Modular electroporation method and system

Abstract

A device is provided for sending electrical signals to an electroporation chamber and for monitoring the quality of the transmitted signals. Specifically, the device is a modular structure including a main module and an audit module. The main module has control electronics with a signal generator configured to generate electrical pulses for electroporation and is electrically connected to the audit module. The audit module is configured to store calibration data, measure current and voltage passed from the main module over time, and pass such measurement data to the main module. The main module is configured to generate an error signal if the voltage and current measured by the audit module do not match the expected values ​​of voltage and current. The audit module is removablely mounted to the main module.

Description

Technical Field

[0001] The present invention relates to an electroporation machine. More specifically, the present invention relates to a modular electroporation system and a method of operating the system.

Background Art

[0002] Electroporation is understood to be the use of electrical pulses that reversibly destabilize biological membranes such as vesicle membranes or cell membranes, and provide a pathway for the introduction of exogenous materials into vesicles or cells during the destabilization period. Usually, such introduced exogenous materials may be genetic materials for obtaining genetically modified cells for research or therapeutic purposes. Both the cells and the exogenous material are supplied as liquid suspensions. Most interesting in recent years is the use of electroporation to introduce mRNA into cells, which is easily converted into proteins that give those cells additional functions. Cell therapy is an emerging field of medicine that requires a sterile and reliable production facility where primary cells from donors or patients can be genetically modified in large quantities according to strict "Good Manufacturing Practice and Quality Control Standards (GMP)". For example, the production of clinical batches of allogeneic CAR-T cells, which requires making the outer receptacle part smaller and several gene editing steps to express an artificial chimeric antigen receptor (CAR) [Non-Patent Document 1], is particularly difficult in that regard.

[0003] Patent Document 1 discloses a system including a pulse voltage waveform generator, a switching device that connects the anode or cathode of the pulse voltage waveform generator to an electroporation chamber, and an electrode array in the electroporation chamber that converts the pulse voltage into a pulse electric field. Also disclosed are the filling cycle of the electroporation chamber with cell suspension and exogenous material suspension, the electroporation of the mixture, and emptying the chamber when the amount of cell suspension to be treated exceeds the limit capacity of the chamber.

[0004] During electroporation, in accordance with GMP, it is desirable to monitor and evaluate the electrical pulses applied to the electroporation chamber to ensure consistent quality of the electroporation results, particularly to ensure quality control of the products. Such evaluation must take into account voltage and current, as well as pulse timing. Regarding timing, both pulse duration and the time interval between consecutive pulses should be understood.

[0005] Typically, electroporation voltage and current data measured over time are stored in non-volatile memory for later evaluation and quality control.

[0006] However, as is well known, the output of electronic circuits drifts over time. This applies not only to the circuit that generates the electroporation pulse, the signal generator, but also to the circuit that monitors the voltage and current that constitute the pulse. Traditionally, external calibration equipment has been connected to the electroporation system to evaluate, in detail, the self-monitoring capability of the electronic circuit. However, this is undesirable for several reasons. Specifically, electroporation equipment must be operated under cleanroom conditions, and therefore, introducing external equipment near where electroporation is performed or removing equipment for maintenance and calibration can lead to contamination problems. Furthermore, from this perspective, calibration without moving / disassembling the device has been impossible. Moving and / or disassembling the device leads to production losses, resulting in wasted time and expense. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2006 / 0089674 [Patent Document 2] U.S. Patent No. 6010613 [Non-patent literature]

[0008] [Non-Patent Document 1] Depil, S. et al. (2020), 'Off-the-shelf' allogeneic CAR T cells: development and challenges, Nat Rev Drug Discov 19: pp. 185-199. [Non-Patent Document 2] Schwartz J. et al., [Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue (2013), J Clin Apher, 28(3):145-284] [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, the object of the disclosed invention is to provide an electroporation system that is low cost to build and operate, ensures good electroporation results, and is free from the drawbacks listed above. [Means for solving the problem]

[0010] Beyond the production of therapeutic-grade cells, applications may extend to various areas of research or industrial processes, including, but are not limited to, the transformation of cell lines, bacteria, yeast, algae, and plant cells, the production of protoplasts for bioproduction, or vaccine production.

[0011] definition A modular system describes a structure based on modules that may or may not be integrated. In this invention, modules include a main module and an audit module.

