Scalable fermentation process
AssigneeKuros Biosciences AG
InventorsMarcel Emmerling, Frank Hennecke, Holger Pfründer, Martin Rhiel, Philipp Steiner
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Abstract
This invention provides a robust fermentation process for the expression of a capsid protein of a bacteriophage which is forming a VLP by self-assembly, wherein the process is scalable to a commercial production scale and wherein the expression rate of the capsid protein is controlled to obtain improved yield of soluble capsid protein. This is achieved by combining the advantages of fed-batch culture and of lactose induced expression systems with specific process parameters providing improved repression of the promoter during the growth phase and high plasmid retention throughout the process.
Claims20
A process for producing a recombinant capsid protein of a RNA bacteriophage being capable of forming a virus-like particle (VLP) by self-assembly, wherein said RNA bacteriophage is RNA bacteriophage Qβ, and wherein said recombinant capsid protein has the amino acid sequence of SEQ ID NO:5, said process comprising the steps of:
- a) introducing an expression plasmid consisting of SEQ ID NO:
- 1 into a bacterial host, wherein said expression plasmid comprises (i) a first nucleotide sequence of SEQ ID NO:6 encoding said recombinant capsid protein, and (ii) a promoter inducible by lactose;
- b) initiating a growth phase by cultivating said bacterial host in a medium comprising a major carbon source;
- wherein said cultivating initiates a batch phase during said growth phase, wherein said cultivating is performed in batch culture and under conditions under which said promoter is repressed by lacI, wherein said lacI is overexpressed by said bacterial host, and wherein no feeding of said batch culture is performed during said cultivating;
- c) ending said batch phase and initiating a feed phase during said growth phase by feeding said batch culture with said major carbon source;
- wherein said feeding of said batch culture is performed with a flow rate, wherein said flow rate increases with an exponential coefficient μ, and
- d) ending said growth phase and initiating a production phase by inducing said promoter with an inducer that is lactose, wherein said lactose and said major carbon source are co-fed to said batch culture in a ratio of 2:1 to 1:4 (w/w) and wherein said cultivating and feeding of said batch culture and said inducing of said promoter is performed at a temperature between 23° C. and 35° C.;
- and
- wherein said steps (b), (c) and (d) are performed without removal of medium, except for analytical purposes, and said steps result in an increase of the density of said bacterial host in said medium;
- thereby producing said recombinant capsid protein of the RNA bacteriophage being capable of forming said VLP.
Dependencies are inferred from the claim text.