Abstract
The invention relates to a device for sampling from bioreactors, comprising a rising line (1) connectable to a bioreactor, a tap line (2), and a withdrawal line (3), which are connected to a T-like connection. The withdrawal line (3) comprises a first shut-off valve (4), the tap line (2) is connected to a positive pressure source (5), and the withdrawal line (3) is connected to a pump (7). In the device for sampling, the positive pressure source (5), the pump (7), and the first shut-off valve (4) are controllable in an automated manner via a control unit (13). The invention furthermore relates to a method for using such a device for sampling.
Claims
1. A device for sampling from a bioreactor, the device comprising: a rising line connectable to a bioreactor; a tap line; and a withdrawal line; wherein the rising line, the tap line, and the withdrawal line are connected to a T-like connection, wherein the withdrawal line includes a first shut-off valve; wherein the tap line is connected to a positive pressure source, wherein the withdrawal line is connected to a pump, and wherein the positive pressure source, the pump, and the first shut-off valve are automatically controlled via a control unit.
2. The device according to claim 1, wherein the tap line includes a sterile filter located between the positive pressure source and the T-like connection.
3. The device according to claim 1, wherein the rising line includes a second shut-off valve, which is automatically controlled via the control unit.
4. The device according to claim 1, wherein the tap line includes a check valve.
5. The device according to claim 1, wherein at least one of the rising line or the withdrawal line includes a sensor unit.
6. The device according to claim 1, wherein the rising line includes a first sensor unit, which is configured to transmit a measurement value to the control unit.
7. The device according to claim 6, wherein the first sensor unit comprises a contactless sensor.
8. The device according to claim 6, wherein the first sensor unit comprises a sensor that comes in contact with a media.
9. The device according to claim 1, wherein the withdrawal line comprises a second sensor unit.
10. The device according to claim 9, wherein the second sensor unit comprises a contactless sensor.
11. The device according to claim 9, wherein the second sensor unit comprises a sensor that comes in contact with a media.
12. The device according to claim 1, wherein at least one of the rising line and the withdrawal line includes a sterile coupling.
13. The device according to claim 1, wherein the withdrawal line includes a mixing chamber.
14. The device according to claim 1, wherein the control unit is configured to control at least the positive pressure source, the pump, and the first shut-off valve at a pre-designated time.
15. The device according to claim 6, wherein the control unit is configured to control at least the positive pressure source, the pump, and the first shut-off valve based on the measurement value of the first sensor unit.
16. A method of sampling a bioreactor, the method comprising: providing a device at a bioreactor containing a sample, the device comprising: a rising line; a tap line; and a withdrawal line; wherein the rising line, the tap line, and the withdrawal line are connected to a T-like connection, wherein the withdrawal line includes a first shut-off valve, wherein the tap line is connected to a positive pressure source, wherein the withdrawal line is connected to a pump, and wherein the positive pressure source, the pump, and the first shut-off valve are automatically controlled via a control unit; when the rising line is connected to the bioreactor, operating the pump; operating the pump, with the first shut-off valve open and a second shut-off valve closed, for sampling; applying a positive pressure to the tap line using the positive pressure source, while operating the pump, and with the first shut-off valve open and the second shut-off valve closed, for blowing out the withdrawal line; and applying a second positive pressure using the positive pressure source, with the second shut-off valve open and the first shut-off valve closed, for blowing out the rising line.
Description
[0068] In the drawings:
[0069] FIG. 1 shows a bioreactor system, a bioreactor, and a device for sampling comprising a first sensor system;
[0070] FIG. 2 shows the bioreactor system comprising a second sensor system;
[0071] FIG. 3 shows the bioreactor system comprising a third sensor system;
[0072] FIG. 4 shows the bioreactor system from FIG. 3, comprising a rinsing line;
[0073] FIG. 5 shows the bioreactor system comprising the first sensor system and a sterile coupling;
[0074] FIG. 6 shows the bioreactor system comprising the second sensor system and the sterile coupling;
[0075] FIG. 7 shows the bioreactor system co rising the third sensor system and the sterile coupling;
[0076] FIG. 8 shows the bioreactor system from FIG. 6, having an alternative arrangement of the sterile coupling;
[0077] FIG. 9 shows the bioreactor system from FIG. 6, additionally comprising a mixing chamber; and
[0078] FIG. 10 shows a tabular presentation of a sequence of steps for use of the bioreactor system.