[0012] The system of the present invention is designed to process vesicles that may be artificial cells or living cells. More specifically, the system is designed for the electroporation of cells such as cell lines or primary cells.

[0013] Cells refer to living cells that have been cultured or placed in a suspension.

[0014] Regarding "primary cells" or "multiple primary cells," the term refers to cells taken from living tissue (e.g., biopsy material) and immobilized for in vitro growth over a limited period, meaning they may experience a limited number of population doublings. Primary cells are opposed to tumorigenic serial cell lines or artificially immortalized serial cell lines. Non-limiting examples of such cell lines include CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, and Molt 4 cells. Primary cells are typically used in cell therapy because they are more functional and less tumorigenic.

[0015] Cells differentiated from stem cells such as umbilical cord blood stem cells, progenitor cells, myeloid stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) are considered primary cells, as described in this invention.

[0016] Generally, primary cells are derived directly or indirectly, preferably directly, from a donor or patient by various methods known in the art, such as the leukocyte apheresis transfusion technique verified in Non-Patent Document 2.

[0017] In the present invention, from the viewpoint of manufacturing a clinical batch of therapeutic cells for cell therapy, special attention is paid to immune cells such as T cells and NK cells, stem cells such as hematopoietic stem cells and hematopoietic progenitor cells, embryonic stem (ES) cells, or induced pluripotent stem (iPS) cells.

[0018] Electroporation means creating temporary permeability in the cell membrane using a pulsed electric field without loss of cell viability. <000^080> An exogenous material is any substance outside of a living cell. For the purposes of the present disclosure, it is a substance that is delivered into the cell. The comprehensive list of substances includes polypeptides, polynucleotides, drugs, polymers, carbohydrates, and combinations of these within the same molecule or different molecules. Examples of polypeptides and polynucleotides include proteins, and DNA or RNA (including mRNA, RNAi), respectively. Exogenous materials for use in electroporation may include combinations such as ribonucleoproteins (protein and RNA complexes).

[0020] PulseAgile protocol: A series of at least three waveforms having one, two, or three of the following characteristics: (1) at least two of at least three waveforms have different waveform magnitudes, (2) at least two of at least three waveforms have different waveform widths, and (3) the first waveform interval of a first set of two of at least three waveforms is different from the second waveform interval of a second set of two of at least three waveforms. Examples of such an agile pulse sequence are described in Patent Document 2.

[0021] Suspension: Insoluble particles such as living cells suspended in an aqueous liquid.

[0022] The present invention relates to an electroporation device, system, and method.

[0023] Specifically, the present disclosure relates to a modular electroporation device including a main module and an auditing module. The names "main" and "auditing" are not intended to limit the present disclosure, and generally are merely for the purpose of explaining the functions of each module. The main module includes a control electronic device having a signal generator configured to generate an electrical pulse for electroporation, a multi-electrode connector connected to the control electronic device, and an output connector for connection to an electroporation chamber. The auditing module includes a multi-electrode connector configured to be connected to the multi-electrode connector of the main module. Further, the auditing module is provided with a non-volatile memory configured to store calibration data, and sensor electronics connected to the multi-electrode connector and configured to measure the voltage and current of the electrical pulse received from the main module with respect to time. The sensor electronics are further configured to transmit digital communication to the main module based on the measured voltage and current and the calibration data stored in the non-volatile memory.

[0024] The control electronic device of the main module is configured to generate an error signal if the current and voltage measured by the sensor electronics considering the calibration data and transmitted by the communication channel do not match the expected current and voltage.

[0025] The auditing module is removably attached to the main module. Optionally, the auditing module is nested within a slot in the main module.

[0026] Optionally, the signal generator is configured to generate an electrical pulse having a series of at least three waveforms including: (a) at least two of the at least three waveforms have different waveform magnitudes from each other; (b) at least two of the at least three waveforms have different waveform widths from each other; and (c) the first waveform interval of the first set of two of the at least three waveforms is different from the second waveform interval of the second set of two of the at least three waveforms.

[0027] Optionally, the audit module is configured to draw power from the main module. Furthermore, the sensor electronics may include an analog-to-digital converter. In addition, the sensor electronics may be configured to transmit the measured voltage and current to the main module upon receiving a trigger signal from the main module.

[0028] A further aspect of the present disclosure is an electroporation system comprising the aforementioned electroporation device, an electroporation chamber, and a calibration device configured to connect to the audit module when the audit module is separated from the main module. The calibration device includes a circuit configured to calibrate the audit module and to store calibration data in non-volatile memory.

