Macrocarriers and methods for high throughput production

Abstract

Provided herein is a method for producing a vector of at least 16 kb in size from a bacterial cell, comprising the following consecutive steps: a) obtaining a bacterial cell comprising a vector of at least 16 kb in size, said vector comprising an inducible origin of replication, b) inoculating a culture medium with the bacterial cell comprising the vector, c) culturing the bacterial cell in the culture medium, the present invention relates to a method for producing plasmids from bacterial cells, comprising the steps of: a) adding one or more inducers of an inducible starting point of replication to a culture medium, d) adding one or more inducers of the inducible starting point of replication to the culture medium when the optical density (OD600) of the bacterial culture at 600 nm reaches at least 20, e) further culturing the bacterial cells in the culture medium, f) optionally isolating the bacterial cells from the culture medium, and g) recovering plasmids from the bacterial cells. Also provided herein are vectors having a size of at least 16 kb comprising an inducible origin of replication for use in such methods.

Description

technical field

[0001] The present invention relates to vectors and methods for high yield production of large vectors. Background technique

[0002] Gene therapy and DNA vaccines are promising tools for the prevention, treatment and cure of diseases such as cancer and acquired immunodeficiency syndrome (AIDS). Gene therapy and DNA vaccines require large quantities of highly purified plasmid DNA (pDNA), which should be homogeneous in terms of structural form and DNA sequence. In addition, gene therapy and DNA vaccines, such as those against complex pathogens, require plasmids that can tolerate large genomic DNA inserts. For biopharmaceutical applications, yield and quality are the main drivers of plasmid manufacturing methods. In pursuit of increased yield, plasmids were developed that produced high copy numbers, meaning they would result in multiple copies of the plasmid per cell (>500 copies per cell, eg pUC18, pUC19 vectors). Such high plasmid copies per cell can be...

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