Modular electroporation method and system

Abstract

An apparatus for delivering an electrical signal to an electroporation chamber and monitoring the quality of the delivered signal is provided. Specifically, the equipment is of a modular structure and comprises a main module and an auditing module. The main module has control electronics with a signal generator configured to generate electrical pulses for electroporation and electrically connected to the auditing module. The auditing module is configured to store calibration data and measure current and voltage delivered from the master module over time and to deliver such measured data to the master module. The master module is configured to generate an error signal if the voltage and current measured by the auditing module do not conform to the expected voltage and current values. The auditing module is detachably attached to the main module.

Description

Technical Field

[0001] This invention relates to an electroporation machine. Specifically, this invention relates to a modular electroporation system and a method of operating the system. Background Technology

[0002] Electroporation is understood as the reversible disruption of the stability of biological membranes (such as vesicle membranes or cell membranes) using electrical pulses, providing a pathway to introduce exogenous material into vesicles or cells during this period of instability. Typically, this introduced exogenous material can be genetic material to obtain transgenic cells for research or therapeutic purposes. Both cells and exogenous material are provided in the form of liquid suspensions. In recent years, significant attention has been paid to the use of electroporation to introduce mRNA into cells, which is readily translated into proteins, thereby conferring additional functions to these cells. Cell therapy is an emerging field in medicine that requires sterile and reliable manufacturing facilities where primary cells from donors or patients can be genetically modified in large quantities according to strict Good Manufacturing Practices (GMP). For example, the production of clinical batches of allogeneic CAR T cells requires multiple gene-editing steps to eliminate external receptors and express chimeric antigen receptors (CARs) [Depil, S., et al. (2020) 'Off-the-shelf' allogeneicCAR T cells: development and challenges]. Nat Rev Drug Discov [19: 185–199], which is particularly challenging in this respect.

[0003] U.S. Patent Application US 2006 / 0089674 discloses a system comprising a pulse voltage waveform generator, a switching device for connecting the anode or cathode of the pulse voltage waveform generator to an electroporation chamber, and an electrode array within the electroporation chamber for converting the pulse voltage into a pulsed electric field within the chamber. The document also discloses a cycle of filling the electroporation chamber with a suspension of cells and exogenous substances, electroporating the mixture, and emptying the electroporation chamber when the volume of the cell suspension to be treated exceeds the capacity of the electroporation chamber.

[0004] During electroporation, to comply with GMP specifications and ensure consistent quality of electroporation results, it is necessary to monitor and evaluate the electrical pulses applied to the electroporation chamber to ensure product quality control, etc. This evaluation must consider voltage and current, as well as pulse timing. Timing should be understood as the duration of the pulses and the time intervals between consecutive pulses.

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

[0006] However, it is well known that the output of electronic circuits drifts over time. This applies not only to circuits and signal generators that generate electroporation pulses, but also to circuits that monitor the voltages and currents that constitute these pulses. Traditionally, external calibration equipment has been connected to the electroporation system to specifically assess the self-monitoring capabilities of electronic circuits. However, this practice is not advisable for several reasons. Specifically, electroporation equipment must operate under cleanroom conditions, so introducing external equipment near the electroporation operation site, or removing the equipment for maintenance and calibration, can lead to contamination problems. Furthermore, given this, calibration is impossible without moving / removing the equipment. Moving and / or removing the equipment results in production losses and associated waste of time and money.

[0007] Therefore, the purpose of this invention is to provide an electroporation system that is inexpensive to construct and operate, ensures good electroporation results, and does not have the aforementioned disadvantages.

[0008] In addition to producing therapeutic-grade cells, their applications can be found in various research fields or industrial processes, such as transforming cell lines, bacteria, yeast, algae, plant cells, protoplasts for bioproduction, or producing vaccines, these are just non-limiting examples.

[0009] definition Modularization describes a module-based construction, which may or may not be integrated. For this invention, the modules include a main module and an audit module.

[0010] The system of this invention is designed for processing vesicles, which can be artificial or living cells. The system is specifically designed for electroporation of cells, such as cell lines or primary cells.

[0011] Cells refer to living cells that have been cultured or placed in a suspension.

[0012] "Primary cells" or "first-generation cells" refer to cells derived from living tissue (such as biopsy material) and established in vitro for a limited period of time, meaning they can undergo a limited number of population multiplications. Primary cells are contrasted with continuous tumorigenic or artificially immortalized 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 commonly used in cell therapy because they are considered more potent and less tumorigenic.