[0029] The system disclosed in the preceding paragraph may also include a container for holding a cell suspension, a container for holding an exogenous material suspension, a container for receiving electroporation products, and a tubing device configured to supply a predetermined amount of cell suspension and a predetermined amount of exogenous material suspension to the electroporation chamber 200 before electroporation, and to deliver the electroporation products from the electroporation chamber to the container for receiving the electroporation products after electroporation. The containers for holding cell suspensions typically contain cell suspensions of cell types selected from CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells, as well as T cells, NK cells, umbilical cord blood stem cells, progenitor cells, myeloid stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPS cells), as well as equivalents. The containers for holding exogenous material suspensions typically contain exogenous material suspensions selected from polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and combinations thereof including ribonucleoproteins.

[0030] The disclosed system may further include a replacement audit module, which has the same functionality as the audit module.

[0031] In a further aspect, the disclosure also relates to a method for operating the electroporation system disclosed in the preceding section. The method includes: a) connecting an audit module to a calibration device; b) performing a calibration routine in the audit module; c) storing calibration information relating to the audit module in non-volatile memory; d) disconnecting the audit module from the calibration device; e) connecting the audit module to a main module; f) supplying energy to the main module to perform an electroporation sequence; g) measuring the electroporation voltage and current over time by the audit module; h) correcting the measured voltage and current using the calibration information stored in non-volatile memory; i) passing the voltage and current information from the audit module to the main module; and j) comparing the corrected voltage and current information in the main module with expected conditions. If the result of the comparison is outside a predetermined tolerance range, an error signal is generated.

[0032] Further steps to the method described above are also disclosed, in which a second audit module is calibrated following steps a) through d) above. The audit module is then connected to the main module, and electroporation steps e) through j) are performed. Steps e) through j) can be repeated for a predetermined time or number of cycles, after which the audit module is replaced with the second audit module. Steps e) through j) can then be performed using a combination of the main module and the second audit module.

[0033] The method described above may also include the step of introducing the following into the electroporation chamber before supplying energy to the main module. A predetermined amount of cell suspension is a cell type selected from CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells, as well as T cells, NK cells, umbilical cord blood stem cells, progenitor cells, myeloid stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPS cells), and equivalents, while a predetermined amount of exogenous material suspension is selected from combinations thereof, including polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and ribonucleoproteins.

[0034] The modular electroporation device and system structure described above allows calibration to be maintained without interrupting the electroporation process for unnecessarily long periods and without introducing external equipment into the electroporation environment. The claimed invention makes it possible to maintain cleanroom conditions while simultaneously maintaining the calibration of a continuously operating electroporation system.

[0035] In the attached diagram, the following figure is given as a non-limiting example. [Brief explanation of the drawing]

[0036] [Figure 1] This is a schematic diagram of a modular electroporation device. [Figure 2a] This is a schematic diagram of one structure of an electroporation system. [Figure 2b] This is a schematic diagram of one structure of an electroporation system. [Figure 2c] This is a schematic diagram of one structure of an electroporation system. [Figure 3] Figures 2a and 2b are flowcharts showing the operation of the electroporation system. [Figure 4] Figures 2a and 2c are flowcharts showing the operation of the electroporation system. [Figure 5] This is a diagram illustrating an example of a modular electroporation device. [Modes for carrying out the invention]

[0037] Figure 1 shows a modular electroporation device 100 connected to an electroporation chamber 200. In the context of this disclosure, the phrase "connected to an electroporation chamber" means that an electrical connection is made to the electrodes in the electroporation chamber, for example, so that an electric field is applied to the contents of the chamber.

[0038] The electroporation device 100 includes a main module 10 and a removable audit module 20. The main module 10 and the audit module 20 are connected by multi-electrode connectors 30a, 30b having a main side 30a and an audit side 30b. Suitable such multi-electrode connectors 30a, 30b are 31-way Harting connectors.

[0039] The main module 10 and the audit module 20 may also be mechanically attached to each other. Furthermore, the audit module 20 may be mechanically attached to the main module 10 by autonomous rails and secured to the rear panel by restraining screws. In some devices, the audit module may be partially enclosed by the main module 10. For example, as shown in the example in Figure 5, the audit module may be fitted into a slot in the main module.

