Cell culture process by intensified perfusion with continuous harvest and without cell bleeding

A method for producing a biological substance comprising:

• (a) culturing a cell culture comprising Chinese Hamster Ovary (CHO) cells and a cell culture medium for culturing the CHO cells, wherein the CHO cells are cultured at a range of about 35° C. to about 37° C., and the cell culture is established by inoculating at about 0.6-0.8×106 viable cells/mL,
• (b) perfusing the cell culture in a bioreactor with a basal medium and a feed medium, and
• (c) harvesting the biological substance,
• wherein the basal medium and the feed medium are fed to the cell culture at different rates, the cell culture is continuously passed through a separation system to harvest the biological substance from the bioreactor, and the cells are retained in the bioreactor without bleeding,
• wherein the basal medium is perfused at a perfusion rate of about 0.3 to about 0.6 working volume per day (VVD) from the first day and the perfusion rate is increased to about 1.5 to about 2.0 VVD between days 4-5 of the culturing,
• wherein the perfusion of the feed medium is started one day after the perfusion rate of the basal medium is increased and is started at a rate ranging from about 1 to 5% of the perfusion rate of the basal medium, then increased to about 7 to 9% between days 12-13 of the culturing and maintained at about 6 to 7% from days 18-19 of the culturing, and
• wherein the method further comprises subjecting the cell culture to a temperature shift to a temperature in the range of about 32° C. to about 33° C. in response to a predetermined peak viable cell density (VCD), wherein the feed medium is a CHO cell culture medium or a combination of CHO cell culture media.