Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography - A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography. - Bio

ADVERTISEMENT

Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography
A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography.


BioPharm International


CONCLUSIONS

The clinical application of gene therapy and DNA immunization will depend not only on efficacy but also on safety and the ease with which the technology may be adapted for large-scale pharmaceutical production. The new purification process described here combines novel and conventional purification technologies as high-throughput alternatives. TFF membranes and a perfusion media were used for concentration and recovery of the pIDKE2 plasmid to reduce process time and to increase the productivity of the downstream process. Although the yields are lower when using this process, the advantages of this method over existing plasmid DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived enzymes. Finally, this process may be used as a simple, scalable, and applicable method for the production of the pIDKE2 plasmid, which is being used in Phase 2 human clinical trials.

Miladys Limonta is the principal researcher, Gabriel Marquez is the head, and Isabel Rey is a technician in the downstream process development department, Martha Pupo is a specialist and Odalys Ruiz is a researcher in the analytical development department, Yalena Amador-Canizares is a researcher and Santiago Duenas-Carrera is the head of the hepatitis C vaccine department, all at the Centre for Genetic Engineering and Biotechnology, Havana, Cuba, +53.7336.008,

REFERENCES

1. Sandberg L, Bjurling A, Busson P, Vasi J, Lemmens R. Thiophilic interaction chromatography for supercoiled plasmid DNA purification. J Biotechnol. 2004;109:193–199.

2. US Food and Drug Administration. Center for Biologics Evaluation and Research. Guidance for industry. Considerations for plasmid DNA vaccines for infectious disease indications. Rockville, MD: 2005 Feb.

3. Diogo M, Queiroz J, Monteiro G, Martins S, Ferreira G, Prazeres D. Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography. Biotechnol Bioeng. 2000;68:576–583.

4. Dueñas-Carrera S, Morales J, Acosta-Rivero N, Lorenzo LJ, García C, Ramos T, et al. Variable level expression of hepatitis C virus core protein in a prokaryotic system. Analysis of the humoral response in rabbit. Biotecnología Aplicada. 1999;16(4):226–231.

5. Lorenzo LJ, Garcia O, Acosta-Rivero N, Dueñas-Carrera S, Martinez G, Alvarez-Obregon J, et al. Expression and immunological evaluation of the Escherichia coli-derived hepatitis C virus envelope E1 protein. Biotechnol Appl Biochem. 2000;32:137–143.

6. Martínez-Donato G, Capdesuñer, Acosta-Rivero N, Rodríguez A, Morales-Grillo J, Martínez E, González M, et al. Multimeric HCV E2 protein obtained from Pichia pastoris cells induces a strong immune response in mice. Mol Biotechnol. 2007;35:225–35.

7. Dueñas-Carrera S. Alvarez-Lajonchere L, Alvarez-Obregón JC, Pérez A, Acosta-Rivero N, Vázquez DM, et al. Enhancement of the immune response generated against hepatitis C virus envelope proteins after DNA vaccination with polyprotein-encoding plasmids. Biotechnol Appl Biochem. 2002;35:205–212.

8. Lis JT, Schleif R. Size fractionation of double stranded DNA by precipitation with polyethylene glycol. Nucleic Acids Res. 1975;2:383–389.

9. Horn NA, Meek JA, Budahazi G, Marquet M. Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials. Human Gene Ther. 1995;6:565–573.

10. Ayazy P. Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA. Biotech Appl Biochem. 2003;37:207–218.

11. Ferreira GNM, Cabral JMS, Prazeres DMF. Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications. Biotechnol Prog. 1999;15:725–731.

12. Lemmens R, Olsson U, Nyhammar T, Stadler J. Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption. J Chromatogr B. 2003;784:291–300.

13. Afeyan NB. Gordon NF, Mazsaroff I, Varady L, Pulton SP. Flow-through particles for the high performance liquid chromatographic separation of biomolecules: perfusion chromatography. J Chromatogr. 1990;519:1–29.

14. Sambrook J, Fritsch EF, Maniatis M. Cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, ed. Cold Spring Harbor, New York; 1989.

15. Stadler J, Lemmens R, Nyhammar T. Plasmid DNA purification. J Gene Med. 2004;6:S54–S66.

16. Eon-Duval A, Burke G. Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process. J Chromatogr B. 2004;804:327–335.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

Lilly to Acquire Novartis Animal Health
April 22, 2014
Novartis and GSK Trade Assets
April 22, 2014
Mallinckrodt to Acquire Questcor Pharmaceuticals
April 16, 2014
EMA Warns of Falsified Herceptin Vials
April 16, 2014
American CryoStem and Rutgers University File Joint Patent on Stem Cell Platform
April 11, 2014
Author Guidelines
Source: BioPharm International,
Click here