[0040] Here, the main module 10 and the audit module 20 are described respectively. The main module includes control electronics 11, which includes a signal generator 5 configured to generate electrical pulses for electroporation. The duration and voltage of the electrical pulses are selected according to the biological material on which electroporation is performed. Furthermore, the shape of the voltage profile may also be controlled. An exemplary signal generator is configured to generate signals according to the PulseAgile protocol.

[0041] Output electrical connectors 18a and 18b are configured to connect the main module 10 to the electroporation chamber 200. In some devices, these electrical connectors 18a and 18b are separable. The signal generated by the signal generator 5 is passed through the output electrical connectors to the electroporation chamber 200.

[0042] The audit module 20 includes a sensor electronic device 15 connected to a multi-electrode connector. The sensor electronic device also includes an analog-to-digital converter and a non-volatile memory 16.

[0043] The electrical connection 12 between the main module and the audit module passes through a multi-electrode connector. This electrical connection includes several components, including wiring that transmits electroporation signals generated by the signal generator 5, and a digital communication channel configured for communication between the audit module 20 and the main module 10. The digital communication channel may be a series bus, such as RS232. The electrical connection 12 is also configured to supply power from the main module to the audit module. In some other devices, the audit module includes its own power supply.

[0044] The sensor electronic device 15 is configured to measure the voltage and / or current of the electroporation signal, which is generated by the signal generator and transmitted to the audit module by the electrical connection unit 12. The voltage and / or current are recorded over time. Preferably, the electroporation signal is sent through the audit module. The sensor electronic device 15 in the audit module 20 is configured to measure the current and voltage of the electroporation signal as it passes through the audit module. That is, the electroporation signal is generated in the main module by the signal generator 5, and the signal is then sent through the audit module and returned to the main module and then to the electroporation chamber 200.

[0045] As described above, a digital communication channel is provided between the audit module 20 and the main module 10. Furthermore, the sensor electronics are configured to indicate the current and voltage measured over time, and to digitally transmit this information to the control electronics of the main module through the digital communication channel.

[0046] Furthermore, as described above, the audit module includes a non-volatile memory 16. Specifically, the non-volatile memory is configured to store calibration information regarding the audit module's sensitivity to measured current and voltage. Therefore, current and voltage measurements are corrected according to the calibration data. Such corrections are preferably performed by the sensor electronics 15 before the corrected voltage and current information is communicated to the main module 10 via a digital communication channel. Alternatively, the uncorrected current and voltage information, as well as the calibration information, are passed through the digital communication channel so that the voltage and current information can be corrected in the control electronics 11 of the main module 10.

[0047] In some devices, the audit module is configured to continuously measure current and voltage and transmit this information to the main module via a digital communication channel. Alternatively, measurement and information transmission are initiated upon receipt of a trigger signal from the main module. Upon receipt of this trigger signal, the sensor electronics are configured to measure the current and voltage of the electroporation signal and to communicate this information digitally to the main module 10. Alternatively, upon receipt of the trigger signal, the audit module is configured to transmit previously measured values ​​of current and voltage measured over time.

[0048] As illustrated, the audit module 20 draws its power from the main module 10, whereas in other devices, the audit module has its own power source.

[0049] Time-based logs of voltage and current measurements, as measured by the audit module, are stored in non-volatile memory. This non-volatile memory may constitute part of the main module 10 or the audit module 20.

[0050] Referring to Figures 2a and 2c, the modular electroporation device 100 described above is now incorporated into the electroporation system. As shown in Figure 2a, the modular electroporation device 100 is connected to the electroporation chamber 200. During electroporation, the voltage and current of the electroporation signal are measured by the sensor electronics 15 of the audit module, and this information is transmitted via a digital communication channel to the control electronics 11 of the main module 10. The measured voltage and current are corrected using calibration information stored in the non-volatile memory 16 of the audit module 20. In the main module, the voltage and current information is compared to expected values. This comparison may include the measured versus expected temporal characteristics of the electrical signal, including pulse width and time between consecutive pulses. Based on this comparison, if the measured value is not within a predetermined tolerance range of the expected value, the control electronics is configured to generate an error signal. If such an error signal is generated, the resulting electroporation product is unusable and must be rejected. If no error signal is generated, the electroporation product may be approved for its intended purpose.

