3D-printed matrices for steric exclusion chromatography of plasmid DNA
Santos, Ana Rita; Silva, Joana; Boto, Patrícia; Rosa, Sara; Conti, Mariachiara; Pullen, James Robert; Dimartino, Simone; Azevedo, Ana Margarida
- Publisher
- Zenodo
- Language
- en
Abstract
Currently, plasmid DNA (pDNA) holds great promise not only for gene therapy and DNA vaccination, but also as a raw material to produce mRNA and viral vaccines. However, achieving high-quality purification remains a significant challenge. Current purification methods, often involving multiple precipitation and chromatographic steps, can compromise the structural integrity and stability of the desired supercoiled (sc) pDNA isoform, which is crucial for effective gene transfer. This work aims to explore the potential of steric exclusion chromatography (SXC) as a novel approach for capturing pDNA, immediately after cell lysis, and using 3D-printed chromatographic matrices. SXC is a size-based separation mode whereby retention is achieved through mutual steric exclusion of polyethylene glycol and a large target solute in a hydrophilic solid phase. SXC shares common mechanistic roots with PEG precipitation, with the main operational difference being that SXC employs a hydrophilic solid phase as a nucleation centre on which biomolecules accrete instead of forming precipitates. The experimental workflow included cultivation of Escherichia coli cells, harboring the model plasmids pUC18 (2,686 bp), pVAX-eGFP (3,685 bp), or pCEP4 (10,410) bp, alkaline lysis, and the use of SXC for pDNA capture and purification on 3D printed hydrophilic supports. The process concludes with concentration of the plasmid-containing fractions through precipitation with high concentrations of PEG. After resuspension, the fractions were analyzed by HIC-HPLC and the protein content was determined by BCA. Applying SXC on 0.70 mL 3D-printed monolithic columns successfully purified and isolated the desired sc pDNA isoform with high yield in a single chromatographic step. The SXC operation was tested with PEGs of different molecular weights (1500 to 8000 Da) and concentration (5 to 15%). Total pDNA retention was achieved when employing short PEG chains at high concentrations, or longer PEGs at lower concentrations. This had the spill over benefit of mitigating backpressures and shear stress effects. Most importantly, under the optimised conditions tested, the total purity of sc pDNA was consistently above 97%, with an 80% HCP reduction.
Full text
3D-printed matrices for steric exclusion chromatography of plasmid DNA Ana Rita Santos(1), Joana Silva(1), Patrícia Boto(1), Sara Sousa Rosa(1), Mariachiara Conti(2), James R. Pullen(3), Simone Dimartino(2), Ana M. Azevedo(1) (1) iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa (2)Institute for Bioengineering, School of Engineering, The University of Edinburgh (3)Fujifilm Biotechnologies
The role of plasmids in G&C therapies Biological Drugs Starting Material DNA vaccines Engineer animal cells Produce mRNA, viral vector, siRNA
Biomanufacturing Host selection Fermentation Downstream processing Formulation Final pDNA product must contain: Minimum of 97 % purity (at least 80 % sc pDNA) Specifically in pDNA vaccines: maximum of 1 % of gDNA, host cell proteins, RNA and endotoxins
Downstream Processing Cell harvest Cell lysis Alkaline lysis Clarification and concentration Precipitation or TFF Capture & Polishing Chromatography (AEX, SEC, HIC) Centrifugation
Challenges in Downstream Processing Most of the impurities present in the lysate extracts share similar physicochemical properties to pDNA Structural susceptibility of sc pDNA to nicking under certain physical and chemical environmental conditions during the current purification procedure Relaxed isoforms become predominant in the final product Negative impact on the purity and yield of sc pDNA
GOAL Cell harvest Cell lysis Alkaline lysis Clarification and concentration Precipitation or TFF Capture & Polishing Steric Exclusion Chromatography Centrifugation
Steric Exclusion Chromatography (SXC) Based on the principle of steric hindrance Retention achieved through mutual steric exclusion of polyethylene glycol (PEG) by alarge target solute and a hydrophilic solid phase PEG preferentially excluded from large solutes over small solutes Solutes separated due to their size and shape Elution achieved through the reduction of PEG concentration
SXC involves a sequential process Steric Exclusion Chromatography (SXC)
Challenges PEG significantly increases buffer viscosity Reduction of solute diffusion Increase of molecular shear forces in the inter-particle (void) space Monoliths Ideal for SXC as it minimizes viscosity effects and ensure efficient mass transfer (convective transport) Its large pores allow high flow rates and low back pressure
pVAX1-GFP Total pDNA recovery yield Total purity of sc pDNA Protein removal efficiency (%) 12% PEG -1500 98.1 89.2 15% PEG -1500 100.0 90.3 10% PEG -3350 97.8 82.3 8% PEG -6000 96.9 88.5 8% PEG -8000 68.9 80.0 ↓PEG molecules (1500 and 3350) ↑Total sc pDNA purity Lower viscosity, and consequently, reduced risk of significant backpressure and shear stress, mitigate plasmid relaxation effects
Influence of plasmid size
pUC18 Total pDNA recovery yield Total purity of sc pDNA Protein removal efficiency (%) 1M NaCl 12% PEG -1500 96 100 15% PEG -1500 96 96 12% PEG -3350 98 97 10% PEG -6000 93 100 12% PEG -8000 94 99 0.5M NaCl 15% PEG -1500 94 99 ↓NaCl concentration More sustainable process. A decrease in ionic strength leads to more charge repulsion that affects the association of the plasmids with the matrix.
pCEP4 Total pDNA recovery yield Total purity of sc pDNA Protein removal efficiency (%) 0.5M NaCl 15% PEG -1500 97 96 20% PEG -1500 95 95 For bigger plasmids, 1 M NaCl leads to conformational destabilization and nicking by shear forces. Even at lower ionic strengths, higher PEG MW lead to conformational changes, and do not allow to obtain more than 50%sc pDNA.
Conclusion Applying SXC as a single chromatographic step using 3D-printed monolithic columns with a 0.70 mL bed volume, the desired sc pDNA isoform was successfully purified and isolated with high yield. It is deducible that this novel approach significantly mitigates the relaxation effects in plasmid observed in current purification procedures required for isolating sc pDNA. As the size of the plasmid increase,the PEG MW and % decreases. This innovative approach holds promise as a starting point for developing a fast, high-capacity, and cost-effective pDNA purification method
iBB –Institute for Bioengineering and Biosciences, University of Lisbon: Ana Azevedo Sara Sousa Rosa Ricardo Silva Joana Silva Patrícia Boto Institute for Bioengineering, University of Edinburgh: Simone Dimartino Mariachiara Conti FUJIFILM Biotechnologies: James R. Pullen Acknowlegments Want to know more?
Any questions? This project has received funding from the European Union's Horizon Europe, grant number 101159993. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or REA. Neither the European Union nor the granting authority can be held responsible for them. Thank you! For important updates on the project: 29
