Growth of bacterial host cells in gas permeable low-density polyethylene bags for production of plasmid DNA or recombinant molecules

Inventors

Chambers, Isabelle Louise

Assignees

Aldevron LLC

Abstract

The present disclosure pertains to methods of growing bacterial host cells in a low-density polyethylene (LDPE) bag to produce plasmid DNA or express recombinant protein. The LDPE bag is filled with media, an antibiotic, and host bacterial cells that have been transformed with plasmid DNA encoding a protein of interest. The LDPE bag is sealable to the external environment and incubated at a growth temperature until a desired concentration of bacteria is achieved. When plasmid DNA is desired, host cells are harvested and plasmid DNA is separated from host cell components. When recombinant protein is desired, expression is induced while host cells are in the LDPE bag, followed by the harvest and separation of the recombinant protein. The LDPE bags are sterile and conducive to bacterial growth equal to or greater than that afforded by conventional shake flasks under similar growth conditions.

Core Innovation

The invention relates to a gas-permeable bag format for growing bacterial host cells to a desired concentration using a starter culture and a main culture. The method includes providing a starter culture of bacterial host cells transformed with plasmids comprising a gene for a selection marker, providing a gas-permeable bag, adding media, a selection factor, and the starter culture host cells to the gas-permeable bag, and incubating so that only host cells expressing the selection marker gene remain viable while the host cells grow to a desired concentration.

The same gas-permeable bag culture approach is used to produce plasmid DNA from bacterial host cells. In this method, after forming the main culture and incubating until the host cells reach a desired concentration, the host cells are harvested, pelleted, and resuspended and lysed to obtain a lysate comprising plasmid DNA produced by the host cells.

The gas-permeable bag culture is further used for expressing a protein in bacterial host cells. A main culture is formed by adding media, a selection factor, and host cells transformed with plasmids comprising a DNA sequence encoding the protein and a gene for a selection marker, and the culture is incubated at a growth temperature to reach a desired host cell concentration. Protein expression is then induced by the host cells of the main culture.

Claims Coverage

The document includes three independent claims, each centered on a gas-permeable bag culture workflow. The inventive features cover growing plasmid-bearing bacterial host cells in a gas-permeable bag with selection, producing plasmid DNA from those bag-grown cells via harvest and lysis, and expressing a protein in bag-grown bacterial host cells by inducing expression from selection-marker plasmids.

Across the independent claims, the shared inventive concept is the use of a gas-permeable bag culture with plasmid selection to support growth of transformed bacterial host cells to a desired concentration, downstream plasmid DNA production from harvested and lysed bag-grown cells, and downstream protein expression via induction in bag-grown cells carrying protein-encoding plasmids with a selection marker.

Stated Advantages

Documented Applications