Patent ID: 8187855
Filing Date: 2012-05-29
Classification: C12N,C12Y

Abstract:
1. A method for producing soluble rHuPH20, comprising: a) inoculating cell medium in a bioreactor with an inoculum of cells that encode soluble rHuPH20 to produce a cell culture, wherein: the cells comprise between 150 and 300 copies of nucleic acid encoding soluble rHuPH20; the bioreactor contains at least 100 liters of cell culture; about 10 the cells are cultured at a set temperature; b) feeding the cells with a first feed medium containing glucose, c) feeding the cells with a second feed medium containing glucose, lowering the temperature compared to the temperature in step a) to a temperature sufficient to increase cell cycle arrest, increase cell viability and stabilize the soluble hyaluronidase; wherein: the amount of the amount of yeast extract is increased compared to the amount of yeast extract in step b); and the feed medium is added to the culture at a volume of 0.5% or about 0.5% to 20% of the cell culture volume; d) feeding the cells with a third feed medium containing glucose, lowering the temperature compared to the temperature in step c) to a temperature sufficient to increase cell cycle arrest, increase cell viability and stabilize the soluble hyaluronidase; wherein: the amount of the amounts of yeast extract, glucose and sodium butyrate are increased compared to the amounts of yeast extract, glucose and sodium butyrate in step c); and the feed medium is added to the culture at a volume of 0.5% or about 0.5% to 20% of the cell culture volume; e) feeding the cells with a fourth feed medium containing glucose, lowering the temperature compared to the temperature in step d) to a temperature sufficient to increase cell cycle arrest, increase cell viability and stabilize the soluble hyaluronidase; wherein: the amount of the amount of sodium butyrate is decreased compared to the amount of sodium butyrate in step d); and the feed medium is added to the culture at a volume of 0.5% or about 0.5% to 20% of the cell culture volume; f) continuing to culture the cells until viability drops below at least or about 50%; g) obtaining the harvest cell culture fluid; and h) purifying the rHuPH20 from the harvest cell culture fluid.