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 provides a sealed gas-permeable bag system for growing bacterial host cells for producing plasmid DNA or expressing recombinant proteins. The system uses a gas-permeable low-density polyethylene (LDPE) bag that allows incubation with an antibiotic selection system while the culture is sealed to the external environment through a sealable bag/opening structure.

Starter and main cultures are described, where the starter culture comprises at least one colony of bacterial host cells transformed with plasmids comprising an antibiotic resistance gene. The main culture is formed by adding media, an antibiotic, and the starter culture host cells to the gas-permeable bag, such that host cells expressing the antibiotic resistance gene remain viable in the presence of the antibiotic, and the bag is incubated until growth reaches a desired concentration.

The disclosed workflow supports downstream recovery of either plasmid DNA or expressed proteins from the bag-grown culture. For plasmid DNA, host cells are harvested when the host cell concentration indicated by OD600 reaches a desired concentration, followed by centrifugation pelleting and resuspending and lysing to obtain a lysate comprising plasmid DNA. For protein expression, the method includes inducing protein expression after reaching the OD600 target, and can further include harvesting, pelleting, resuspending and lysing, and purifying the expressed protein from the lysate.

Claims Coverage

The document includes three independent claims, each defining inventive subject matter around bag-based antibiotic-selected growth of plasmid-transformed bacterial host cells, OD600-monitored plasmid DNA production with LDPE-bag culture followed by harvesting, pelleting, and lysis, and OD600-monitored protein expression in an LDPE-bag culture followed by induction and optional downstream purification workflow.

Across the independent claims, the core claim coverage is centered on cultivating plasmid-transformed bacterial host cells in a gas-permeable bag, specifically an LDPE bag, using antibiotic selection and OD600-based determination of the culture reaching a desired concentration, followed by either harvesting, pelleting, resuspending and lysing to obtain plasmid DNA, or inducing protein expression at the OD600 target and, in further dependent processing, harvesting, pelleting, resuspending and lysing, and purifying the expressed protein.

Stated Advantages

Documented Applications