Drain down and re-feed of microcarrier bioreactor

Abstract

Disclosed is a method of increasing product yield per culture in a population of bound product-secreting cells in a bioreactor, the method comprising: semi-harvesting product by removing a volume of the culture medium with a first-secreted product concentration; re-feeding the bound population of product-secreting cells by adding an amount of a fresh culture medium sufficient to increase the volume of the culture medium to approximately the original volume of the culture medium; agitating the culture medium under sufficient conditions and for a sufficient time period to allow the bound population of product-secreting cells to grow and to release a second-secreted product concentration into the culture medium; and harvesting product.

1 . A method of increasing product yield per culture in a population of product-secreting cells bound to a scaffold, which can optionally be a microcarrier, at least partially immersed in an original volume of a culture medium in a bioreactor, the method comprising:
 semi-harvesting product by removing from the bioreactor a first portion of the original volume of the culture medium with a first-secreted product concentration from the bioreactor while leaving the scaffold with the bound population of product-secreting cells in the bioreactor;   re-feeding the bound population of product-secreting cells by adding to the bioreactor an amount of a fresh culture medium sufficient to increase the volume of the culture medium in the bioreactor to approximately the original volume of the culture medium;   agitating the culture medium in the bioreactor under sufficient conditions and for a sufficient time period to allow the bound population of product-secreting cells to grow and to release a second-secreted product concentration into the culture medium; and harvesting product by removing from the bioreactor at least a portion of the culture medium with the second-secreted product concentration from the bioreactor while leaving the scaffold with the bound population of product-secreting cells in the bioreactor.   
     
     
         2 . The method of  claim 1 , wherein the product is chosen from a virus, an antibody, a growth factor, a protein, a peptide, and a hormone. 
     
     
         3 . The method of  claim 1 , wherein the product is a protein and the protein is an interferon. 
     
     
         4 . The method of  claim 1 , wherein the product is a virus and the virus is optionally a flavivirus. 
     
     
         5 . A method of increasing virus yield per culture in cells growing on a conditioned microcarrier in a bioreactor, the method comprising:
 transferring a plurality of seed cells into the bioreactor containing a microcarrier and an original volume of a culture medium,   cultivating the seed cells to bind to the microcarier and form a bound cell population that is from about 35 percent to about 95 percent confluent on the microcarrier;   removing from the bioreactor from about 30 percent to about 88 percent of the original volume of the culture medium in the bioreactor, while leaving the microcarrier with the bound cell population in the bioreactor, to form a first reduced volume of culture medium;   infecting the bound cell population with a virus, which can optionally be a flavivirus;   adding fresh culture medium to the bioreactor to maintain the bound cell population;   culturing the bound cell population for a sufficient time period to allow the infected, bound cell population to release a shed virus concentration into the culture medium in the bioreactor; and   harvesting virus by removing from the bioreactor at least a portion of the culture medium with shed virus therein.   
     
     
         6 . The method of  claim 5 , wherein the method further comprises:
 semi-harvesting virus by removing from the bioreactor a portion of the culture medium with a first-shed virus concentration, the portion of the culture medium removed equal to from about 50 percent to about 90 percent of the original volume of the culture medium, while leaving the microcarrier with the infected, bound cell population, and a second reduced volume of the culture medium in the bioreactor;   re-feeding the bound cell population by adding to the bioreactor a second amount of a fresh culture medium sufficient to increase the second reduced volume of the culture medium in the bioreactor to approximately the original volume of the culture medium;   culturing the bound cell population in the bioreactor under sufficient conditions and for a sufficient time period to allow the virus to continue to allow the infected, bound cell population to release a second-shed virus into the culture medium in the bioreactor; and   harvesting virus by removing from the bioreactor at least a portion of the culture medium with the second-shed virus while leaving the microcarrier with the infected, bound cell population in the bioreactor.   
     
     
         7 . The method of  claim 6 , wherein the semi-harvesting of virus comprises removing about 65 percent to about 75 percent of the culture medium with the first-shed virus from the bioreactor. 
     
     
         8 . The method of  claim 5 , wherein the infecting of the bound cell population with a virus is performed when the bound cell population is about 85 percent confluent on the microcarrier. 
     