[0079] FIG. 1 shows a bioreactor system, wherein a bioreactor 11, which is designed as an autoclavable or as a steam-sterilizable vessel, for example, is connected to a rising line 1 of a device for sampling. On the side facing away from the bioreactor 11, the rising line 1 is connected in a T-like manner to a tap line 2 and a withdrawal line 3, for example by means of a T piece. The withdrawal line comprises a pump 7, for example a roller pump or a peristaltic pump, by which negative pressure can be generated in the withdrawal line 3. The withdrawal line 3 furthermore comprises a first shut-off valve 4. The rising line 1 comprises a second shut-off valve 8, which can be used to close the rising line 1. A first sensor unit 10, which is equipped with one or more sensors, is located on the side of the second shut-off valve 8 which faces the bioreactor 11. In this embodiment, the first sensor unit is preferably equipped with contactless sensors, for example with optical sensors. The device according to the application is not limited to the effect that one or more sensors have to be provided in a single first sensor unit 10. It is also possible to distribute multiple sensors among multiple sensor units. On the side facing away from the T-like connection, the tap line 2 comprises a positive pressure source 5 for generating positive pressure in the tap line 2. Such a positive pressure source 5 can be designed as a simple syringe or another gas source, such as a pressurized gas receptacle or a receptacle comprising a movable stud, which can be pressurized by lowering the stud, similarly to a syringe, preferably in an automatically controllable manner. The gas used for generating the positive pressure can, for example, be a process gas, nitrogen or air. It may stem from a sterile gas source, for example. The gas stemming from the positive pressure source 5 can be filtered by an additional possible sterile filter 6, which is arranged in the tap line, before it comes in contact with the organisms present in the bioreactor 11 or in the remaining lines 1, 2. A check valve 9, which is arranged in the tap line 2 on the side of the sterile filter 6 which faces the T-like connection, is provided for protecting the sterile filter 6. This check valve serves as an additional safety measure, but may also be dispensed with since negative pressure preferably does not arise in the tap line, which causes organisms to find their way into the tap line, but preferably positive pressure for blowing out is generated in the tap line 2, while negative pressure is preferably only generated in the withdrawal line 3. The withdrawal line 3 comprises a pump 7 for generating negative pressure in the withdrawal line 3, in particular in relation to the rising line 1 and in relation to the bioreactor 11. The withdrawal line 3 furthermore comprises a first shut-off valve 4. The withdrawal line 3 can optionally furthermore comprise a check valve, which is not shown and allows fluid to flow only from the T-like connection in the direction of the pump 7, but not in the opposite direction.
[0080] It is preferably possible to sterilize the lines together with the bioreactor 11. For example, the bioreactor 11 can be autoclaved together with the lines 1, 2, 3 designed as flexible tubing, wherein tubing ends are preferably kept closed so as to prevent contamination after sterilization. The sterile coupling 6 also helps to prevent the contamination. After the sterilization, the shut-off valves 4, 8 can be mounted. The first sensor unit 10 can also preferably be attached to the outside of the rising line 1 designed as flexible tubing and, for example, measure optical density through the tubing.
[0081] After sterilization, the device can be set up as described above, and a reaction can be started in the bioreactor.
[0082] The device according to the application can then be used to carry out different methods for sampling during the reaction, during which the individual elements of the device, such as the shut-off valves 4, 8, the pump 7, and the positive pressure source 5 are activated, or opened and/or closed, according to a particular pattern.
