Scalable process for therapeutic cell concentration and residual clearance

Abstract

Apparatus and corresponding method for concentration and washing of live mammalian cells, for preparation of human cell therapy products. Optimized parameters for a temperature regulated, completely closed, fully disposable and scalable counterflow centrifugation separation system having integrated disposables designed for both the input cells and output cells are provided.

What is claimed is: 
     
       1. A method for reducing volume of a first cell suspension using counterflow centrifugation, said method comprising the steps of:
 introducing the first cell suspension having an initial volume into a cell collection chamber of a counter-flow centrifugation apparatus via an inlet at a first flow rate; 
 introducing a cell collection fluid into the cell collection chamber at the first flow rate to allow the cells of the first cell suspension to form a cell bed; 
 distributing the cells in the cell bed into a plurality of groups of cells within the cell collection chamber by adjusting the first flow rate of the cell collection fluid to follow a predetermined flow profile, wherein the flow profile increases the first flow rate to a second flow rate over a predetermined time period at a ramp-up rate, wherein said ramp up rate is between one to two ml/minute/minute; 
 increasing the concentration of the cells within each of the plurality of groups of cells distributed within the cell collection chamber; 
 introducing a predetermined amount of washing fluid into the cell collection chamber, wherein the washing fluid replaces the cell collection fluid within the cell collection chamber; and 
 harvesting a second cell suspension, from each of the plurality of groups of cells distributed within the cell collection chamber; 
 wherein at least 80 percent of cells from said first cell suspension are recovered in said second cell suspension; 
 wherein said second cell suspension is at least ten fold more concentrated than said first cell suspension: and 
 wherein said second cell suspension maintains at least 80 percent cell viability. 
 
     
     
       2. The method of  claim 1 , wherein the direction of the flow of the cell suspension is opposite to a centrifugal force generated within the cell collection chamber, wherein the centrifugal force is balanced by the counter flow of the cell suspension at the first flow rate. 
     
     
       3. The method of  claim 1 , wherein the plurality of groups of cells are distributed within the cell collection chamber based on the cell size and density, wherein the group comprising cells having a first size and density is distributed in close proximity to an exit to the cell collection chamber and the group comprising cells having a second size and density is distributed in close proximity to the inlet to the cell collection chamber, wherein the second size and density is greater than the first size and density, wherein the first flow rate of the cell collection fluid is modified to follow the first profile, and wherein the first profile increases the first flow rate to the second flow rate over the second predetermined time period at the ramp-up rate. 
     
     
       4. The method of  claim 1 , wherein the concentration of the cells within each of the plurality of groups of cells is increased by maintaining the cell collection fluid at the second flow rate for a third predetermined time period. 
     
     
       5. A method comprising:
 introducing a first cell suspension having an initial volume into a cell collection chamber of a counter-flow centrifugation apparatus via an inlet at a first flow rate, wherein the direction of the flow of the first cell suspension is counter to a centrifugal force generated within the cell collection chamber, wherein the centrifugal force is balanced by the counter flow of the first cell suspension at the first flow rate; 
 introducing a cell collection fluid into the cell collection chamber at the first flow rate for a first predetermined time period to allow cells of the first cell suspension to form a cell bed; 
 distributing the cells in the cell bed into a plurality of groups of cells within the cell collection chamber based on cell size and density by modifying the first flow rate of the cell collection fluid, wherein the group comprising cells having a first size and density is distributed in close proximity to an exit to the cell collection chamber and the group comprising cells having a second size and density is distributed in close proximity to the inlet to the cell collection chamber, wherein the first flow rate of the cell collection fluid is modified to follow a first profile, wherein the first profile increases the first flow rate to a second flow rate over a second predetermined time period at a ramp-up rate, wherein said ramp up rate is between one to two ml/minute/minute; 
 increasing the concentration of the cells within each of the plurality of groups of cells distributed within the cell collection chamber by maintaining the cell collection fluid at the second flow rate for a third predetermined time period; 
 introducing a washing fluid into the cell collection chamber at a third flow rate for a fourth predetermined time period, wherein the washing fluid replaces the cell collection fluid within the cell collection chamber; and 
 harvesting a second cell suspension, from each of the plurality of groups of cells distributed within the cell collection chamber; 
 wherein at least 80 percent of cells from said first cell suspension are recovered in said second cell suspension; 
 wherein said second cell suspension is at least ten fold more concentrated than said first cell suspension; and 
 wherein said second cell suspension maintains at least 80 percent cell viability. 
 
