用于生物制药应用的超螺旋多聚体质粒DNA的生产。

Production of supercoiled multimeric plasmid DNA for biopharmaceutical application.

作者信息

Voss Carsten, Schmidt Torsten, Schleef Martin, Friehs Karl, Flaschel Erwin

机构信息

Chair of Fermentation Engineering, Faculty of Technology, University of Bielefeld, P.O. 100 131, Bielefeld D-33501, Germany.

出版信息

J Biotechnol. 2003 Nov 6;105(3):205-13. doi: 10.1016/j.jbiotec.2003.07.004.

DOI:10.1016/j.jbiotec.2003.07.004

PMID:14580792

Abstract

Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral vectors like plasmid DNA are currently investigated in various clinical trials. Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors. A process for the preparation of Escherichia coli strains replicating dimers, trimers, and tetramers of a 4.6 kb plasmid is presented. Cultivation of these clones on semi-defined glycerol medium in a 7 l bioreactor shows structural stability of dimers and trimers during the whole cultivation process. Plasmid concentrations and selectivities are compared to the corresponding cultivation with the plasmid monomer. Cultivation of the tetramer replicating strain shows a disintegration of the plasmid multimer and reconstitution of the monomer and smaller multimers.

摘要

在基因治疗和基因疫苗接种中，核酸作为活性药物成分（API）的生产正变得越来越重要。非病毒载体如质粒DNA目前正在各种临床试验中进行研究。超螺旋多聚体质粒因其包含治疗基因的多个拷贝，因此可能是更有效的载体，在制药领域具有特别的吸引力。本文介绍了一种制备能够复制4.6 kb质粒二聚体、三聚体和四聚体的大肠杆菌菌株的方法。在7升生物反应器中的半限定甘油培养基上培养这些克隆，结果表明二聚体和三聚体在整个培养过程中具有结构稳定性。将质粒浓度和选择性与相应的质粒单体培养进行比较。培养四聚体复制菌株时，质粒多聚体出现解体，单体和较小的多聚体重新形成。