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Process for plasmid DNA fermentation

a technology of plasmid dna and fermentation process, which is applied in the direction of fertilization, genetic therapy composition manufacture, vector-based foreign material introduction, etc., can solve the problems of undesirable acetate production, unacceptable use of animal products, in particular bovine products, in plasmid manufacturing, etc., to improve plasmid dna quality, improve plasmid dna production yield, and reduce impurities in purified plasmid

Inactive Publication Date: 2011-11-17
NATURE TECH CORP (US)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach significantly increases plasmid DNA yield, improves purity, and reduces impurities, achieving yields of up to 1100 mg / L with high-quality, super-coiled plasmid DNA, addressing the limitations of existing methods and enhancing regulatory compliance.

Problems solved by technology

However, in the future, international standards for plasmid DNA purity are likely to be the same or very similar to those that are used for recombinant protein products similarly produced from E. coli fermentation, and such standards exceed the current purity attainable from established methods.
However, high glucose concentrations cause undesirable acetate production due to metabolic overflow (known as the Crabtree effect).
The use of animal-derived products, and in particular bovine products, in plasmid manufacture is unacceptable due to the risk of prion or virus contamination.
High growth rates have been associated with acetate production, plasmid instability, and lower percentages of super-coiled plasmid.
29: 85-91) found that a single drop in dissolved oxygen concentration to 5% of air saturation led to rapid loss in plasmid stability.
41: 666-670) showed that fluctuations in oxygen input lead to plasmid instability.
This low yield imposes a cost and purity burden on commercialization of plasmid DNA production processes.

Method used

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  • Process for plasmid DNA fermentation
  • Process for plasmid DNA fermentation
  • Process for plasmid DNA fermentation

Examples

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example 1

NTC3018 and NTC3019 Fermentation Media

[0072]The following criteria were established for evaluation of an optimized fermentation process fo manufacturing plasmid DNA:

1) High specific yield of plasmid (mg plasmid DNA per g cell mass);

2) High biomass yield;

3) It will retain high degrees of super-coiled plasmid;

4) It will cause minimal problems during downstream processing;

5) It will meet regulatory requirements; and

6) It will retain plasmid structure (e.g. no deletions or other rearrangements).

[0073]Culture media was formulated to support high specific plasmid yield, high biomass yield, and high plasmid quality. The batch fermentation medium was designed to reduce the specific growth rate. The use of a reduced growth rate has been associated with higher plasmid copy number and better plasmid stability. During fed-batch fermentations the growth rate was controlled at 0.12 hr−1 by feeding of the limiting nutrient. The host strain, DH5α, has the endA1, recA, and relA mutations, all import...

example 2

NTC3019 Media Fed-Batch Culture with pBR322-Derived Plasmids

[0075]Fed-batch fermentations were carried out in a New Brunswick BioFlo 110 fermentor at 37° C. pH was controlled by automatic addition of 30% ammonium hydroxide or 10% phosphoric acid. The dissolved oxygen probe was calibrated to 0% by nitrogen gas sparging and 100% with air saturation. The vessel was aerated at 1 VVM and dissolved oxygen was maintained at 30% by proportional-integral control of agitation. At cell densities above about 20 OD600, O2 supplementation was also required to maintain 30% saturation.

[0076]Seed cultures were started from single isolated colonies inoculated into LB plus 50 μg / ml kanamycin and grown at 37° C. At mid-exponential phase (0.5-1.5 OD600) the seed cultures were used to provide 1% inoculums for the fermentor.

[0077]During fed-batch cultures a semi-defined feed nutrient was added according to a carbon limiting exponential feeding strategy. Briefly, an initial amount of carbon substrate is co...

example 3

NTC3018 Medium Batch Fermentation with High-Copy Gene Therapy Plasmid

[0088]NTC3018 medium batch culture with pUC origin plasmids was performed. The following pUC origin containing plasmids were utilized:

1) pW2.0, a derivative of pUC19 that has an altered polylinker sequence.

2) pMaxGFP

3) pEGFP-C1

[0089]Batch fermentations were carried out in a New Brunswick BioFlo 110 fermentor at 37° C. pH was controlled by automatic addition of 30% ammonium hydroxide or 10% phosphoric acid. The dissolved oxygen probe was calibrated to 0% by nitrogen gas sparging and 100% with air saturation. The vessel was aerated at 1 VVM and dissolved oxygen was maintained at 30% by proportional-integral control of agitation. At cell densities above about 20 OD600, O2 supplementation was also required to maintain 30% saturation.

[0090]Seed cultures were started from single isolated colonies inoculated into LB plus 50 μg / ml kanamycin or 100 μg / ml ampicillin and grown at 37° C. At mid-exponential phase (0.5-1.5 OD600...

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Abstract

Improvements in plasmid DNA production technology are needed to insure the economic feasibility of future DNA vaccines and DNA therapeutics. General methods are described, by means of which it is possible to dramatically increase plasmid DNA productivity in a fermentor. These processes feature fed-batch fermentation strategies, combined with novel growth and induction phase temperature shifts.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 11 / 573,825, filed on Feb. 16, 2007, in the United States Patent and Trademark Office, currently pending, which is a National Phase of International Patent Application No PCT / US05 / 29238, filed Aug. 16, 2005, which claims the benefit of U.S. Provisional Patent Application No. 60 / 603,000, filed Aug. 19, 2004, the disclosures of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]Not ApplicableFIELD OF THE INVENTION[0003]The present invention relates to the production of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly is a method for production of the said DNA molecules at high levels in fermentation culture.BACKGROUND OF THE INVENTION[0004]The present invention re...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P19/34
CPCA61K48/0091C12N15/69C12P19/34C12N15/74C12N15/70
Inventor CARNES, AARON E.WILLIAMS, JAMES A.
Owner NATURE TECH CORP (US)
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