     
         9 . The method of  claim 1 , wherein the bioreactor is a bench-top bioreactor, spinner flask or a disposable bioreactor having a volume of from about 25 liters to about 200 liters. 
     
     
         10 . The method of  claim 1 , wherein the cells are anchorage-dependent mammalian cells and optionally are VERO cells. 
     
     
         11 . A method of making a product from a cell culture of product-secreting cells bound to a scaffold, which can optionally be a microcarrier, at least partially immersed in an original volume of a culture medium in a bioreactor, the method comprising:
 semi-harvesting product by removing from the bioreactor a first portion of the original volume of the culture medium with a first-secreted product concentration from the bioreactor while leaving the scaffold with the bound population of product-secreting cells in the bioreactor;   re-feeding the bound population of product-secreting cells by adding to the bioreactor an amount of a fresh culture medium sufficient to increase the volume of the culture medium in the bioreactor to approximately the original volume of the culture medium;   agitating the culture medium in the bioreactor under sufficient conditions and for a sufficient time period to allow the bound population of product-secreting cells to grow and to release a second-secreted product concentration into the culture medium; and   harvesting product by removing from the bioreactor at least a portion of the culture medium with the second-secreted product concentration from the bioreactor while leaving the scaffold with the bound population of product-secreting cells in the bioreactor.   
     
     
         12 . The method of  claim 11 , wherein the product is chosen from a virus, an antibody, a growth factor, a protein, a peptide, and a hormone. 
     
     
         13 . The method of  claim 11 , wherein the product is a protein and the protein is an interferon. 
     
     
         14 . The method of  claim 11 , wherein the product is a virus and the virus is optionally a flavivirus. 
     
     
         15 . A method of making a virus in cells growing on a conditioned microcarrier in a bioreactor, the method comprising:
 transferring a plurality of seed cells into the bioreactor containing a microcarrier and an original volume of a culture medium,   cultivating the seed cells to bind to the microcarier and form a bound cell population that is from about 35 percent to about 95 percent confluent on the microcarrier;   removing from the bioreactor from about 30 percent to about 88 percent of the original volume of the culture medium in the bioreactor, while leaving the microcarrier with the bound cell population in the bioreactor, to form a first reduced volume of culture medium;   infecting the bound cell population with a virus, which can optionally be a flavivirus; adding fresh culture medium to the bioreactor to maintain the bound cell population;   culturing the bound cell population for a sufficient time period to allow the infected, bound cell population to release a shed virus concentration into the culture medium in the bioreactor; and   harvesting virus by removing from the bioreactor at least a portion of the culture medium with shed virus therein.   
     
     
         16 . The method of  claim 15 , wherein the method further comprises:
 semi-harvesting virus by removing from the bioreactor a portion of the culture medium with a first-shed virus concentration, the portion of the culture medium removed equal to from about 50 percent to about 90 percent of the original volume of the culture medium, while leaving the microcarrier with the infected, bound cell population, and a second reduced volume of the culture medium in the bioreactor;   re-feeding the bound cell population by adding to the bioreactor a second amount of a fresh culture medium sufficient to increase the second reduced volume of the culture medium in the bioreactor to approximately the original volume of the culture medium;   culturing the bound cell population in the bioreactor under sufficient conditions and for a sufficient time period to allow the virus to continue to allow the infected, bound cell population to release a second-shed virus into the culture medium in the bioreactor; and   harvesting virus by removing from the bioreactor at least a portion of the culture medium with the second-shed virus while leaving the microcarrier with the infected, bound cell population in the bioreactor.   
     
     
         17 . The method of  claim 16 , wherein the semi-harvesting of virus comprises removing about 65 percent to about 75 percent of the culture medium with the first-shed virus from the bioreactor. 
     
     
         18 . The method of  claim 15 , wherein the infecting of the bound cell population with a virus is performed when the bound cell population is about 85 percent confluent on the microcarrier. 
     
     
         19 . The method of  claim 1 , wherein the bioreactor is a bench-top bioreactor, spinner flask or a disposable bioreactor having a volume of from about 25 liters to about 200 liters. 
     
     
         20 . The method of  claim 1 , wherein the cells are anchorage-dependent mammalian cells and optionally are VERO cells.