Fed-batch fermentation process and culture medium for the production of plasmid DNA in E. coli on a manufacturing scale

Abstract

A process for producing plasmid DNA E. coli cells comprises a pre-culture and fed-batch process. The culture media of the batch phase and the culture medium added during the feeding phase are chemically defined. The culture medium of the feeding phase contains a growth-limiting substrate and is added, for at least a fraction of the feeding phase, at a feeding rate that follows a pre-defined exponential function, thereby controlling the specific growth rate at a pre-defined value. The process results in high yield and homogeneity of plasmid DNA.

Description

[0001] This Application claims priority benefit from U.S. Provisional 60 / 568,857, filed May 7, 2004 and from EP 04 008 556.5, filed Apr. 8, 2004 the contents of which are incorporated herein.FIELD OF THE INVENTION [0002] The invention relates to the fermentation of Escherichia coli for the production of plasmid DNA (pDNA), in particular for pDNA intended for the use in gene therapy and DNA vaccination. Introduction [0003] The requirement for industrial fermentation of pDNA came up by the clinical success of gene therapy and DNA vaccination during the last decade. [0004] Gene therapy is the treatment or prevention of disease by the administration, delivery and expression of genes in mammalian cells. The ultimate goal of gene therapy is to cure both inherited and acquired disorders by adding, correcting, or replacing genes. Basically, there are two types of gene therapy vectors to achieve these goals, i.e. viral vectors based on inactivated viruses and non-viral vectors based on plas...

AI Technical Summary

Benefits of technology

[0040] In fermentations operated at low growth rates, due to amino acid starvation, uncharged tRNAs arise which increase the plasmid copy number. When re / A-negative E. coli strains are used, the cells cannot respond to amino acid starvation in terms of metabolic down-regulation, consequently plasmid replication is enhanced. The additional supply with isoleucine supports the disturbance of the amino acid metabolism and increases the pDNA yield even further.

[0041] It has been surprisingly found that the process of the invention results in an outstandingly high specific and volumetric yield of plasmid DNA, accompanied by a high homogeneity of the pDNA throughout the fermentation time.

Problems solved by technology

For pDNA production on a laboratory scale, cultivation of plasmid-bearing cells in shake flasks is the simplest method, which however normally achieves low yields.

In shake flask cultivations, several drawbacks such as poor oxygen transfer and the lack of possibility for pH value control, limit the pDNA yield.

Culture media containing complex components have the disadvantage that these components originate from biological materials; therefore, the composition of the medium underlies normal natural deviations that make the cultivation process less reproducible.

Further disadvantages of using complex medium components are the uncertainty about the exact composition (presence of undesired substances), the impossibility to do stoichiometric yield calculations, the formation of undesired products upon sterilization, difficult handling due to poor dissolution, formation of dust as well as clumping during medium preparation.

Complex components of animal origin (meat extracts, casein hydrolysates) are in particular undesired for pDNA production due to the risk of transmissible spongiform encephalopathy and their use is therefore restricted by pharmaceutical authorities (CBER 1998).

Although batch fermentations are usually simple and short, they have fundamental disadvantages that result in limited plasmid DNA yields.

Furthermore, the growth rate in batch fermentations cannot be controlled directly; it is therefore unlimited, while steadily changing during fermentation, and ceases only when one or more nutrients are depleted or if metabolic by-products (such as acetate) inhibit growth of the cells.

For instance, when an antifoam agent has to be added, the DOT changes (normally decreases), which results in a lower feeding rate.

This makes the fermentation process less reproducible.

Further difficulties arise during scale-up of the process, since fermenters of different geometry or size show different oxygen transfer rates.

Another disadvantage of feed-back control is that the specific growth rate can not be exactly predefined nor controlled, resulting in suboptimal yields in processes, where the product formation is dependent on growth.

Batch fermentations that are widely applied for pDNA production are associated with technological and economical drawbacks.

In general, many pDNA fermentation processes suffer from poor homogeneity (i. e. percentage of supercoiled plasmid).

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