 |
PROTEIN PURIFICATION AND PRODUCTION
Scale up of cost-effective and generic purification process for plasmid DNA
Download PDF
L. M. Sandberg1*, P. Nilsson2, E. Håkansson2, A. Andersson1, J.Vasi1, and R. Lemmens1
1 Amersham Biosciences AB, Uppsala, Sweden; 2 GIN Laboratories, Rudbeck Laboratory, Uppsala, Sweden
*Corresponding author
The production of highly purified plasmid DNA in large quantities for gene therapy is vital to the successful use of plasmid DNA as a therapeutic. The shift from small-scale plasmid production for cell transfections at the benchtop to large-scale plasmid production for gene therapy and DNA vaccines poses new demands on the bacterial fermentation, bacterial lysate processing and the final purification of plasmid DNA. This study describes a process for the scale-up of a cost-effective and generic purification process for plasmid DNA.
Introduction
Gene therapy and vaccination with nucleic acids are two of the most striking innovations in medical sciences. Non-viral, direct delivery of DNA to target cells is often preferred in clinical vaccine application because of the minimal risk of viral infections (1). However, this technique is less effective in transfecting cells than viral vectors (2). Consequently, large amounts of plasmids must be used in treatment. To satisfy strict health guidelines, the genetic material must consist of highly purified, homogenous preparations of supercoiled plasmid DNA (3–5).
To meet the needs of purifying such large quantities of plasmids, a purification method was developed. The use of a three-step chromatographic purification scheme resulted in high yield of a final product with purity that exceeds current regulatory requirements.
Materials and Methods
A high copy-number plasmid (pUC 19 derivative, pNGVL-3-VEGF, 4583 bp) was cultivated in E. coli. Bacteria were harvested prior to alkaline lysis and then the lysate was clarified using centrifugation. All chromatographic media, systems, columns, and ultrafiltration cartridges were from Amersham Biosciences.
Experimental
The purification method was developed on the laboratory scale by using a three-step chromatographic purification protocol. In the first step, group separation was used to remove RNA contaminants and for buffer exchange. In the second step, supercoiled covalently closed circular (ccc) plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, which was specifically designed for plasmid purification (7). An anion exchange step was utilized for final endotoxin removal and polishing. The scale-up of this purification protocol was performed by increasing column diameters.
Laboratory Scale
The clarified alkaline lysate was separated on Sepharose™ 6 Fast Flow in an XK50/30 column to remove RNA. The plasmid DNA containing fractions were collected and the capture step was run on PlasmidSelect in an XK 16/20 column. The ccc plasmid DNA was then polished on SOURCE™ 30Q in an XK 16/20 column as the final purification step. All chromatographic steps were performed on an ÄKTApurifier™ 100 system.
Pilot Scale
The clarified alkaline lysate was concentrated five times on a hollow fiber ultrafiltration cartridge (nominal molecular weight cutoff: 100 000, 1400 cm2) with a flux of 13 l/m2/h (LMH) and a transmembrane pressure (TMP) of 0.88 bar (13 psi). The lysate (480 g cell paste in 10 l) was divided in two parts, both of which were separated on Sepharose 6 Fast Flow in a BPG™ 100 column to remove RNA. The plasmid DNA-containing fractions were pooled. Half of the fractions were stored for later use. Supercoiled plasmid DNA was purified from the other half using PlasmidSelect in a FineLine™ 70 column. The DNA was polished using SOURCE 30Q in a FineLine 35 column. A similar ultrafiltration technique, as above, was used for final concentration and buffer change of the plasmid DNA. All chromatographic steps were performed on an ÄKTApilot™ system (Fig 1).
Fig 1. Pilot-scale purification of plasmid DNA. (A) Group separation using Sepharose 6 Fast Flow. (B) Capture using PlasmidSelect. (C) Polishing using SOURCE 30Q.
Results
The quality of the purified plasmid DNA was determined by several analytical techniques at PlasmidFactory GmbH, Bielefeld, Germany (www.plasmidfactory.com). The pilot scale yield was calculated after each purification step as indicated in Table 1. Final product purity is described in Table 2. The results show the desired outcome of both high yield and purity in a process that meets the regulatory requirements for cGMP production of plasmid therapies.
Table 1. Pilot Scale Yield Data*
 | Yield (%) |
| Sepharose 6 Fast Flow† | 92 |
| PlasmideSelect | 72 |
| SOURCE 30Q | 98 |
| Ultrafiltration | 81 |
*The yield was calculated using UV absorption (A260)
†The DNA concentration in the UF clarified lysate was measured with PicoGreen™ fluorescence assay (Molecular Probes Inc.).
Table 2. Pilot Scale Purity Data
| Final product purity |
| ccc plasmid DNA (%), CGE* | 95 |
| ccc plasmid DNA (%), AGE† | 99 |
| plasmide DNA (mg/ml) | 3.9 |
| endotoxins (E.U./mg plasmid DNA) | 0.05 |
| RNA (µg/mg plasmid DNA) | 0.02 |
| gDNA (µg/mg plasmid DNA) | 0.97 |
| protein (µg/ml) | <5‡ |
*Capillary gel electrophoresis
†Agarose gel electrophoresis
‡Below detection limit
Conclusions
As the production of purified plasmid DNA for gene therapy and vaccination is relatively easy and as health problems with other methods of gene therapy remain unresolved, the need to produce large quantities of highly purified plasmids is expected to increase. The ability to create a robust and scalable process at the laboratory scale is a cost-effective way to advance into the large-scale production of plasmids.
References:
1. Jolly, D. Viral vector systems for gene therapy. Cancer Gene Therapy 1,51–64 (1994).
2. Crystal. R. G. The gene as drug. Nature Medicine 1, 15–17 (1995).
3. Committee for Proprietary Medicinal Products. Note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products. CPMP/BWP/3088/99. European Agency for the Evaluation of Medicinal Products (2001). [Report, pdf, 130 KB]
4. Center for Biologics Evaluation and Research. Points to consider on plasmid DNA vaccines for preventative infectious disease indications. United States Food and Drug Administration (1996). [Report, pdf, 50 KB]
5. Center for Biologics Evaluation and Research. Guidance for human somatic cell therapy and gene therapy. United States Food and Drug Administration (1998) [Report, pdf, 80 KB]
6. Lemmens, R. et al. Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption. J. Chromatog. B 784, 291–300 (2003). [PubMed abstract]
Acknowledgement
This work was carried out at GIN Laboratories, a cGMP contract manufacturing facility at Uppsala University, Sweden, which is approved for the production of injectable pharmaceuticals and vaccines.
Ordering Information
| UFP-100-C-4x2MA | Xampler Cartridge, 50 kD, 0.5 mm lumen, size 4x2M, autoclavable | POR | |
| QSM-03SP | QuixStand System with PRP-09WM perstaltic pump | POR | |
| UFP-300-E-H22LA | MidGee Hoop Cartridge, 300 kD, 1.0 mm lumen, 60 cm | POR | |
| MDG-4SP | Advanced MidJet System | POR | |
|
|