     
     
       6. The method of  claim 5 , wherein harvesting said second cell suspension further comprises:
 modifying flow rate of the washing fluid to a fourth flow rate; and 
 collecting the plurality of groups of cells to yield a plurality of harvest volumes, and wherein the second cell suspension comprises the plurality of harvest volumes. 
 
     
     
       7. The method of  claim 6 , wherein each of the plurality of harvest volumes is determined using the relationship: y=37.865x+10, wherein y is the harvest volume in milliliters and x is the number of cells processed in billions. 
     
     
       8. The method of  claim 5 , further comprising the step of priming the counter-flow centrifugation apparatus with a media fluid. 
     
     
       9. The method of  claim 5 , further comprising the step of maintaining the cell collection chamber at a temperature of about 37° C. 
     
     
       10. The method of  claim 5 , wherein the second size is greater than the first size, and wherein the second density is greater than the first density. 
     
     
       11. The method of  claim 5 , wherein the first flow rate is from about 30 ml/min to about 60 ml/min. 
     
     
       12. The method of  claim 5 , wherein the first profile increases the first flow rate of the cell collection fluid to the second flow rate of from about 70 ml/min to about 155 ml/min. 
     
     
       13. The method of  claim 5 , wherein the first profile increases the first flow rate of the cell collection fluid to the second flow rate over the second predetermined time period of from about 3 minutes to about 150 minutes. 
     
     
       14. The method of  claim 5 , wherein each of the plurality of groups of cells distributed within the cell collection chamber is harvested at the fourth flow rate of from about 50 ml/min to about 250 ml/min. 
     
     
       15. The method of  claim 5 , wherein the centrifugal force generated within the cell collection chamber has a relative centrifugal force value of from about 500 to about 1000. 
     
     
       16. The method of  claim 15 , wherein the harvested cells of said second cell suspension are substantially free of mycoplasma, endotoxin and microbial contamination. 
     
     
       17. The method of  claim 15 , wherein said harvesting said second cell suspension is less than 2 hours from said introducing the cell suspension into a cell collection chamber. 
     
     
       18. The method of  claim 15 , wherein said harvesting said second cell suspension is less than 1 hour from said introducing the cell suspension into a cell collection chamber. 
     
     
       19. The method of  claim 15 , further comprising monitoring the plurality of groups of cells using an automation device, the device comprising:
 a viable cell density sensor means, for monitoring harvest density; 
 a flow meter monitoring means, for monitoring harvest volume; 
 a control logic software means, to control or achieve the desired final concentration of product cells in collection bag, and for operating valves, wherein said control logic software means are pneumatically or electrically activated and wherein the flow of fluid can be diverted from one collection bag to the other. 
 
     
     
       20. The method of  claim 15 , wherein the harvested cells of said second cell suspension are in a pharmaceutical-grade solution suitable for human administration. 
     
     
       21. The method of  claim 5 , wherein the total number of viable harvested cells is 2 billion to 30 billion. 
     
     
       22. The method of  claim 5 , wherein said cells are in a final harvest volume between 75 milliliter to 1200 milliliter. 
     
     
       23. The method of  claim 5 , wherein the first profile comprises operating parameters for a dynamic flow rate operation to follow a predetermined increase in net force for cell retention, wherein said operating parameters comprises:
 the cell collection fluid first flow rate of between 30-50 ml/min; 
 the cell collection fluid second flow rate of between 120-160 ml/min; 
 a centrifugal force value of between 500-1000 ref; and 
 introducing the washing fluid at a wash flow rate of between 100-160 ml/min. 
 
     
     
       24. The method of  claim 5 , further comprising the step of maintaining the cell collection chamber at less than 28° C. 
     
     
       25. The method of  claim 5 , wherein the harvested cells of said second cell suspension are in a pharmaceutical-grade solution suitable for human administration. 
     
     
       26. The method of  claim 5  wherein harvest volume y in milliliters is determined using the relationship y=37.865x +10 for product concentration of 20 million cells/ml, wherein x is the number of total viable cells in billions. 
     
     
       27. The method of  claim 5 :
 wherein at least 90 percent of cells from said first cell suspension are recovered; 
 wherein said second cell suspension is at least 50 fold more concentrated than said first cell suspension; and 
 wherein said second cell suspension maintains at least 90 percent cell viability. 
 
     
     
       28. The method of  claim 27 :
 wherein at least 95 percent of cells from said first cell suspension are recovered; and 
 wherein said second cell suspension is at least 100 fold more concentrated than said first cell suspension.