[0013] According to the present invention, cells differentiated from stem cells, such as umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSs), are considered primary cells.

[0014] Primary cells are typically derived directly or indirectly (preferably directly) from donors or patients using a variety of methods known in the art, such as leukocyte separation techniques reviewed by 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].

[0015] This invention focuses in particular on immune cells, such as T cells and NK cells, and stem cells, such as hematopoietic stem cells and progenitor cells, embryonic stem (ES) cells, or induced pluripotent stem (iPS) cells, to produce clinical batches of therapeutic cells for cell therapy.

[0016] Electroporation refers to the process of creating transient permeability in the cell membrane using a pulsed electric field without losing cell viability.

[0017] Exogenous substances are any substances outside of living cells. For the purposes of this disclosure, they are substances to be delivered into cells. A non-exclusive list of substances includes polypeptides, polynucleotides, drugs, polymers, carbohydrates, and combinations of these substances in the same or different molecules. Examples of polypeptides and polynucleotides are proteins and DNA or RNA (including mRNA, RNAi), respectively. Exogenous substances used for electroporation may include combinations of the above substances, such as ribonucleoproteins (protein and RNA complexes).

[0018] PulseAgile protocol: A sequence of at least three waveforms having one, two, or three of the following characteristics: (1) at least two of the at least three waveforms are different from each other in waveform amplitude; (2) at least two of the at least three waveforms are different from each other in waveform width; and (3) a first waveform interval of two waveforms in a first group of the at least three waveforms is different from a second waveform interval of two waveforms in a second group of the at least three waveforms. An example of such a flexible pulse sequence is described in U.S. Patent US 6010613.

[0019] Suspension: Insoluble particles (such as living cells) suspended in an aqueous liquid. Summary of the Invention

[0020] This invention relates to electroporation equipment, systems, and methods.

[0021] Specifically, this disclosure relates to a modular electroporation apparatus comprising a main module and an audit module. The terms "main" and "audit" are not intended to limit this disclosure, but merely to broadly describe the function of each module. The main module includes control electronics with a signal generator configured to generate electrical pulses for electroporation; a multi-electrode connector connected to the control electronics; and an output connector for connection to the electroporation chamber. The audit module includes a multi-electrode connector configured to mate with the multi-electrode connector of the main module. Furthermore, the audit module is equipped with non-volatile memory configured to store calibration data, and sensor electronics connected to the multi-electrode connector, the sensor electronics being configured to measure the voltage and current of the electrical pulses received from the main module relative to time. The sensor electronics are also configured to send digital communications to the main module based on the measured voltage and current and the calibration data stored in the non-volatile memory.

[0022] The control electronics of the main module are configured to generate an error signal if the current and voltage measured by the sensor electronics, transmitted via the communication channel, and referenced to calibration data do not match the expected current and voltage.

[0023] The approval module can be detachably attached to the main module. Alternatively, the approval module can be nested in a slot within the main module.

[0024] Optionally, the signal generator is configured to generate an electrical pulse sequence having at least three waveforms, the sequence comprising: (a) at least two of the at least three waveforms being different from each other in waveform amplitude; (b) at least two of the at least three waveforms being different from each other in waveform width; and (c) a first waveform interval of a first group of two waveforms of the at least three waveforms being different from a second waveform interval of a second group of two waveforms of the at least three waveforms.

[0025] Optionally, the audit module is configured to draw power from the main module. Furthermore, the sensor electronics may include an analog-to-digital converter. Moreover, the sensor electronics may be configured to send the measured voltage and current to the main module upon receiving a trigger signal from the main module.

[0026] Another aspect of this disclosure is a system for electroporation, the system including the aforementioned electroporation apparatus, 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 circuitry configured to calibrate the audit device and store calibration data in a non-volatile memory.

[0027] The disclosed system may further include: a container for containing a cell suspension; a container for containing a suspension of exogenous substances; a container for receiving electroporation products; and a piping arrangement configured to supply predetermined amounts of cell suspension and predetermined amounts of exogenous substance suspension to the electroporation chamber (200) before electroporation, and to transport the electroporation products from the electroporation chamber to the container for receiving the electroporation products after electroporation. The container for containing the cell suspension typically comprises a cell suspension, wherein the cell types are 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, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) and equivalents. The container for containing a suspension of exogenous substances typically comprises a suspension of exogenous substances selected from: peptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and various combinations of these substances, including ribonucleoproteins.