[0051] Figure 2b shows how the audit module 20 is not connected to the main module 10, but instead to a calibration device 50. The calibration device 50 contains a circuit 51 configured to evaluate the current and voltage measurement capabilities of the audit module and to write calibration information to a non-volatile memory 16. This calibration information is used to correct the voltage and current measurements performed by the audit module, as described above.

[0052] Figure 2c shows additional equipment, with a replacement audit module 20a attached to the main module 10. This can occur simultaneously with the installation of the audit module 20 on the calibration device 50.

[0053] Typically, the electroporation chamber 200 would be fluidly connected to a tubing device configured to supply a predetermined amount of cell suspension from a container holding the cell suspension and a predetermined amount of exogenous material suspension from a container holding the exogenous material suspension. After electroporation, the tubing device is further configured to deliver the electroporation products from the electroporation chamber 200 to a container for receiving the electroporation products.

[0054] Containers for holding cell suspensions typically contain cell suspensions of cell types selected from CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells, as well as T cells, NK cells, umbilical cord blood stem cells, progenitor cells, myeloid stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPS cells), as well as equivalents.

[0055] Containers for holding exogenous material suspensions typically contain exogenous material suspensions selected from combinations thereof, including polypeptides, polynucleotides (including DNA and RNA), drugs, polymers, carbohydrates, and ribonucleoproteins.

[0056] Here, we will describe a method 1000 for operating the electroporation system shown in Figures 2a and 2b above.

[0057] In connection step 1001, the audit module 20 is connected to the calibration device 50. The calibration device is then powered by the audit module to perform a calibration routine 1002, for example. During this step, the calibration device supplies voltage and current of known magnitude to the audit module, and the output of the audit module is evaluated by the calibration device. Based on this, the calibration device determines the calibration information necessary to correct any errors in the measurements performed by the audit module 20.

[0058] The calibration information thus determined by the calibration device 50 is then stored in the non-volatile memory 16 of the audit module. After this step, the audit module is disconnected from the calibration device 1004.

[0059] Collectively, steps 1001, 1002, 1003, and 1004 can be considered a calibration step 1050 that provides the necessary calibration information for the non-volatile memory. In some examples, the first such calibration is performed during the manufacturing process.

[0060] Subsequently, in connection step 1005, the audit module is connected to the main module 10. The main module is then given energy 1006 to perform an electroporation sequence. As part of this electroporation sequence, an electroporation signal is generated by the main module and supplied to the electroporation chamber 200. During this electroporation sequence, the voltage and current supplied to the electroporation chamber 200 are measured by the audit module 20.

[0061] After measurement, the voltage and current measurements are corrected based on calibration information stored in the non-volatile memory 16 1008 and transmitted from the audit module 20 to the main module 10 via a communication channel 1009. Calibration is preferably performed in the audit module before the voltage and current information is communicated to the main module 10. Alternatively, the raw voltage and current data may be communicated from the audit module 20 to the main module 10 along with the calibration information, and the main module 10 corrects the voltage and current information based on the calibration data. In a suitable device, a timestamp is attached to each current and voltage measurement. The data is communicated from the audit module to the main module when it is measured and / or when the audit module receives a trigger signal from the main module.

[0062] In step 1010, the main module compares the modified voltage and current information with the expected values. This comparison concerns both the magnitude of the current and voltage, as well as the temporal aspects of the pulse width and distance between pulses. If the result of this comparison falls outside a predetermined tolerance range, the main module generates an error signal. In this case, the result of the electroporation sequence does not meet the requirements of GMP and is therefore rejected. After such rejection, the electroporation device must be subjected to inspection, for example.

[0063] In summary, steps 1006, 1007, 1008, 1009, and 1010 can be considered the electroporation step 1060, during which electroporation occurs, the electroporation signal is audited and compared to the expected value, and an error signal is generated if necessary.

[0064] To ensure appropriateness with respect to this method, the material to be electroporated is introduced into the electroporation chamber 200 prior to step 1006, which energizes the main module 10. Typically, this consists of a predetermined amount of cell suspension, which is a cell type selected from CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells, as well as T cells, NK cells, umbilical cord blood stem cells, progenitor cells, myeloid stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPS). A predetermined amount of the exogenous material suspension is selected from polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and combinations thereof including ribonucleoproteins.