[0083] The pump 7, the positive pressure source 5, as well as the first and second shut-off valves 4, 8 can be activated via a control unit 13. The communication link between the individual components and the control unit 13 is indicated by dotted lines in the figure. For example, the control unit 13 can be programmed in such a way that the withdrawal process is carried out at established time intervals. For this purpose, the user can predefine, for example, a starting point in time, an interval between the samplings, and a number of samplings. However, the control unit 13 can also be in communication with the sensor 10, so that sampling by means of a pre-programmed sequence of steps, which is described in more detail hereafter, is triggered when a measurement value measured by the sensor of the first sensor unit 10 meets an established criterion. It is also possible that a sampling process that is intended at an established point in time is only carried out when the measured value meets such a criterion. In this way, it is possible, for example, to start a sampling process at an established point in time, wherein sample material is pumped from the bioreactor 11 into the rising line, at least up to the first sensor unit 10. Based on the measurement values recorded by the sensor of the first sensor unit 10, the sample is either pumped onward and withdrawn, or pumped back into the bioreactor.
[0084] In this way, it is made possible in particular to carry out multiple sampling processes or measurements distributed over a longer period of time, and to minimize the need for human interaction to the extent possible.
[0085] Possible steps for taking a sample or collecting measurement values, which are advantageous for the use of the shown sampling process, are described hereafter.
[0086] The execution of all steps is controlled via the control unit 13, which receives signals from the individual components or transmits signals to the individual components.
[0087] During a sampling process, sterile compressed air can be introduced into the tap line 2 in a preparatory manner by means of the positive pressure source, in conjunction with the sterile filter 6, wherein the first shut-off valve is preferably kept closed, while the second shut-off valve is opened. As a result, sample material remaining in the rising line 1 connected to the tap line 2 is forced back into the bioreactor. The second shut-off valve can then be closed.
[0088] Thereafter, the pump 7 is started up, wherein the first shut-off valve 4 is preferably initially kept closed. Thereafter the first shut-off valve 4 is opened, and the second shut-off valve 8 is kept closed, so as to generate negative pressure in the withdrawal line 3, or establish positive pressure in the bioreactor 11, and at least a portion of the rising line 1, compared to the withdrawal line 3. In a later step, the second shut-off valve 8 is then also opened, and during continued operation of the pump 7 an established amount of the organisms present in the bioreactor 11 is pumped or drawn out of the bioreactor 11 in to the rising line 1. These organisms pass the first sensor unit 10 arranged in the rising line 1 and are measured by means of the one sensor, or by means of at least one of the multiple sensors, of the first sensor unit 10, and the measurement values are transmitted to the control unit 13.
[0089] For example as a function of the measurement values thus recorded, which are, for example, compared to user-defined threshold values in the control unit 13, the organisms can then be forced back into the bioreactor 11 in that the first shut-off valve 4 is closed by the control unit 13 and the positive pressure source 5 is activated. After the sample has been forced back into the bioreactor, both shut-off valves 4, 8 can be closed. The steps that relate to forcing back the sample after the measurement are provided in the devices according to the application in which one or more sensors are arranged in the rising line 1. As is described hereafter in connection with other figures, not all devices provide sensors in the rising line 1, so that the steps for forcing back are typically not used in methods for using device having no sensors in the rising line 1.
[0090] As an alternative, it is possible to withdraw the sample, as a function of the result of the comparison of the measurement values to the threshold values, by pumping the sample, after it has passed the first sensor unit 10, into the withdrawal line 3 by continuing to operate the pump 7, with the shut-off valves 4, 8 open, and through the withdrawal line 3, for example into a sample vessel (not shown).
[0091] For rinsing the pumping path, the second shut-off valve 8 is then closed again, and positive pressure is generated in the tap line by the positive pressure source 5. The withdrawal line is blown out by simultaneously continuing to operate the pump 7 and keeping the first shut-off valve 4 open. Thereafter, the first shut-off valve 4 can be closed, and the second shut-off valve 8 can be opened, while keeping the positive pressure source 5 switched on. The pump 7 can be switched off in the process. In this way, the rising line 1 is blown out. The duration of this blowing-out step of the rising line 1 can be selected in such a way that only a small amount of gas is introduced into the bioreactor. So as to avoid contamination, the steps for blowing out the withdrawal line 3 and the rising line 1 can be carried out not only after, but also before a sampling process.
[0092] FIG. 2 shows the bioreactor system, wherein a second sensor unit 10′ is arranged in the withdrawal line 3, instead of the first sensor unit 10 shown in FIG. 1. The second sensor unit 10′ can comprise contactless sensors and/or sensors that come in contact with media, provided the sensors coming in contact with media are suitable to be sterilized together with the bioreactor.
