SINGLE-USE CELL CULTURE CONTAINER WITH ONE OR MORE IN-SITU ONLINE SENSORS

Abstract

Herein is reported a bioreactor comprising a cultivation vessel and a reactor head plate, wherein the cultivation vessel has a working volume of from 20 ml to 350 ml and comprises two or more in-situ sensors, wherein the reactor head plate comprises an in-situ sensor port, wherein to the in-situ sensor port at least one in-situ glucose sensor and one in-situ pH sensor are connected.

Claims

1. A bioreactor comprising a cultivation vessel that has a working volume of from 20 ml to 350 ml, comprises a stirrer shaft with at least one impeller affixed thereto, comprises a feed pipe comprising i) a sparging tube connected to a sparger at its end, and ii) at least one feed line with an opening at its end, comprises two or more baffles extending from the wall of the cultivation vessel perpendicular in the direction to the center of the cultivation vessel, and a reactor head plate, wherein the reactor head plate comprises a fitting for connecting the drive axis of a motor to the stirrer shaft, a sparger gas inlet connected to the sparging tube in the feed pipe, optionally a gas inlet connected to the headspace, a gas outlet connected to the headspace of the cultivation vessel, at least one inlet for liquids of a feed line being part of the feed pipe, one in-situ sensor port with a pH electrode mounted thereto, and a supply port area comprising the sparger gas inlet and the inlet of the at least one feed line, wherein the cultivation vessel and the reactor head plate are both substantially made of non-metal material, characterized in that the bioreactor comprises an in-situ glucose sensor.

2. The bioreactor according to claim 1, wherein the small volume bioreactor is a single-use small volume bioreactor.

3. The bioreactor according to claim 1, wherein the bioreactor is a radiation sterilized small volume bioreactor.

4. The bioreactor according to claim 1, wherein the bioreactor is a sterilized small volume bioreactor and has been sterilized two times using radiation.

5. The bioreactor according to claim 3, wherein the radiation is beta radiation or/and gamma radiation.

6. The bioreactor according to claim 4, wherein the first radiation is beta radiation and the second radiation is gamma radiation or vice versa.

7. The bioreactor according to claim 1, wherein the glucose sensor is a screen-printed electrode coated with an immobilized enzyme.

8. The bioreactor according to claim 1, wherein the glucose sensor base material is polymer USP class VI.

9. The bioreactor according to claim 1, wherein the glucose sensor determines the glucose concentration every 20 seconds and/or determines the change of the glucose concentration.

10. The bioreactor according to claim 1, wherein the determined glucose concentration value is transmitted wireless or by cable from the glucose sensor to a computer.

11. The bioreactor according to claim 1, wherein the reactor head plate further comprises a sampling port.

12. The bioreactor according to claim 1, wherein the in-situ glucose sensor passes the head plate in or at the supply port area.

13. A method for cultivating a mammalian cell using a bioreactor according to claim 1.

14. A method for determining cultivation conditions using a bioreactor according to claim 1.

Description

DESCRIPTION OF THE FIGURES

[0192] FIG. 1: Side view of an exemplary single use small volume bioreactor (SUSVB) according to the current invention containing a glucose sensor (111).

[0193] FIG. 2: Side view of an exemplary SUSVB according to the invention with dimension and volume annotations.

[0194] FIG. 3: Top view of an exemplary SUSVB according to the invention showing the glucose sensor (111) fitted to the supply port area (133).

[0195] FIG. 4: Top-down view of an exemplary SUSVB according to the current invention containing a glucose sensor (111).

[0196] FIG. 5: Enlarged view of the part of the SUSVB according to the invention comprising the glucose sensor (111).

[0197] FIG. 6: Side view of the lower part of an exemplary SUSVB according to the invention comprising a glucose sensor (111) embedded in the cultivation medium (129).

[0198] FIG. 7: Schematic view of an exemplary glucose sensor (111).

[0199] FIG. 8: Temporal plot of on-line determined glucose concentration (thin line trending downward), off-line determined glucose concentrations (black dots); added glucose solution volume (thick line starting just before Thu 9 May) and glucose pump action (closely-spaced lines also starting just before Thu 9 May) of the cultivation RE02.

[0200] FIG. 9: Temporal plot of on-line determined glucose concentration (thin line trending up and down), off-line determined glucose concentrations (black dots); added glucose solution volume (thick line starting just before Thu 9 May) and glucose pump action (closely-spaced lines also starting just before Thu 9 May) of the cultivation RE06.

[0201] FIG. 10: Temporal plot of on-line determined glucose concentration (thin line trending up and down), off-line determined glucose concentrations (black dots); added glucose solution volume (thick line starting just before Thu 9 May) and glucose pump action (closely-spaced lines also starting just before Thu 9 May) of the cultivation RE10.

[0202] FIG. 11: Temporal plot of antibody concentration of the fermentations RE01-RE02, and RE04-RE12.

[0203] FIG. 12: Schematic diagram of various embodiments of the combination stirrer according to the invention; b: width of the stirrer blade; d: stirrer diameter; d.sub.w: diameter of the rotary shaft; h: height of the stirrer blade of the radially-conveying stirrer; h.sub.m: height of the fastening sleeve; h.sub.SB: height of the axially-conveying stirrer; h.sub.u: height of the reducer; l: length of the stirrer blade of the axially-conveying stirrer; α: blade pitch of the axially-conveying stirrer; z: number of stirrer blades per stirrer; d.sub.i: inner distance between the stirrer blades of the radially-conveying stirrer; h.sub.4/5: 4/5 height from above of h; K: stirrer head.

EXAMPLE 1

Cultivation Conditions for RE01-RE12

[0204] The cultivations were performed with a starting cell density of about 1.5*10E7 cells/ml in a total volume of 170 ml. The cultivation medium was a serum-free chemically defined medium. The cultivation temperature was set to 35° C., the gassing rate was set to 5-5.5 ml/min, the agitation rate was set to 450-500 rpm, and the pH was set to pH 7. PH-control was performed by adding a 1 M sodium carbonate solution or CO.sub.2 on top of the gassing rate. Defoamer was added at the beginning and during the cultivation when needed. Feed 1 containing glucose was added continuously with a pre-defined rate until stopped. Feed 2 not containing glucose was added as bolus-feed on days 1, 3 and 6.

[0205] In cultivations with an in-situ glucose sensor according to the invention sensor-dependent glucose feeding was started (feed 3) after the end of feed 1 once the determined in-situ glucose concentration dropped below a threshold value. The threshold value was lowered during the process.

[0206] FIG. 8 to FIG. 10 show plots of exemplary cultivation and FIG. 11 depicts the product concentration profile.