[0065] Figure 4 shows a method 1000 for operating the electroporation system described above and further incorporating the apparatus shown in Figure 2c. Specifically, the use of a second audit module 20a is provided. This method is identical to that provided previously, in that calibration step 1050 is performed in the audit module. Furthermore, the second audit module 20a is also calibrated in a manner similar to that of step 1050a described above with respect to the audit module 20. The second audit module 20a may be calibrated while the audit module 20 is connected to the main module, or at any other time that coincides with the availability of the second audit module 20a when necessary.

[0066] This method is initiated by performing step 1005, which connects the audit module 20 to the main module, followed by electroporation step 1060, which uses the audit module to audit the electroporation signal supplied to the electroporation chamber 200 and rejects the electroporation sequence if the voltage and current are not within the permitted range, as if an error signal has been generated.

[0067] Assuming no such rejection occurs, the voltage and current are determined to be within the permitted tolerance range, and the electroporation step 1060 may be repeated for a predetermined time until a predetermined number of electroporation cycles are reached or the logic in step 1010 returns an error. Based on these determinations, the decision box 1070 decides whether the cycle should be repeated.

[0068] When a predetermined time or number of cycles is reached, the audit module 20 is disconnected from the main module 10 1011 and replaced by a second audit module 20a 1005a. The electroporation step 1060a is then performed using the combination of the main module 10 and the second audit module 20a. The audit module, now freed from its duties, can be calibrated without the need to suspend the operation of the electroporation device, which continues to operate with the combination of the main module and the second audit module 20a. The electroporation cycle continues with this combination until a predetermined time or number of cycles is achieved 1070a, similar to the combination of the main module 10 and the audit module 20.

[0069] Although not shown herein, calibration may, of course, be performed in the audit module 20 while the second audit module 20a is connected to the main module 10. Similarly, further audit modules may be calibrated and replaced as needed. In this way, consistency in the quality of results can be ensured while minimizing the total downtime of the electroporation device.

[0070] Figure 5 shows a typical example of a system having a main module 10 to which an audit module 20 is connected via a multi-pin connector. In this depiction, the audit module is located in a shallow recess in the main module. Naturally, the audit module can be easily removed for calibration or replacement.

[0071] The reader will understand that the aforementioned electroporation devices, systems, and operating methods represent a welcome improvement in the cell industry. The disclosed devices, systems, and methods enable continuous, high-quality calibration and, therefore, electroporation results over long-term production processes without the need to bring out electroporation instruments for calibration. [Explanation of Symbols]

[0072] 5. Signal Generator 10 Main Modules 11 Control Electronic Equipment 12 Electrical connection section 15 Sensor Electronic Devices 16 Non-volatile memory 18a, 18b Output electrical connectors, output connectors 20 Audit Modules 20a Replacement audit module, second audit module 30a Main side section, multi-electrode connector 30b Audit side, multi-electrode connector 50 Calibration Devices 51 circuits 100 Modular Electroporation Devices 200 Electroporation Chambers 1000 (How to operate an electroporation system)

Claims

1. A modular electroporation device (100), The main module (10) is Control electronic equipment including a signal generator (5) configured to generate electrical pulses for electroporation, A multi-electrode connector (30a) connected to the control electronic equipment, which includes at least one conduit for electrical wires and a digital communication channel, electrically connected to the signal generator, Output connectors (18a, 18b) for connecting to the electroporation chamber to transmit the electrical pulses generated by the signal generator. The main module (10), The audit module (20) A multi-electrode connector (30b) is configured to be connected to the multi-electrode connector (30a) of the main module in order to receive the electrical pulse generated by the signal generator and to transmit digital communication between the audit module (20) and the main module (10) via the digital communication channel. Non-volatile memory (16) configured to store calibration data, A sensor electronic device (15) is connected to the multi-electrode connector (30b) and is configured to measure the voltage and current of the electrical pulse received from the main module (10) over time. The audit module (20) includes, Includes, The sensor electronic device (15) is further configured to transmit communications via the digital communication channel based on the measured voltage and current, and based on the calibration data stored in the non-volatile memory (16). The control electronic device is configured to generate an error signal if the current and voltage over time, measured by the sensor electronic device and transmitted through the communication channel, taking the calibration data into consideration, do not match the expected current and voltage over time. The audit module (20) is detachably attached to the main module (10) in a modular electroporation device (100).

2. The modular electroporation device (100) according to claim 1, wherein the error signal is generated if the time between continuous electrical pulses does not match the expected time, or if the time width of the electrical pulse does not match the expected time width.