[0093] In this configuration, the steps for sampling are carried out similarly to the manner described in connection with FIG. 1, wherein here, however, no option exists for pumping the sample back into the reactor after the measurement. As mentioned above, typically no steps for forcing the sample back are thus carried out. Instead, the sample is withdrawn as described above, wherein measurement values are collected during the withdrawal which can be associated with the withdrawn sample. The advantage of this configuration is that the second sensor unit 10′ can be disconnected from the bioreactor by the shut-off valves 4, 8, so that the sensors of the second sensor unit 10′ can be cleaned by blowing out the withdrawal line 3, and undesirable contact between the sensors and the organisms from the bioreactor can be avoided between the measurements.
[0094] FIG. 3 shows the bioreactor system, wherein the device for sampling, as a combination of the embodiments shown in FIGS. 1 and 2, comprises both the first sensor unit 10 and the second sensor unit 10′. In this way, the above-described advantages of both devices are combined. The device can accordingly be operated as described in connection with FIG. 1. However, additional advantages arise from the combination of the first sensor unit 10 and the second sensor unit 10′. For example, the second sensor unit 10′ can be configured to transmit a signal to the control unit 13 when the sample arrives at or passes the second sensor unit 10′ during the sampling process. The sampling process can then be terminated, for example, as soon as the sample arrives at the second sensor unit 10′, for example in that the second shut-off valve 8 is closed by the control unit. In this way, it is possible to control the volume of the withdrawn samples. For example, it can be ensured that every sample has the same volume or approximately the same volume. In a possible method in which the second shut-off valve is closed as soon as the sample reaches the second sensor unit 10′, the volume of the sample approximately corresponds to a line volume of the section of the withdrawal line 3 located between the T piece and the second sensor unit 10′. In some cases, such volume control is preferable to time control, in particular when the viscosity of the sample or the pressure conditions in the bioreactor system are not constant and, for example, change during the reaction or during the withdrawal. A combination of the described volume control with time control is likewise possible.
[0095] FIG. 4 shows the bioreactor system from FIG. 3, wherein a rinsing line 14 is provided in the withdrawal line 3 between the first shut-off valve 4 and the second sensor unit 10′. Sterile gas and/or liquids can be introduced into the flow in the withdrawal line 3 via the rinsing line 14, for example. This can be used, for example, for cleaning the withdrawal line 3 itself, in which case the second sensor unit 10′ does not have to be present in the sampling device. However, this can also be used to clean the sensors of the second sensor unit 10′ or be necessary for the use of the sensors of the second sensor unit 10′, for example when buffers that are used to reduce the impact on enzymatic sensors or to extend the service life thereof are fed into the withdrawal line via the rinsing line 14. In addition, it is possible for solutions for calibrating the sensors of the second sensor unit 10′ to be introduced into the flow. For introducing such solutions into the flow, these are supplied, for example, between two withdrawal processes via the rinsing line 14, and thereafter, with the first shut-off valve 4 open and the second shut-off valve 8 closed, the withdrawal line is blown out using sterile gas by way of the positive pressure source 5, so as to have no residue of the introduced solution, to the extent possible, present during the subsequent measurement which could adversely affect a measurement. The method steps described here which relate to the rinsing line can also be carried out with modified devices; for example, a first sensor unit 10 is also not necessary for this purpose.
[0096] FIG. 5 shows the bioreactor system as in FIG. 1, wherein additionally an optional rinsing line 14 is arranged at the withdrawal line, and a sterile coupling is provided between the first sensor unit 10 and the bioreactor 11. The sterile coupling comprises two coupling unions 111, 12.2, which can be connected to one another so as to establish a fluid connection, which is sealed to the outside, between two tubing or line ends at which the coupling unions are arranged. In the process, a first coupling union 12.1 is arranged in the rising line 1 on the side of the first sensor unit 10, and a second coupling union 12.2 is arranged in the rising line on the side of the bioreactor 11, so that the bioreactor 11, together with a portion of the rising line 1 and the second coupling union 12.2, can be disconnected from the remaining components of the sampling device. The shown embodiment comprising the sterile coupling is in particular of advantage when the sensor, or one of the sensors, of the first sensor unit 10″ is designed as a sensor coming in contact with media. Sensors coming in contact with the media are frequently not steam-sterilizable and can accordingly not be sterilized together with the bioreactor. The arrangement of the sterile coupling shown in the figure allows the section of the device comprising the sensor or sensors coming in contact with media to be sterilized separately, for example by gamma sterilization, plasma sterilization, or by means of ethylene oxide. So as to be able to sterilize the two sections of the device or of the bioreactor accordingly separately, the particular coupling unions connected to the sections are accordingly designed to be sterilizable. For example, the first coupling union 12.1 located on the side of the first sensor unit 10 can be gamma-sterilizable and made of plastic or steel, and the second coupling union 12.2 located on the side of the bioreactor 11 can be steam-sterilizable and made of plastic or metal.
