Fermentation process

摘要

The present invention provides a process for periplasmic expression of a bacterial toxoid comprising the steps of:
a) growing a culture of a gram negative host cell in a fermentation medium, wherein the host cell is transformed with a polynucleotide, and wherein the polynucleotide encodes the bacterial toxoid and a periplasmic signal sequence; or providing a gram negative host cell wherein the host cell is transformed with a polynucleotide, the polynucleotide encodes the bacterial toxoid and a periplasmic signal sequence and wherein the gram negative host cell comprises the bacterial toxoid expressed in the periplasm;a(i)) inducing expression of the bacterial toxoid;b) maturing the host cell, wherein the maturing step comprises:
I) subjecting the host cell to a pH shock;II) incubating the host cell with no feed addition; and/orIII) subjecting the host cell to a temperature below −20° C.; andc) extracting the bacterial toxoid from the host cell wherein the extraction process comprises osmotic shock.

主权项

1. A process for periplasmic expression of a bacterial toxoid comprising the steps of:
(a) growing a culture of a gram negative host cell in a fermentation medium, wherein the host cell is selected from the group consisting of E. Coli, Pseudomonas and Moraxella, and is transformed with a polynucleotide, and wherein the polynucleotide encodes a bacterial toxoid and a periplasmic signal sequence; (a(i)) inducing expression of the bacterial toxoid and collecting the host cells from the culture by centrifugation; and then (b) maturing the host cells, wherein the maturing step comprises:
I) subjecting the host cells to a pH shock; andII) incubating the host cells in a fermentation medium with no feed addition for at least two hours; (c) harvesting the host cells using centrifugation; and (d) extracting the bacterial toxoid from the host cells wherein the extraction process comprises osmotic shock;wherein the host cells are alive during step (b) and wherein the process is carried out in a fermentor which contains 10-5000 liters of culture.