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Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA

Paper ID Volume ID Publish Year Pages File Format Full-Text
25787 43601 2006 12 PDF Available
Title
Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA
Abstract

As part of a program to develop DNA vaccines for pharmaceutical applications, we recently established a manufacturing process for the production of clinical grade plasmid DNA. In an evaluation of two cell separation methods, the cell culture experienced a temperature spike in a new tangential flow filtration rig, resulting in an aberrant plasmid HPLC peak. Analysis by agarose gel electrophoresis and HPLC demonstrated that the aberrant plasmid material's overall primary structure, methylation pattern and topological integrity was indistinguishable from that of reference material. Transmission electron microscopy and high-resolution agarose gel electrophoresis revealed that the unknown plasmid form exhibited a very low level of supercoiling, whereas the normal supercoiled fraction contained highly twisted DNA. We hypothesized that an enzymatic process, induced by stress during the temperature spike, caused the distinct plasmid topology. This idea was supported by a lab-scale fermentation experiment, where plasmid topology was shown to be similarly altered by conditions designed to induce metabolic stress.

Keywords
DNA vaccine; Characterization; Plasmid; Topology; Supercoiling
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Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 121, Issue 1, 2 January 2006, Pages 1–12
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us