[0028] The disclosed system may also include a replacement audit module, which has the same functions as the audit module.

[0029] In another aspect, this disclosure also relates to a method of operating the electroporation system disclosed above. The method includes: a) connecting an audit module to a calibration device; b) performing a calibration procedure on the audit module; c) storing calibration information associated with the audit module in a non-volatile memory; d) disconnecting the audit module from the calibration device; e) connecting the audit module to a main module; f) starting the main module to execute an electroporation sequence; g) measuring the electroporation voltage and current relative to time by the audit module; h) correcting the measured voltage and current using the calibration information stored in the non-volatile memory; i) transferring the voltage and current information from the audit module to the main module; and j) comparing the corrected voltage and current information with desired conditions at the main module. If the comparison result exceeds a predetermined tolerance, an error signal is generated.

[0030] Further steps of the above method are also disclosed, wherein the second audit module is calibrated according to steps a) to d). The audit module is then connected to the main module, and electroporation steps e) to j) are performed. Steps e) to 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) to j) can then be performed using a combination of the main module and the second audit module.

[0031] The above method may further include, before starting the main module, introducing the following substances into the electroporation chamber: a predetermined amount of cell suspension, wherein the cell type 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; and T cells, NK cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSs) and equivalents; and a predetermined amount of exogenous substance suspension, wherein the exogenous substance is selected from: peptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and various combinations of these substances including ribonucleoproteins.

[0032] Thanks to the modular configuration of the aforementioned electroporation equipment and system, calibration can be maintained without prolonged interruption of the electroporation process or the introduction of external equipment into the electroporation environment. Thanks to the claimed invention, a continuously operating electroporation system can be calibrated while maintaining cleanroom conditions.

[0033] The accompanying drawings are provided by way of non-limiting examples: Figure 1 This is a schematic diagram of a modular electroporation device; Figure 2a , Figure 2b and Figure 2c A schematic diagram of three configurations of the electroporation system is shown; Figure 3 It shows Figure 2a and Figure 2b A flowchart of the operation of the electroporation system; Figure 4 It shows Figure 2a and Figure 2c A flowchart of the operation of the electroporation system; Figure 5 This is an example of a modular electroporation device. Detailed Implementation

[0034] Figure 1 A modular electroporation device 100 connected to an electroporation chamber 200 is shown. In the context of this disclosure, the term “connected to an electroporation chamber” means establishing an electrical connection with electrodes inside the electroporation chamber in order to apply an electric field to the contents of the electroporation chamber.

[0035] The electroporation device 100 includes a main module 10 and a detachable audit module 20. The main module 10 and the audit module 20 are connected via multi-electrode connectors 30a, 30b having a main side portion 30a and an audit side portion 30b. Suitable such multi-electrode connectors 30a, 30b are 31-channel Harting connectors.

[0036] The main module 10 and the audit module 20 can also be further attached to each other mechanically. Furthermore, the audit module 20 can be mechanically attached to the main module 10 via a self-guiding rail and secured to the rear panel with additional screws. In some arrangements, the audit module can be partially surrounded by the main module 10. For example, as... Figure 5 As shown in the example, the audit module can be installed into the slot of the main module.

[0037] The main module 10 and the audit module 20 will now be described sequentially. 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 based on the biomaterial being electroporated. Furthermore, the shape of the voltage curve can also be controlled. The example signal generator is configured to generate signals conforming to the PulseAgile protocol.

[0038] Output electrical connectors 18a and 18b are configured to connect the main module 10 to the electroporation chamber 200. In some arrangements, these connectors 18a and 18b are detachable. The signal generated by the signal generator 5 is transmitted to the electroporation chamber 200 via the output electrical connectors.

[0039] The audit module 20 includes sensor electronics 15 connected to a multi-electrode connector. The sensor electronics also include an analog-to-digital converter and non-volatile memory 16.

[0040] An electrical connection 12 between the main module and the audit module passes through a multi-electrode connector. The electrical connection includes several components, including wiring carrying an electroporated signal generated by the signal generator 15; and a digital communication channel configured for communication between the audit module 20 and the main module 10. The digital communication channel may be, for example, a serial bus, such as RS232. The electrical connection 12 can also be configured to supply power from the main module to the audit module. In some other arrangements, the audit module itself includes a power supply.

