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Methods Mol Biol. 2014;1143:197-217. doi: 10.1007/978-1-4939-0410-5_13.

Plasmid fermentation process for DNA immunization applications.

Author information

1
Nature Technology Corporation, 4701 Innovation Drive, Lincoln, NE, 68521, USA, acarnes@natx.com.

Abstract

Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

PMID:
24715290
DOI:
10.1007/978-1-4939-0410-5_13
[Indexed for MEDLINE]

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