Process for the culturing of cells

Abstract

The invention relates to a process for the culturing of cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells in a reactor in suspension in a cell culture medium, wherein the cells produce a biological substance, preferably an antibody, wherein at least one cell culture medium component is fed to the cell culture and wherein the cell culture comprising the cells, the biological substance and cell culture medium is circulated over a separation system and wherein the separation system separates the biological substance from substances having a lower molecular weight than the biological substance and wherein the biological substance is retained in or fed back into the reactor. Preferably part of the substances of lower molecular weight is continuously removed from the cell culture.

Claims

1. Process for the culturing of cells in a reactor in suspension in a cell culture medium, comprising: circulating a cell culture through a separation system, wherein the cell culture comprises eukaryotic cells, a biological substance produced by the cells, and a cell culture medium; wherein the biological substance is an IgG; wherein at least one cell culture medium component is fed to the cell culture; wherein the cell culture is circulated through the separation system in a flow substantially parallel to a surface of said separation system resulting in a liquid outflow and a flow wherein cell culture contents are retained or fed back into the reactor; wherein the separation system comprises a filter having a pore size characterized by a molecular weight cut-off smaller than the molecular weight of the biological substance to separate the biological substance from substances having a lower molecular weight than the biological substance; wherein the liquid outflow consists essentially of components having molecular weight lower than that of the biological substance and the flow wherein cell culture contents are retained or fed back into the reactor consists essentially of components having molecular weight equal to or higher than that of the biological substance; wherein the cell culture is removed at least once from the reactor, and wherein the biological substance is harvested from the cells and/or from the cell culture.

2. Process according to claim 1, wherein the filter comprises a membrane filter.

3. Process according to 2, wherein the membrane filter comprises a hollow fiber filter.

4. Process according to claim 2 or 3 wherein the filter has a pore size with a molecular weight cut-off of at most 100 kDa.

5. Process according to claim 4, wherein the filter has a pore size with a molecular weight cut-off of at most 50 kDa.

6. Process according to claim 5, wherein the filter has a pore size with a molecular weight cut-off of at most 30 kDa.

7. Process according to claim 1, wherein the molecular weight cut-off is smaller by at least a factor of 2 than the molecular weight of the IgG.

8. Process according to claim 1, wherein the molecular weight cut-off is smaller by at least a factor of 3 than the molecular weight of the IgG.

9. Process according to claim 1 wherein the cell culture is circulated through the filter in an alternating tangential flow.

10. Process according to claim 1 wherein the cell culture is circulated through the filter in a unidirectional tangential flow.

11. Process according to claim 1, wherein part of the substances of lower molecular weight is continuously removed from the cell culture.

12. Process according to claim 1, wherein the cells are mammalian cells.

13. Process according to claim 12, wherein the mammalian cells are selected from the group consisting of Chinese hamster ovary (CHO) cells, hybridomas, baby hamster kidney (BHK) cells, myeloma cells, human cells, or mouse cells.

14. Process according to claim 13, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

15. Process according to claim 1, wherein the harvesting comprises subjecting the cell culture to affinity chromatography and/or ion exchange chromatography.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. shows the viable cell density Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).

(2) FIG. 2. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).

(3) FIG. 3. shows the viable cell density Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C2 (process of the invention).

(4) FIG. 4. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C2 (process of the invention).

(5) FIG. 5. shows the viable cell density Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C3 (process of the invention).

(6) FIG. 6. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A3) versus the process time X (days) for process A, and C3.

(7) FIG. 7. shows the cumulative yield Q (% as compared to yield in process A, per L reactor volume) plotted versus the process time X (days) for process A, B and C3.

(8) FIG. 8. shows the cell number Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for C4 (process of the invention).

(9) FIG. 9. shows the IgG concentration in the reactor Z (% as compared to the maximum IgG concentration reached versus the process time X (days) for process C4 (one embodiment of process of the invention)

EXAMPLES

Example 1

(10) Comparison between a Batch Process, a Fed Batch Process and the Process according to the Invention.

(11) In this example the performance of the process according to the present invention was compared to batch and fed-batch processes.

(12) FIG. 1 shows the viable cell density Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).

(13) FIG. 2 shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).

(14) All fermentations were performed using a Sartorius Biostat B controller to control the temperature at 36.5 C., the pH between 7.2 and 6.8 and the DO at 50% air saturation and at 200 rpm. The same IgG producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.

(15) Batch Process A

(16) The batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 310e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.

(17) Fed-Batch Process B

(18) The fed-batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 310e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose and glutamine were added to keep the concentration above respectively 15 mM and 1 mM. Amino acids and peptides were added from day 5 to replenish the consumed amino acids.