[0041] Sensor electronics 15 are configured to measure the voltage and / or current of an electroporation signal generated by a signal generator and transmitted to the audit module via electrical connection 12. The voltage and / or current are recorded relative to time. Preferably, the electroporation signal is routed via the audit module. Sensor electronics 15 of 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 by signal generator 5 in the main module; then the signal is routed via the audit module; returns to the main module, and then to the electroporation chamber 200.

[0042] 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 represent current and voltage measured relative to time, and this information is digitally transmitted to the control electronics of the main module via the digital communication channel.

[0043] As described above, the audit module includes a non-volatile memory 16. Specifically, the non-volatile memory is configured to store calibration information related to the audit module's sensitivity to the measured current and voltage. Therefore, the measured current and voltage values ​​are corrected based on the calibration data. Preferably, this correction is 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, along with the calibration information, is transmitted via the digital communication channel so that the voltage and current information can be corrected at the control electronics 11 of the main module 10.

[0044] In some arrangements, 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, it may begin measuring and transmitting information upon receiving a trigger signal from the main module. Upon receiving this trigger signal, the sensor electronics are configured to measure the current and voltage of the electroporation signal and communicate this information digitally to the main module 10. Alternatively, the audit module may be configured to transmit previously measured current and voltage values ​​relative to time upon receiving the trigger signal.

[0045] As shown in the figure, the audit module 20 obtains its power from the main module 10, although in other arrangements, the audit module has its own power supply.

[0046] The time-based logs of voltage and current measured by the audit module are stored in non-volatile memory. This non-volatile memory may form part of the main module 10 or the audit module 20.

[0047] Reference Figures 2a-2c The following describes how to integrate the modular electroporation device 100 described above into an electroporation system. For example... Figure 2a As shown, the modular electroporation device 100 described above 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 to the control electronics 11 of the main module 10 via a digital communication channel. The measured voltage and current are calibrated using calibration information stored in the non-volatile memory 16 of the audit module 20. At the main module, the voltage and current information is compared with expected values. This comparison may include the time characteristics of the measured electrical signal and the expected electrical signal, including pulse width and the time between consecutive pulses. Based on this comparison, if the measured value is not within a predetermined tolerance of the expected value, the control electronics are configured to generate an error signal. In the case of generating such an error signal, the resulting electroporation product is unusable and must be discarded. In the case of not generating an error signal, the electroporation product can be accepted for its intended purpose.

[0048] Figure 2bThe illustration depicts a scenario where the audit module 20 is not connected to the main module 10, but rather to a calibration device 50. The calibration device 50 includes a circuitry 51 configured to evaluate the audit module's current and voltage measurement capabilities and write calibration information into a non-volatile memory 16. As described above, this calibration information is used to correct the voltage and current measurements performed by the audit module.

[0049] exist Figure 2c Another arrangement is shown, in which the replacement audit module 20a is attached to the main module 10. This can occur simultaneously with the attachment of the audit module 20 to the calibration device 50.

[0050] Typically, the electroporation chamber 200 is fluidly connected to a piping arrangement configured to supply a predetermined amount of cell suspension from a container containing cell suspension, and a predetermined amount of exogenous substance suspension from a container containing exogenous substance suspension. Following electroporation, the piping arrangement is also configured to transport the electroporation product from the electroporation chamber (200) to a container for receiving the electroporation product.

[0051] The container for containing the cell suspension typically contains a cell suspension of the 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, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSs) and equivalents.

[0052] Containers for containing suspensions of exogenous substances typically contain suspensions of exogenous substances selected from: peptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and various combinations of these substances, including ribonucleoproteins.

[0053] The operation will now be described. Figures 2a-2b Method 1000 of the electroporation system shown.

[0054] In connection step 1001, the audit module 20 is connected to the calibration device 50. Thereafter, the calibration device is activated to perform calibration procedure 1002 on the audit module. For example, during this step, the calibration device provides the audit device with voltage and current of known magnitudes, and the calibration device evaluates the output of the audit device. Based on this, the calibration device determines the calibration information needed to correct any errors in the measurements performed by the audit device 20.

[0055] The calibration information thus determined by calibration device 50 is then stored in the non-volatile memory 16 of audit device. After this step, the audit module is disconnected from calibration device 1004.

[0056] In summary, steps 1001, 1002, 1003, and 1004 can be considered as calibration step 1050, which equips the non-volatile memory with the necessary calibration information. In some cases, such calibration is performed for the first time during the manufacturing process.