[0097] FIG. 6 shows the bioreactor system, wherein the sterile coupling is arranged in the same location as in the example of FIG. 5, however, as is also shown in FIG. 2, only the second sensor unit 10′ is provided, instead of the first sensor unit 10. This configuration is particularly advantageous when the second sensor unit comprises sensors coming in contact with media. On the one hand, the rinsing line 14, as described above, allows the sensors to be cleaned and/or calibrated, and on the other hand, the sterile coupling makes it possible to separately sterilize sensors coming in contact with media. Otherwise, such a system is used as described in connection with FIG. 2.
[0098] As mentioned, the sterile coupling allows the sensors of the second sensor unit 10′ which come in contact with media to be sterilized separately from the bioreactor 11. The sterile filter 6 and the check valve 9 can then, for example, be gamma-sterilized together with the second sensor unit 10′, and thereafter can be connected to the autoclaved bioreactor 11. The shut-off valves 4, 8 can be configured as pinch valves, for example, and be installed after the sterilization.
[0099] FIG. 7 shows a combination of the embodiments from FIGS. 5 and 6. The sterile coupling is still arranged as the first component downstream of the bioreactor 11 in the rising line 1. In addition, the device comprises both the first sensor unit 10 and the second sensor unit 10′, and a rinsing line. In this way, both sensor units 10, 10′ can be sterilized separately from the bioreactor, for example when both sensor units comprise sensors that come in contact with media. Otherwise, the shown system is used as described in connection with FIG. 3, wherein additionally the above-described advantages of the rinsing line can be taken advantage of.
[0100] FIG. 8 shows the bioreactor system, wherein the sterile coupling is arranged in the withdrawal line 3, instead of in the rising line 1. In the process, the sterile coupling is positioned between the first shut-off valve 4 and the rinsing line 14. The bioreactor 11 can then be steam-sterilized, for example together with the check valve 9 and the sterile filter 6 as well as the rising line 1, the tap line 2, a portion of the withdrawal line 3, and the first coupling union 12.1, wherein the shut-off valves 4, 8 can be installed again after sterilization. The second sensor unit and the rinsing line 14, as well as the section of the withdrawal line 3 connected thereto and the second coupling union 12.2, can either be sterilized separately or be unsterile.
[0101] In other embodiments, such a configuration can also comprise a first sensor unit 10 and/or a sterile coupling as arranged in FIGS. 5 to 7.
[0102] FIG. 9 shows the bioreactor system, wherein the sensors and the sterile coupling are arranged as in FIG. 6, however a mixing chamber 16 is provided in the withdrawal line 3 between the second sensor unit and the first shut-off valve 4. A rinsing line 15 is connected to the mixing chamber 16 and furthermore comprises a mixing chamber drain 17 including a valve.
[0103] A sensor coming in contact with media, such as a pH sensor, can also be present in the mixing chamber 16.
[0104] During sampling by means of a system comprising such a mixing chamber 16, essentially the same steps are carried out as in the other described systems. The withdrawn sample first reaches the mixing chamber, before passing the second sensor unit 10′, and can then be left in the mixing chamber 16 for a certain period of time by accordingly briefly shutting off the pump 7 and/or by actuating valves. For this purpose, further valves can also be provided in possible embodiments. In the mixing chamber, the sample can be pretreated before being pumped further along the withdrawal line, so as to be measured by the second sensor unit 10′ and then be withdrawn or discarded.
