The Purification of Plasmid DNA for Clinical Trials Using Membrane Chromatography - Membrane chromatography ensures purity at high flow rates. - BioPharm International


The Purification of Plasmid DNA for Clinical Trials Using Membrane Chromatography
Membrane chromatography ensures purity at high flow rates.

BioPharm International
Volume 23, Issue 2


Our process produces plasmid DNA (pDNA) with 95% purity, and the process fulfills all regulatory requirements and renders pharmaceutical-grade pDNA The content of genomic DNA is lower than 5 ng per dose, RNA is not detectable by agarose gel electrophoresis; endotoxin content is 0.77 EU per kg body weight, and the protein content is 4.1 μg per dose, which is lower than the limit established.

In conclusion, this process, which combines size exclusion and membrane chromatography, met the criteria of purity, robustness, and reproducibility required for manufacturing pharmaceutical-grade pDNA for human clinical trials.

Miladys Limonta is a principal researcher and Gabriel Márquez is the department head, both in downstream process development, Martha Pupo is a specialist in analytical development, and Odalys Ruíz is a researcher in fermentation development, all at the Centre for Genetic Engineering and Biotechnology, Havana, Cuba,
+(53-7) 271-6013.


1. Urthaler J, Schleg R, Pogdornik A, Strancar A, Jungbauer A, Necina R. Application of Monoliths for plasmid DNA purification development and transfer to production. J Chrom A. 2005;1065: 93–106.

2. Ayazy P. Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA. Biotech Appl Biochem. 2003;37:207–18.

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

4. Limonta M, Márquez G, Rey I, Pupo M, Ruíz O, Amador-Cañizare Y. Plasmid DNA recovery using size exclusion and perfusion chromatography. BioPharm Int. 2008;21:38–48.

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

6. US Food and Drug Administration. Guidance for Industry: Considerations for Plasmid DNA vaccines for Infectious Disease Indications. Rockville, MD: 2005 Feb.

7. Dueñas-Carrera S, Morales J, Acosta-Rivero N, Lorenzo LJ, García C, Ramos T. Variable level expression of hepatitis C virus core protein in a prokaryotic system. Analysis of the humoral response in rabbit. Biotecnología Aplicada. 1999;16: 226–31.

8. Lorenzo LJ, Garcia O, Acosta-Rivero N, Dueñas-Carrera S, Martinez G, Alvarez-Obregon J. Expression and immunological evaluation of the Escherichia coli-derived hepatitis C virus envelope E1 protein. Biotechnol Appl Biochem. 2000;32:137–43.

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

10. Dueñas-Carrera S, Alvarez-Lajonchere L, Alvarez-Obregón JC, Pérez A, Acosta-Rivero N, Vázquez DM. Enhancement of the immune response generated against hepatitis C virus enveloped proteins after DNA vaccination with polyprotein-encoding plasmids. Biotechnol Appl Biochem. 2002;35:205–12.

11. Acosta-Rivero N, Aguilera Y, Falcon V, Poutou J, Musacchio A, Alvarez-Lajonchere A. Ultrastructural and immunological characterization of hepatitis C core protein–DNA plasmid complexes. Am J Immunol. 2006;2:67–72.

12. Birnboim HC. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymology. 1983;100:243–55.

13. Duval E, Burke G. Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process. J Chromatogr B.2004;804:327–35.

14. Meacle FJ, Lander R, Ayazi Shamlou P, Titchener-Hooker NJ. Impact of Engineering Flow Conditions on Plasmid DNA Yield and Purity in Chemical Cell Lysis Operations. Biotechnol Bioeng. 2004;87:293–302.

15. Endres HN, Johnson JAC, Ross CA, Welp JK, Etzel MR. Evaluation of an ion-exchange membrane for the purification of plasmid DNA, Biotechnol Appl Biochem. 2003;37:259–66.

16. Zhang S, Krivosheyeva A, Nochumson S. Large scale capture and partial purification of plasmid DNA using anion-exchange membrane capsules. Biotechnol Appl Biochem. 2003;37:245–9.

17. Gottschalk U, Fischer-Fruehholz S, Reif O. Membrane adsorbers—A cutting edge process technology at the threshold. Bioprocess Int. 2004 May;(5):56–65.

blog comments powered by Disqus



GPhA Issues Statement on Generic Drug Costs
November 20, 2014
Amgen Opens Single-Use Manufacturing Plant in Singapore
November 20, 2014
Manufacturing Issues Crucial to Combating Ebola
November 20, 2014
FDA Requests Comments on Generic Drug Submission Criteria
November 20, 2014
USP Joins Chinese Pharmacopoeia Commission for Annual Science Meeting
November 20, 2014
Author Guidelines
Source: BioPharm International,
Click here