[0057] Subsequently, in connection step 1005, the audit module is connected to the main module 10. Then, in step 1006, the main module is started to execute the electroporation sequence. As part of this electroporation sequence, electroporation signals are generated by the main module and applied 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.

[0058] After measurement, the voltage and current measurements are corrected 1008 based on calibration information stored in non-volatile memory 16, and transmitted 1009 from audit module 20 to main module 10 via communication channel. Preferably, calibration is performed at the audit module before the voltage and current information is communicated to the main module 10. Alternatively, the raw voltage and current data can be communicated from audit module 20 to main module 10 along with calibration information, and the voltage and current information is corrected at main module 10 based on the calibration data. In a preferred arrangement, a timestamp is appended to each current and voltage measurement. Data is communicated from audit module to main module at the time of measurement and / or after audit module receives a trigger signal from main module.

[0059] In step 1010, at the main module, the corrected voltage and current information are compared with the expected values. The comparison involves the amplitude of the current and voltage, as well as the timing aspects of the pulse width and the distance between pulses. If the result of this comparison exceeds a predetermined tolerance, an error signal is generated by the main module. In this case, the result of the electroporation sequence does not meet GMP requirements and will therefore be rejected. For example, following such a rejection, the electroporation equipment must be sent for repair.

[0060] In summary, steps 1006, 1007, 1008, 1009, and 1010 can be considered as electroporation step 1060, during which electroporation occurs, and the electroporation signal is reviewed and compared with the expected value, and an error signal is generated as appropriate.

[0061] The applicable method involves introducing the material to be electroporated into the electroporation chamber 200 before starting the main module 1006. Typically, this consists of a predetermined amount of cell suspension and a predetermined amount of exogenous substance suspension. The cell types are 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, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs). The exogenous substances are selected from: polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates and various combinations thereof, and various combinations of these substances including ribonucleoproteins.

[0062] Figure 4 The operation of the above-described electroporation system and its further integration are illustrated. Figure 2c The arrangement method shown is (1000). That is, the use of a second audit module 20a is provided. Here, the method is the same as described above, that is, calibration step 1050 is performed on the audit module. In addition, the second audit module 20a is calibrated by a method similar to step 1050a of the audit module 20 described above. The second audit module 20a can be calibrated when the audit module 20 is connected to the main module or at any other time consistent with when the second audit module 20a is ready for use when needed.

[0063] The method audits the electro-poration signal supplied to the electro-poration chamber 200 by performing step 1005 of connecting the audit module 20 to the main module, and then using the audit module to perform electro-poration step 1060, and rejects the electro-poration sequence when the voltage and current are not within the allowable tolerance and thus generate an erroneous signal.

[0064] Assuming no rejection occurs and the voltage and current are determined to be within acceptable tolerances, the electroporation step 1060 may be repeated for a predetermined amount of time, until a predetermined number of electroporation cycles is reached, or until a logic error is returned at step 1010. Decision box 1070 determines whether to repeat the cycle based on these considerations.

[0065] After the predetermined time or number of cycles is reached, audit module 20 is disconnected from main module 10 (1011) and replaced (1005a) with second audit module 20a. Then, the electroporation step (1060a) is performed using the combination of main module 10 and second audit module 20a. The audit module thus released from service can now be calibrated without stopping the operation of the electroporation equipment, which continues to operate due to the combination of main module and second audit module 20a. The electroporation cycle continues with this combination until the predetermined time or number of cycles (1070a) is reached, as with the combination of main module 10 and audit module 20.

[0066] Although not depicted here, it will be understood that when the second audit module 20a is connected to the main module 10, calibration can be performed on audit module 20. Similarly, more audit modules can be calibrated and replaced as needed. In this way, downtime of the electroporation equipment can be minimized while ensuring consistency in result quality.

[0067] Figure 5 A representative example of a system with a main module 10 is shown, in which an audit module 20 is connected via a multi-pin connector. In this representative example, the audit module is located within a shallow recess in the main module. It is understood that the audit module can be easily removed for calibration or replacement.

[0068] Readers will recognize that the electroporation apparatus, system, and operating method described above represent a welcome improvement in the cell industry. The disclosed apparatus, system, and method allow for continuous high-quality calibration during extended production runs, resulting in high-quality electroporation results without requiring the electroporation equipment to be withdrawn for calibration.

