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Design and operation of the preparative chromatography of bio-products

Collaborations / Fundings

Amersham Biosciences

KTI

Plasmid DNA production

Interest in producing large quantities of plasmid DNA has been increasing during the last decade as a result of the developments in gene therapy and DNA vaccines. Although the way to clinical tests has been in many cases more difficult than expected, gene therapy remains a very promising way of therapy for sicknesses like cancer, diabetes and certain inherited diseases. In addition, a new and powerful method of vaccination involving the administration of pathogen’s genes may become the new generation of vaccines getting more efficiently over emerging disease threats.
Plasmid DNA (pDNA) vectors are preferred over viral based vectors because they minimize the risk of viral infection. However, targeting of pDNA vectors is less efficient, meaning that larger amounts of pharmaceutical-grade plasmid are required to carry out a treatment. These amounts will probably be in the order of milligrams. A large scale pDNA production process needs to be established in order to supply the demanded amounts of pharmaceutical grade pDNA for clinical trials and eventually to commercialize. The manufacturing process must fulfill all the requirements dictated by the international agencies (namely the FDA). Summarizing, gene therapy research needs to go together with process development and gmp considerations.
The production of pDNA starts with the fermentation of the selected bacterial strain. In order to harvest the plasmid, a lysis is performed to destroy the wall and cellular membrane. Lysis yields an heterogeneous mixture containing solids that are separated by centrifugation. Most of the chromosomal DNA (cDNA) is separated with the cell debris during centrifugation. Even in the case of high copy number plasmid, pDNA represents less than 3% of the cleared lysate. The main contaminants to be eliminated are RNA, cDNA fragments, proteins and endotoxins. The supercoiled covalently closed form of pDNA (ccc) is thought to be therapeutically more effective than the open-circular, linear, or multimeric forms, and therefore these forms have to be separated from the final product. The classical lab protocol for pDNA purification is not scalable and uses reagents that are not suited for clinical production; as a result this does not constitute any basis for process development. Chromatographic techniques are preferred alone or in combination with other techniques, generally related to membrane technology. Plasmid purification schemes include in general several chromatographic steps, often called capture, intermediate purification steps and polishing.

Purification of Plasmid DNA

Simulated moving bed (SMB) technology could play an important role in the upscaling of biomolecules purification processes. It is known from other fields of application that SMB allows to increase productivity while reducing the solvent consumption. SMB was first used in the petrochemical industry, and then successfully downscaled to be used in the food industry and lately in the purification of chiral molecules. In the new field of bio-separations the challenges to be faced are related to the differences with traditional SMB applications: the nature of the molecules and often the high density of the samples, the requirement of aqueous mobile phases, typically buffer solutions, and the compressible chromatographic media typically used in bio-separations.

This work studies the first purification step based on size exclusion chromatography and using the simulated moving bed technology (SMB). The idea of starting the purification scheme by a size exclusion step has already been proposed in the frame of column chromatography processes, and is exploited here as a proof-of-concept for the SEC-SMB. SMB ensures a continuous and fast processing of the cleared lysate from which the pDNA has to be purified. A fast capture step is convenient because it gives stability to the sample. The main objective is to eliminate the RNA, the proteins and most of the cDNA fragments. Some impurities may be left in the product in order to guarantee complete recovery of the plasmid. Later purification steps will involve complete elimination of RNA, cDNA and protein, further reduction of the endotoxin levels and separation of the pDNA forms other than ccc.

The technical implementation of a CIP step into the SMB has been addressed recently [2]. The implementation of the CIP step into the SMB is technically possible. The details of the cleaning procedure (frequency, cleaning agent, contact time, etc) require the establishment of the so-called cleaning protocol. This issue is being studied and will be reported in the future.

Further work addresses the eventual performance improvement of SMB with respect to column chromatography for the capture step. The optimization of both, the batch and the SMB processes is investigated using a Genetic Algorithm and detailed simulations of the two processes [6].

 

Publications

[1] S. Abel, M. Bäbler, C. Arpagaus, M. Mazzotti, and J. Stadler. Two-fraction and three-fraction continuous SMB separation of nucleosides. J. Chromatogr. A, submitted for publication, 2003.

[2] Paredes, G., Abel, S., Mazzotti, M., Morbidelli, M. and Stadler, J. Analysis of a simulated moving bed operation for three-fraction separations (3F-SMB). Ind. Eng. Chem. Res., 2004, 43, 6157-6167.

[3] Paredes, G., Mazzotti, M., Stadler, J., Makart, S., Morbidelli, M. SMB operation for three-fraction separations: purification of plasmid DNA. Adsorption, 2005, 11, 841-845.

[4] Paredes, G., Makart, S., Stadler, J., Mazzotti, M. Simulated Moving Bed Operation for Size Exclusion Plasmid Purification. Chem. Eng. Technol., 2005, 43(19), 6157-6167.

[5] Paredes, G., Rhee, H.K. and Mazzotti, M. Design of Simulated Moving Bed chromatography with enriched extract operation (EE-SMB): Langmuir isotherms. Ind. Eng. Chem. Res., 2006, 45, 6289-6301

[6] Paredes, G. and Mazzotti, M. SMB and column chromatography process comparison for the first purification step of plasmid DNA. J. Chromatogr. A, 2006, submitted for publication

 

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