[0105] A medium for diluting the sample may be added via the rinsing line 15. It is also possible, however, to add chemicals to the mixing chamber 16, for example, via the rinsing line 15, which enable a sensor measurement. For example, acids or lyes can be added, which shift the pH value of the sample in such a way that a measurement by way of a sensor of the second sensor unit 10′ is possible.
[0106] The mixing chamber can be emptied, for example, after use via the mixing chamber drain 17 and be cleaned by way of the rinsing line 15.
[0107] FIG. 10 shows, in table form, a possible sequence of steps for use of the bioreactor system according to the application, which are automatically triggered by the control unit, for example. In the process, steps S1 to S10 are carried out in the order listed here. A duration of each step is generally a few seconds. The status of the valves 4, 8 or of the pump 7 and the positive pressure source 5 during each step is indicated with “0” or “1” in the corresponding columns. In the process, “0” denotes “closed” or “off” and “1” denotes “open” or “on.” In a starting position S1, the valves 4, 8 are closed, and the pump 7 and the positive pressure source 5 are switched off. At an established point in time, for example at a point in time programmed in advance or in an event-controlled manner, triggered by a signal that is applied based on a value measured by a sensor 10, 10′, step S2 is initiated. In S2, pressure is applied to the tap line 2 by switching on the positive pressure source 5, while all other components remain in the “0” position. In S3, in addition to the positive pressure source 5 being switched on, the second shut-off valve 8 is opened, and the 1 is purged. Thereupon, in S4, the positive pressure source 5 is switched off so as to reduce the pressure in the rising line 1, while the second shut-off valve 8 remains open. In step S5, all valves 4, 8 are then closed again, and the positive pressure source 5 is left in the switched-off state, while the pump 7 is started up. In 56, positive pressure is generated in the rising line 1 in relation to the withdrawal line 3 by opening the first shut-off valve 4, while operating the pump at the same time. By subsequently additionally opening the second shut-off valve 8, in S7 the entire fluid path from the bioreactor 11 to the pump 7 is released, so that organisms can be withdrawn. The duration of this withdrawal process S7 varies as a function of the volume to be withdrawn. After the withdrawal has been completed, in S8 the second shut-off valve 8, and thus the rising line 1, are closed again so that the withdrawal line 3 can be blown out and cleaned. For this purpose, the positive pressure source 5 is additionally switched on. This step is carried out until it is ensured that the entire sample has reached the sample vessel. Thereafter, in S9, the rising line 1 is blown out by closing the first shut-off valve 4, and opening the second shut-off valve 8 again, while continuing to operate the positive pressure source 5. After having carried out steps 8 and 9, organisms that remained in the rising line 1 after the withdrawal process were recirculated to the reactor and organisms that remained in the withdrawal line were recirculated to the withdrawn sample, so that the process has no or a negligibly small dead volume. Thereafter, in the final position S10=S1 all elements can be returned to the “0” position until a new signal for sampling is issued.
[0108] In addition to the steps described here, it is possible, depending on the design and intended use of the design, as mentioned in connection with the other figures and in the description, to carry out additional steps, for example for forcing back the sample or for collecting measurement values by means of the first and/or the second sensor units, as well as for rinsing the second sensor unit, just to mention a few options. The listed possible steps thus do not represent an exhaustive list of the usage options of a device according to the application, or of a bioreactor system according to the application, but rather form a basis proceeding from which numerous further application options, which include further steps, for example, open up to a person skilled in the art.
LIST OF REFERENCE NUMERALS
[0109] 1 rising line [0110] 2 tap line [0111] 3 withdrawal line [0112] 4 first shut-off valve [0113] 5 positive pressure source [0114] 6 sterile filter [0115] 7 pump [0116] 8 second shut-off valve [0117] 9 check valve [0118] 10 first sensor unit [0119] 10′ second sensor unit [0120] 11 bioreactor [0121] 12.1 first coupling union [0122] 12.2 second coupling union [0123] 13 control unit [0124] 14 rinsing line [0125] 14 first rinsing line [0126] 15 second rinsing line [0127] 16 mixing chamber [0128] 17 mixing chamber drain