Claims

1. A modular electroporation device (100), comprising: Main module (10), the main module (10) includes: Control electronics, including a signal generator (5) configured to generate electrical pulses for electroporation; A multi-electrode connector (30a) connected to the control electronics includes: at least one conductive conduit electrically connected to the signal generator; and a digital communication channel. Output connectors (18a, 18b) are used to connect to the electroporation chamber to transmit the electrical pulses generated by the signal generator; The review module (20) includes: A multi-electrode connector (30b) is configured to mate with the multi-electrode connector (30a) of the main module (10) in order to: receive the electrical pulses generated by the signal generator; and conduct 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 electronics (15) connected to the multi-electrode connector (30b) is configured to measure the voltage and current of the electrical pulses received from the main module (10) relative to time; The sensor electronics (15) is also configured to transmit communication via the digital communication channel based on the measured voltage and current and the calibration data stored in the non-volatile memory (16); If the current and voltage relative to time measured by the sensor electronics, transmitted via the communication channel, and referenced to the calibration data do not match the expected current and voltage relative to time, the control electronics are configured to generate an error signal. The feature is that the audit module (20) is detachably attached to the main module (10).

2. The modular electroporation device (100) according to claim 1, wherein, The error signal is generated if the time between consecutive 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 in the slot of the main module (10).

4. The modular electroporation device (100) according to any one of claims 1 to 3, wherein, The signal generator (5) is configured to generate an electrical pulse sequence having at least three waveforms, the sequence comprising: (a) At least two of the at least three waveforms are different from each other in terms of waveform amplitude. (b) At least two of the at least three waveforms are different from each other in waveform width, and (c) The first waveform interval of the first group of two waveforms of the at least three waveforms is different from the second waveform interval of the second group of two waveforms of the at least three waveforms.

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

6. The modular electroporation device 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, on non-volatile memory, a record of the current and voltage of the electrical pulses measured by the audit module and transmitted to the main module relative to time.

7. The modular electroporation device according to any one of claims 1 to 6, wherein: The main module (10) is configured to generate a signal trigger; The audit module (20) is configured to receive the signal trigger; and The sensor electronics (15) is configured to transmit communication of the measured voltage and current upon receiving the signal trigger.

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

9. The system according to claim 8, further comprising: Containers used to hold cell suspensions; Containers used to contain suspensions of exogenous substances; Container for receiving electroporation products; The piping arrangement is configured to supply a predetermined amount of the cell suspension and a predetermined amount of the exogenous substance suspension to the electroporation chamber (200) before electroporation, and to transport the electroporation product from the electroporation chamber to the container for receiving the electroporation product after electroporation.

10. The system according to claim 9, wherein: The container for containing the cell suspension contains a cell suspension, wherein the cell types are 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, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs). The container for containing the suspension of the exogenous substance contains the suspension of the exogenous substance, which is selected from: polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and various combinations of these substances including ribonucleoproteins.

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

12. A method (1000) of operating the electroporation system according to any one of claims 8 to 10, comprising: a) Connect the audit module (1001) to the calibration device; b) Perform the (1002) calibration procedure on the audit module; c) Store (1003) the calibration information related to the audit module in the non-volatile memory (16); d) Disconnect the audit module (20) from the calibration device (50) (1004); e) Connect the audit module (20) (1005) to the main module (10); f) Start (1006) the main module (10) to execute the electroporation sequence; g) The electroporation voltage and current relative to time are measured (1007) by the audit module; h) Correct the voltage and current measured by (1008) using the calibration information stored in the non-volatile memory; i) Transmit the voltage and current information from the audit module (20) (1009) to the main module (10); j) The corrected voltage and current information are compared with the expected values ​​at the main module (1010), wherein: if the comparison result exceeds the predetermined tolerance, an error signal is generated.

13. A method (1000) of operating the electroporation system of claim 11, comprising: The electroporation system according to claim 11 is operated using the audit module described above; Replace the audit module (20) with the replacement audit module; The electroporation system is operated using the replacement audit module.

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

15. The method according to any one of claims 12 to 14, further comprising introducing the following substance into the electroporation chamber (200) before activating the main module (10): A predetermined amount of cell suspension, wherein the cells are 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, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSs) and equivalents; A predetermined amount of exogenous substance suspension, wherein the exogenous substance is selected from: polypeptides, polynucleotides including DNA and RNA, drugs, polymers, carbohydrates, and various combinations of these substances including ribonucleoproteins.

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