Seed train processes and uses thereof

Abstract

Provided herein are seed train processes and methods of producing a recombinant protein that include the use of these seed train processes.

1 .- 33 . (canceled) 
     
     
         34 . A method of producing a recombinant protein comprising:
 (a) disposing a plurality of recombinant mammalian cells into a first culture medium comprised within a vessel to provide a first cell culture;   (b) batch culturing the first cell culture to a cell density range of about 1.0×10 6  cells/mL to about 5.0×10 6  cells/mL;   (c) disposing a volume of the first cell culture medium of (b) into a second culture medium comprised within a perfusion bioreactor to provide a second cell culture with an initial cell density in a range of about 0.25×10 6  cells/mL to about 0.5×10 6  cells/mL;   (d) perfusion culturing the second cell culture to a cell density range of between about 5×10 6  cells/mL to about 60×10 6  cells/mL;   (e) disposing a volume of the second cell culture of (d) into a third culture medium comprised within a production bioreactor to provide a production cell culture with an initial cell density in a range of about 0.25×10 6  cells/mL to about 8×10 6  cells/mL;   (f) perfusion culturing the production cell culture under conditions that allow the recombinant mammalian cells to secrete a recombinant protein;   (g) harvesting the recombinant protein from the production cell culture; and   (h) isolating the recombinant protein from the third culture medium.   
     
     
         35 . The method of  claim 34 , wherein disposing the plurality of recombinant mammalian cells in (a) comprises:
 thawing a frozen cell bank; and   disposing a volume of the thawed cell bank into the first culture medium.   
     
     
         36 .- 38 . (canceled) 
     
     
         39 . The method of  claim 34 , wherein disposing the plurality of recombinant mammalian cells in (a) comprises disposing a volume of a third cell culture comprising the plurality of recombinant mammalian cells into the first culture medium. 
     
     
         40 . The method of  claim 39 , further comprising:
 (1) disposing a plurality of the recombinant mammalian cells into a fourth culture medium comprised within a vessel to provide the third cell culture;   (2) batch culturing the third cell culture in (1) to a cell density range of about 1.0×10 6  cells/mL to about 5.0×10 6  cells/mL,   wherein a volume of the third cell culture in (2) is disposed into the first culture medium in (a).   
     
     
         41 .- 42 . (canceled) 
     
     
         43 . The method of  claim 40 , wherein disposing the plurality of the recombinant mammalian cells in (1) comprises:
 thawing a frozen cell bank; and   disposing a volume of the thawed cell bank into the fourth culture medium.   
     
     
         44 .- 47 . (canceled) 
     
     
         48 . The method of  claim 34 , wherein the first cell culture in (a) has a volume range of about 1.0 L to about 50 L. 
     
     
         49 . (canceled) 
     
     
         50 . The method of  claim 34 , wherein the second cell culture in (c) has a volume range of about 5 L to about 600 L. 
     
     
         51 . (canceled) 
     
     
         52 . The method of  claim 34 , wherein the production cell culture in (e) has a volume range of about 50 L to about 20,000 L. 
     
     
         53 . (canceled) 
     
     
         54 . The method of  claim 40 , wherein the fourth culture medium in (1) has a volume range of about 500 mL to about 20 L. 
     
     
         55 . (canceled) 
     
     
         56 . The method of  claim 34 , wherein the vessel in (a) has an internal volume range of about 1.5 L to about 100 L. 
     
     
         57 . (canceled) 
     
     
         58 . The method of  claim 34 , wherein the perfusion bioreactor in (c) has an internal volume range of about 7.5 L to about 1,000 L. 
     
     
         59 . (canceled) 
     
     
         60 . The method of  claim 34 , wherein the production bioreactor in (e) has an internal volume range of about 150 L to about 25,000 L. 
     
     
         61 . (canceled) 
     
     
         62 . The method of  claim 40 , wherein the vessel in (1) has an internal volume range of about 1 L to about 40 L. 
     
     
         63 . (canceled) 
     
     
         64 . The method of  claim 34 , wherein the perfusion culturing in (c) is performed using a perfusion bioreactor equipped with an alternating tangential flow filtration device. 
     
     
         65 . The method of  claim 34 , wherein the initial cell density in (e) is in a range of about 2.0×10 6  cells/mL to about 8×10 6  cells/mL 
     
     
         66 . The method of  claim 34 , wherein the initial cell density in (e) is at least 10% of the steady state production cell density. 
     
     
         67 . (canceled) 
     
     
         68 . The method of  claim 66 , wherein the steady state production cell density is between 5×10 6  cells/mL to about 50×10 6  cells/mL. 
     
     
         69 . (canceled) 
     
     
         70 . The method of  claim 34 , wherein the perfusion culturing in (f) results in the production cell culture reaching the steady state production cell density in a period of between about 1 day to about 10 days. 
     
     
         71 .- 74 . (canceled) 
     
     
         75 . The method of  claim 34 , wherein the isolating is performed using an integrated and continuous process. 
     
     
         76 . The method of  claim 34 , further comprising formulating the isolated recombinant protein into a pharmaceutical agent.