(19) Process of the Invention C1

(20) The process of the invention was performed in a 2 L Applikon vessel. A 100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane obtained from General Electric (GE) operated in ATF flow mode with an ATF-2 system (Refine Technology) was used to retain the cells and the IgG product. The culture was started with 310e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture medium (SAFC) supplemented with 6 mM L-Glutamin was perfused through the suspension cell culture using a Specific Flow Rate (SFR) between 0.05 and 0.2 nL/cell/day. The highest product concentration obtained was 1.4 g/L.

(21) The process of the invention resulted in increased viable cell densities and increased product concentrations compared to mentioned cultivation modes in less time, as can be seen from FIG. 1 and FIG. 2 below.

Example 2

(22) Comparison between a Batch Process, a Fed Batch Process and the Process according to the Invention.

(23) In this example the process according to the present invention is again compared to batch and fed-batch processes; in process C2, the CO.sub.2 pressure is controlled and a 50 kDa separation system was used.

(24) FIG. 3 shows the viable cell density Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C2 (process of the invention).

(25) FIG. 4 shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C2 (process of the invention)

(26) All fermentations were performed using a Sartorius Biostat B controller to control temperature at 36.5 C., pH between 7.2 and 6.8 and DO at 50% air saturation and at 200 rpm. The same IgG (of approximately 150 kDa) producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.

(27) Batch Process A

(28) The batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3.10.sup.5 cells.mL.sup.1 in VPRO medium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.

(29) Fed-Batch Process B

(30) The fed-Batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3.10.sup.5 cells.mL.sup.1 in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose and glutamine were added to keep the concentration above respectively 15 mM and 1 mM. Amino Acids and peptides were added from day 5 to replenish the consumed amino acids.

(31) Process of the Invention C2

(32) The process of the invention was performed in a 2 L Applikon vessel. A 50 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane (GE) operated in ATF flow mode with an ATF-2 system (Refine Technology) was used to retain the cells and the IgG product. The culture was started with 310e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture medium (SAFC) supplemented with 6 mM L-Glutamin is perfused through the suspension cell culture using an SPR between 0.05 and 0.2 nL.cell.sup.1.day.sup.1. The CO.sub.2 pressure was controlled below 15%.

(33) Result

(34) As can be seen from FIG. 3 and from FIG. 4, the process according to the invention results in significantly increased viable cell densities and increased product concentrations (2415%Batch yield; 690%Fed-Batch yield) in equal or less time (100% Batch time; 81% Fed-Batch time).

(35) The overall productivity increase in g.L.sup.1.day.sup.1 of the process of the invention is 23.9 times the Batch productivity in g.L.sup.1.day.sup.1) and 8.5 times the Fed-batch productivity in g.L.sup.1.day.sup.1. In the process of the invention C2, 11.1 g product/L was produced. Clogging of the retention device did not occur during 17 days, even with very high cell density.

Example 3

(36) Comparison between a Batch Process, a Fed Batch Process and the Process according to the Invention.

(37) In this example the performance of the process according to the present invention with cell culture removal and again compared to batch and fed-batch processes; in process C3 cell culture has been removed.

(38) FIG. 5 shows the viable cell density Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C3 (process of the invention).

(39) FIG. 6 shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A3) versus the process time X (days) for process A, and C3.

(40) FIG. 7 shows the cumulative yield Q (% as compared to yield in process A, per L reactor volume) plotted versus the process time X (days) for process A, B and C3.

(41) All fermentations were performed using a Sartorius Biostat B controller to control temperature at 36.5 C., pH between 7.2 and 6.8 and DO at 50% air saturation and at 200 rpm. The same IgG (of approximately 150 kDa) producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.

(42) Batch Process A

(43) The batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3.10.sup.5 cells.mL.sup.1 in VPRO medium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.

(44) Fed-Batch Process B

(45) The fed-Batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3.10.sup.5 cells.mL.sup.1 in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose and glutamine were added to keep the concentration above respectively 15 mM and 1 mM. Amino Acids and peptides were added from day 5 to replenish the consumed amino acids.

(46) Process of the Invention C3

(47) The process of the invention was performed in a 2 L Applikon vessel. A 100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane (GE) operated in ATF flow mode with an ATF-2 system (Refine Technology) was used to retain the cells and the IgG product. The culture was started with 310e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture medium (SAFC) supplemented 6 mM L-Glutamin is perfused through the suspension cell culture using an SPR between 0.05 and 0.2 nL.cell.sup.1.day.sup.1. Cell culture is removed at 10% of the working volume per day above 10.10.sup.6 cells.mL.sup.1 and at 30% of the working volume per day when the viable cell density exceeds 30.10.sup.6 cells.mL.sup.1 and onwards.

