Microcarrier perfusion culturing methods and uses thereof

Abstract

Provided herein are methods of culturing a mammalian cell and various methods that utilize these culturing methods.

1 .- 110 . (canceled) 
     
     
         111 . A method for testing a manufacturing process for making a recombinant protein, the method comprising:
 providing a shake tube containing an adherent Chinese hamster ovary (CHO) cell containing a nucleic acid encoding a recombinant protein disposed in a first liquid culture medium, wherein the first liquid culture medium occupies about 10% to about 30% of the volume of the shake tube and contains a plurality of microcarriers at a concentration of about 1.0 g/L to about 15.0 g/L;   incubating the shake tube for a period of time at about 32° C. to about 39° C. and with a rotary agitation of about 130 revolutions per minute (RPM) to about 150 RPM;   after about the first 48 to 96 hours of the period of time, continuously or periodically removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal, and the method achieves a viable cell density of greater than 2.0×10 6  cells/mL in the first liquid culture medium or a combination of the first and second liquid culture medium at some point during the period of time;   detecting the recombinant protein in the cell or in the first and/or second culture medium; and   comparing the amount of recombinant protein present in the adherent CHO cell or in the first and/or second culture medium to a reference level of recombinant protein.   
     
     
         112 . The method of  claim 111 , wherein the reference level of recombinant protein is a level of recombinant protein produced using a different culturing method. 
     
     
         113 . The method of  claim 112 , wherein the different culturing method utilizes a different first or second liquid culture medium, a different adherent CHO cell, a different temperature, a different level of agitation, a different shake tube, or a different microcarrier. 
     
     
         114 . The method of  claim 112 , wherein the different culturing method utilizes a different raw material, anti-clumping agent, or chemically-defined liquid culture medium. 
     
     
         115 . The method of  claim 111 , wherein the method is used to perform high throughput cell culture experiments to perform a design-of-experiment (DOE) or a quality-by- design (QBD) study. 
     
     
         116 . The method of  claim 111 , wherein the shake tube has a volume of between about 10 mL to about 100 mL. 
     
     
         117 . The method of  claim 111 , wherein the adherent CHO cell is suspended in about 2 mL to about 20 mL of the first liquid culture medium. 
     
     
         118 . The method of  claim 111 , wherein the recombinant protein is a secreted immunoglobulin, a secreted enzyme, a secreted growth factor, a secreted protein fragment, or a secreted engineered protein, and wherein the recombinant protein is recovered from the first or second culture medium. 
     
     
         119 . The method of  claim 111 , wherein the recombinant protein is recovered from the adherent CHO cell. 
     
     
         120 . The method of  claim 111 , wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein. 
     
     
         121 . The method of  claim 111 , wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed simultaneously. 
     
     
         122 . The method of  claim 111 , wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed continuously. 
     
     
         123 . The method of  claim 111 , wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed periodically. 
     
     
         124 . The method of  claim 111 , wherein the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added are increased over time. 
     
     
         125 . The method of  claim 111 , wherein the first liquid culture medium and/or second liquid culture medium is selected from the group consisting of: a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, and a protein-free medium. 
     
     
         126 . The method of  claim 111 , wherein the shake tube is incubated at a reactor angle of about 25 degrees to about 90 degrees from horizontal. 
     
     
         127 . A method of optimizing a manufacturing process of producing a recombinant protein, the method comprising:
 providing a shake tube containing an adherent Chinese hamster ovary (CHO) cell containing a nucleic acid encoding the recombinant protein disposed in a first liquid culture medium, wherein the first liquid culture medium occupies about 10% to about 30% of the   volume of the shake tube and contains a plurality of microcarriers at a concentration of about 1.0 g/L to about 15.0 g/L;   incubating the shake tube for a period of time at about 32° C. to about 39° C. and with a rotary agitation of about 130 revolutions per minute (RPM) to about 150 RPM; and   after about the first 48 to 96 hours of the period of time, continuously or periodically removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal, and the method achieves a viable cell density of greater than 2.0×10 6  cells/mL in the first liquid culture medium or a combination of the first and second liquid culture medium at some point during the period of time;   detecting the recombinant protein in the adherent CHO cell or in the first and/or second culture medium;   comparing the amount of recombinant protein present in the adherent CHO cell or in the first and/or second culture medium to a reference level of recombinant protein produced by a different method; and   identifying and removing or altering in a manufacturing process any culture components or parameters that are associated with a decrease in the amount of recombinant protein produced as compared to the reference level, or identifying and adding to a manufacturing process any culture components or parameters that are associated with an increase in the amount of recombinant protein produced as compared to the reference level.   
     
     
         128 . The method of  claim 127 , wherein the shake tube has a volume of between about 10 mL to about 100 mL. 
     
     
         129 . The method of  claim 127 , wherein the adherent CHO cell is suspended in about 2 mL to about 20 mL of the first liquid culture medium. 
     
     
         130 . The method of  claim 127 , wherein the shake tube is incubated at a reactor angle of about 25 degrees to about 90 degrees from horizontal.