of urea, particularly for the linear RNA. Ribonucleoprotein (RNP) complexes formed from a single guide RNA and the Cas9 protein can be used for CRISPR-based gene editing. Experiments performed with the guide RNA and Cas9 alone were used to identify filtration conditions that could provide high selectivity for the removal of excess guide RNA from the RNP. Actual separations were performed using a diafiltration process, providing greater than 90% yield of the desired RNP whith significant removal of the free RNA. These studies also demonstrated the importance of shear-induced aggregation of the Cas9, which led to significant fouling that could complicate the application of this technology for large-scale purification of these RNP products. Experimental studies were performed to evaluate the effects of polyamines (multivalent cations) and membrane orientation on the ultrafiltration of different DNA isoforms. The addition of small quantities of spermine had a very strong effect on DNA transmission, with the sieving coefficient of the supercoiled plasmid decreasing by more than an order of magnitude upon addition of only micromolar levels due to reduction in elongational flexibility of the DNA. A comparable change in DNA transmission required >20X of the amount of trivalent spermidine. The polyamines significantly increased the selectivity for the separation of DNA from a model protein, but they were unable to provide a significant increase in the selectivity for separating DNA isoforms. Membranes with gradients in pore size can potentially be used to pre-condition (or pre-stretch) the plasmid into an extended conformation, increasing DNA transmission and reducing membrane fouling. We evaluated the potential of using hollow fiber membranes in the reverse orientation for the separation of the linear and supercoiled isoforms. The supercoiled isoform was selectively retained within the microporous support structure at low filtrate flux but could then be recovered by increasing the flux to increase the elongation of the DNA. Overall, the results presented in this dissertation provide important new insights into the factors controlling the ultrafiltration behavior of nucleic acids while providing a framework for the design and application use of this unit operation for the purification of RNA and DNA-based therapeutics