3. The modular electroporation device (100) according to claim 1 or 2, wherein the audit module (20) is nested within a slot in the main module (10).

4. The signal generator (5) is (a) At least two of the three waveforms have different magnitudes, (b) At least two of the three waveforms have different waveform widths, (c) The first waveform interval of two first sets of the at least three waveforms is different from the second waveform interval of two second sets of the at least three waveforms. A modular electroporation device (100) according to any one of claims 1 to 3, configured to generate an electrical pulse having a series of at least three waveforms, including

5. The modular electroporation device (100) according to any one of claims 1 to 4, wherein the audit module (20) is configured to draw power from the main module (10).

6. The modular electroporation device (100) according to any one of claims 1 to 5, wherein the audit module or the main module includes an analog-to-digital converter and is further configured to store in a non-volatile memory a record of the current and the voltage over time of the electrical pulses measured by the audit module and transmitted to the main module.

7. The main module (10) is configured to generate a signal trigger. The audit module (20) is configured to receive the signal trigger, The modular electroporation device (100) according to any one of claims 1 to 6, wherein the sensor electronic device (15) is configured to transmit the measured voltage and current when the signal trigger is received.

8. An electroporation device (100) according to any one of claims 1 to 7, Electroporation chamber (200) and When the audit module is separated from the main module (10), a calibration device (50) configured to connect to the audit module (20) is provided. Includes, The system for electroporation includes a circuit (51) configured to calibrate the audit module (20) and store calibration data in the non-volatile memory (16), wherein the calibration device (50) includes a circuit (51) configured to calibrate the audit module (20) and store calibration data in the non-volatile memory (16).

9. A container for holding the cell suspension, A container for holding an exogenous material suspension, A container for receiving electroporation products, A tubing device configured to supply a predetermined amount of the cell suspension and a predetermined amount of the exogenous material suspension to the electroporation chamber (200) before electroporation, and to deliver the electroporation product from the electroporation chamber to a container for receiving the electroporation product after electroporation, The system according to claim 8, further comprising:

10. The container for holding the cell suspension contains a cell suspension of cell types selected from CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells, as well as T cells, NK cells, umbilical cord blood stem cells, progenitor cells, myeloid stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPS cells). The system according to claim 9, wherein the container for holding an exogenous material suspension contains an exogenous material suspension selected from combinations thereof, including polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and ribonucleoproteins.

11. The system according to any one of claims 8 to 10, further comprising a replacement audit module (20a), wherein the replacement audit module (20a) has the same function as the audit module (20).

12. A method (1000) for operating an electroporation system according to any one of claims 8 to 10, a) The step of connecting the audit module to the calibration device (1001), b) A step (1002) in which the audit module performs a calibration routine, c) Step (1003) of storing calibration information relating to the audit module in the non-volatile memory (16), d) Step (1004) of disconnecting the connection from the calibration device (50) to the audit module (20), e) The step (1005) of connecting the audit module (20) to the main module (10), f) The step of supplying energy to the main module (10) (1006) and performing an electroporation sequence, g) The step (1007) of measuring the voltage and current of the electroporation with respect to time using the audit module, h) A step (1008) of correcting the measured voltage and current using calibration information stored in the non-volatile memory, i) The step (1009) of passing the voltage and current information from the audit module (20) to the main module (10), j) A step (1010) in the main module of comparing corrected voltage and current information with expected values, wherein if the result of the comparison is outside a predetermined tolerance range, an error signal is generated. A method (1000) including the following.

13. A method (1000) for operating the electroporation system according to claim 11, comprising the steps of: operating the electroporation system according to claim 11 using the audit module; replacing the audit module (20) with the replacement audit module; and operating the electroporation system using the replacement audit module.

14. The method according to claim 12 or 13, wherein steps f) to j) are repeated until one of a predetermined time or a predetermined number of cycles is reached.

15. Prior to the step of supplying energy to the main module (10), the further step includes introducing it into the electroporation chamber (200), A predetermined amount of cell suspension is selected from CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-h7 cells, Huvec cells, Molt 4 cells, as well as T cells, NK cells, umbilical cord blood stem cells, progenitor cells, myeloid stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPS cells), and equivalents. The method according to any one of claims 12 to 14, wherein a predetermined amount of the exogenous material suspension is selected from a combination thereof, including polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and ribonucleoproteins.

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