Disposable Process for cGMP Manufacture of Plasmid DNA - A low cost, disposable process for the manufacture of pDNA will aid in the development of vaccines. - BioPharm International

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Disposable Process for cGMP Manufacture of Plasmid DNA
A low cost, disposable process for the manufacture of pDNA will aid in the development of vaccines.


BioPharm International Supplements


Formulation and Quality Control


Figure 6
Purified pDNA is formulated into the buffer of choice by a disposable scaled-down version of the previous hollow fiber membrane assembly (Figure 6). The pDNA is formulated at the desired concentration in the appropriate solution (normally isotonic) requested by the sponsor. The choice of filter membrane is also critical to avoid unnecessary losses of plasmid by nonspecific binding.


Table 1. Final QC release testing and specifications for clinical grade pDNA
The final product is fully tested, and satisfies current recommended guidelines (Table 1). Limited in-process QC testing is performed because of the continuous nature of the process.

Conclusion

The continuous process is suitable for the rapid turnaround required for the preparation of these vaccines at a low cost. It is aseptic, contained, and completely disposable to avoid product cross contamination. It has been estimated that on current demand, with two operators in two cleanrooms, up to 40 individual vaccines could be manufactured annually.

By careful design, it is possible to eliminate some of the main obstacles and limitations normally associated with conventional processes (e.g., to reduce the reliance on high-tech equipment and to eliminate the use of column chromatography). The process has been shown to achieve a recovery of pDNA of greater than 45% from bacterial lysate with the quality of the final product satisfying current regulatory requirements.

Paul Lloyd-Evans, PhD is an operations manager, Denise A. Phillips is a production supervisor, Antony C.C. Wright is a production scientist, and R. Keith Williams is a business development manager, all at the Clinical Biotechnology Centre, Bristol, UK, 011.792.89388,

References

1. Schleef M, et al. DNA Pharmaceuticals. Wiley-VCH Verlag GmBH & Co KGaA, Weinheim;2005.

2. Stevenson FK, et al. DNA vaccines to attack cancer. PNAS. 2004;10(2):14646–14652.

3. Werner RG, et al. pDNA—from process science to commercial manufacture. Pharm Technol Eur. 2002;March;34–40.

4. European Pharmacopoeia Supplement 5.6. Gene transfer of medicinal products;2007.

5. Rules and guidance for pharmaceutical manufacturers and distributors. Pharmaceutical Press, London;2007.

6. Lee SY. High cell density culture of Escherichia coli. TIBTECH. 1996;14:94–105.

7. Birnboim HC, Doly JA. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. 1979;7:1513–1523.

8. Eon-Duval A, Gumbs K, Ellet C. Precipitation of RNA impurities with high salt in a plasmid DNA purification process: use of experimental design to determine reaction conditions. Biotech Bioeng. 2003;83:544–553.

9. Eon-Duval A, et al. Anal Biochem. 2003;316:66–73.

10. Shukla AA, et al. Process scale bioseparations for the biopharmaceutical industry. CRC Press Florida. 2007.

11. Shiying Z, Krivosheyeva A, Nochumson S. Large-scale capture and purification of plasmid DNA using anion-exchange membrane capsules. Biotechnol Appl Biochem. 2003;37:245–249.

12. Endres HN, et al. Evaluation of an ion-exchange membrane for the purification of plasmid DNA. Biotechnol. Appl. Biochem. 2003;37:259–266.


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