(48) Result

(49) As can be seen from FIG. 5 with the process of the invention higher viable cell densities are reached fast. Furthermore, FIG. 5 also shows that the viability of the cells can be maintained longer with the process of the invention as process C3 was maintained in operation over a period of nearly 40 days, because no clogging of the retention device occurred even with high cell densities.

(50) FIG. 6 shows that product concentrations for the process of the present invention are much higher than the product concentration in the batch process. The product flow containing the product was harvested from process C3 at approximately 200% to 250% times the final concentration in the batch process A.

(51) FIG. 7 shows that most product is formed by the process of the present invention and that the process of the invention can be maintained longer than the batch process A or the fed-batch B. At day 17, the cumulative yield of process C3 is 8.1 times the cumulative yield of the batch process (A3) and 2.1 times the cumulative yield of the fed-batch process (B). Also, at day 17, the batch process ended. At day 21, the cumulative yield of process C3 is 3.0 times the cumulative yield of the fed-batch process B. At day 21, the fed-batch process ended. After 39 days, the overall cumulative yield of process C3 is 25 times the cumulative yield of the batch process A and 6 times the yield of the fed-batch process B.

(52) It can be concluded from this experiment that the overall yield of a desired biological material in the process according to the present invention can be further improved by applying a bleed of the cell culture when the cell density exceeds a certain high level.

Example 4

(53) Culturing of and Production with CHO Cells.

(54) In this example the process according to the present invention has been performed with an IgG producing CHO cell line and includes a temperature drop to decrease cell growth.

(55) FIG. 8 shows the cell number Y (10.sup.6.ml.sup.1) plotted versus the process time X (days) for C4 (process of the invention).

(56) FIG. 9 shows the IgG concentration in the reactor Z (% as compared to the maximum IgG concentration reached versus the process time X (days) for process C4 (one embodiment of process of the invention)

(57) The fermentation was performed using a Sartorius Biostat B controller to control temperature at 36.5 C., pH between 7.1 and 6.9 and DO at 40% air saturation and at 100 rpm. The temperature was dropped to 32 C. on day 5.

(58) Process of the Invention C4

(59) The process of the invention was performed in a 2 L Applikon vessel. Cell and product retention device is a 50 kD Molecular Weight Cut-Off (MWCO) hollow fiber membrane (General Electric) operated in ATF flow mode with an ATF-2 system (Refine Technology). The culture was started with 5.10.sup.6 cells.mL.sup.1 in MTCM-49 culture medium (Hyclone). The medium was perfused through the suspension cell culture using an SPR between 0.1 and 0.4 nL.cell.sup.1.day.sup.1. The CO.sub.2 pressure was controlled below 15%.

(60) Result

(61) The data show that the process of the invention also works when using a protein producing CHO cell line. The achieved cell density and the product concentrations are increased compared to batch culture. The data also show that in the process according to the present invention cell growth can be arrested (e.g. by a temperature drop), whereas the product accumulation in the culture system continues.

Example 5

(62) Process of the Invention Performed with a Myeloma Cell Line.

(63) The process according to the present invention can also be applied to myeloma cell lines. To this end the fermentation is performed using a Sartorius Biostat B controller to control temperature at 36.5 C., pH between 7.2 and 6.8 and DO at 40% air saturation and at 100 rpm. The cell culturing starts with inoculating the myeloma cells at 310e5 cells/ml in SFM4Mab culture medium (Hyclone) in a 5 L Sartorius vessel. The cell and product retention device is a 30 kD Molecular Weight Cut-Off (MWCO) hollow fiber membrane (General Electric) operated in ATF flow mode with an ATF-4 system (Refine Technology). SFM4Mab culture medium (Hyclone) is perfused through the suspension cell culture using an SPR between 0.1 and 0.4 nL.cell-1.day-1. The CO2 pressure is controlled below 15%.

Example 6

(64) Process of the Invention Performed with an MDCK Cell Line.

(65) The process according to the present invention can also be applied to transformed MDCK cell lines in suspension. To this end the fermentation is performed using a Sartorius Biostat B controller to control temperature at 36.5 C., pH between 7.2 and 6.8 and DO at 40% air saturation and at 100 rpm. The cell culturing starts with inoculating the transformed MDCK cells at 310e5 cells/ml in VP-SFM culture medium (Invitrogen) in a 5 L Sartorius vessel. The cell and product retention device is a 30 kD Molecular Weight Cut-Off (MWCO) hollow fiber membrane (General Electric) operated in ATF flow mode with an ATF-4 system (Refine Technology). VP-SFM culture medium (Invitrogen) is perfused through the suspension cell culture using an SPR between 0.1 and 0.4 nL.cell-1.day-1. The CO2 pressure is controlled below 15%.