Electroporation with active compensation

Abstract

An apparatus for electroporating cells with a cargo includes electrodes defining a path for the flow of a fluid containing the cells and cargo, a power source coupled across the electrodes, and a control circuit. In some examples, the control circuit is configured to detect a decrease in induced current due to an increase in resistance between the electrodes and control the power source to increase the induced current and maintain an electric field between the electrodes. Based on a previous value of the resistance, a future value of the resistance between the electrodes may be predicted. In other examples, the control circuit is configured to detect a parameter of the fluid flowing between the electrodes and control the power source to generate an electric pulse or stop generating an electric pulse in response to the detection of the parameter. Other example apparatus and methods for electroporating cells with a cargo are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 120 from U.S. patent application Ser. No. 16 / 935,987, filed July 22, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to electroporation with active compensation. [Background technology]

[0003] This section provides background information related to the present disclosure that is not necessarily prior art.

[0004] Transfection is commonly used to introduce nucleic acids into cells to generate genetically modified cells. Various physical, chemical, and viral methods exist for transfecting cells, including photoporation, calcium phosphate-based polymer-based methods, microinjection, electroporation, viral transduction, and lipid-mediated methods (e.g., using liposome-DNA complexes). When electroporation is used to transfect cells, a controlled direct current (DC) electric pulse is applied to the cells to induce a membrane potential difference. This membrane potential difference causes reversible disruption of the ordered structure of the cell membrane, forming pores in the membrane. Molecules of interest can then enter the cell through the pores. Pore formation can be controlled by adjusting various parameters, including the distance between the parallel electrodes used in the electroporation process, the characteristics of the electric pulse, and the electric field strength across the electrodes. Summary of the Invention [Means for solving the problem]

[0005] This section provides an overview of the disclosure and is not an exhaustive disclosure of the entire scope of the disclosure or all of its features.

[0006] According to one aspect of the present disclosure, an apparatus for electroporating cells with a cargo is provided. The apparatus includes two electrodes spaced apart from one another and defining a pathway for a fluid containing cells and cargo to flow between them, a DC power supply coupled across the electrodes, and a control circuit. The electrodes have a resistance between them as the fluid flows through the pathway. The control circuit is configured to control the DC power supply to provide a plurality of electrical pulses at a voltage to the electrodes to generate an electric field between the electrodes at a defined value, induce a current through the electrodes, detect a decrease in the induced current due to an increase in resistance between the electrodes, and control the DC power supply to increase the induced current to maintain the electric field between the electrodes at the defined value.

[0007] According to another aspect of the present disclosure, an apparatus for electroporating cells with a cargo is provided, the apparatus including two electrodes spaced apart from one another and defining a pathway for a fluid containing the cells and the cargo to flow therebetween, a DC power supply coupled across the electrodes and configured to provide a plurality of electrical pulses to induce a current through the electrodes to generate an electric field between the electrodes, and control circuitry configured to: detect a first parameter associated with the fluid, control the DC power supply to generate the plurality of electrical pulses in response to detecting the first parameter associated with the fluid, and detect a second parameter associated with the fluid, control the DC power supply to stop generating the plurality of electrical pulses in response to detecting the second parameter associated with the fluid.

[0008] According to another aspect of the present disclosure, an apparatus for electroporating cells with a cargo is provided. The apparatus includes two electrodes spaced apart from one another and defining a pathway for a fluid containing cells and a cargo to flow therebetween, a DC power supply coupled across the electrodes, and a control circuit. The electrodes have a resistance therebetween as the fluid flows through the pathway. The control circuit is configured to control the DC power supply to provide a plurality of electrical pulses at a voltage to the electrodes, induce a current through the electrodes to generate an electric field between the electrodes at a defined value, detect a decrease in the induced current due to an increase in the resistance between the electrodes, identify a plurality of values ​​of resistance between the electrodes over a period of time, predict future values ​​of the resistance between the electrodes based on the identified plurality of values ​​of resistance, and increase the induced current based on the future value of the resistance to control the DC power supply to maintain the electric field between the electrodes at the defined value.

[0009] According to another aspect of the present disclosure, a method for electroporating cells with a cargo is provided. The method includes flowing a fluid containing cells and a cargo through a path defined by two electrodes spaced apart from one another, the electrodes having a resistance therebetween as the fluid flows through the path. The method further includes controlling a DC power supply to provide a plurality of electrical pulses at a voltage to the electrodes to generate an electric field between the electrodes at a defined value, inducing a current through the electrodes, detecting a decrease in the induced current due to an increase in the resistance between the electrodes, and controlling the DC power supply to increase the induced current to maintain the electric field between the electrodes at the defined value.

[0010] According to another aspect of the present disclosure, a method for electroporating cells with a cargo is provided, the method including flowing a fluid containing cells and the cargo through a path defined by two spaced apart electrodes, detecting a first parameter associated with the fluid, and in response to detecting the first parameter associated with the fluid, controlling a DC power supply to generate a plurality of electrical pulses to induce a current through the electrodes to generate an electric field between the electrodes, detecting a second parameter associated with the fluid, and in response to detecting the second parameter associated with the fluid, controlling the DC power supply to stop generating the plurality of electrical pulses.

[0011] According to another aspect of the present disclosure, a method for electroporating cells with a cargo is provided. The method includes flowing a fluid containing cells and a cargo through a pathway defined by two electrodes spaced apart from one another, the electrodes having a resistance therebetween as the fluid flows through the pathway. The method further includes controlling a DC power supply to provide a plurality of electrical pulses at a voltage to the electrodes to generate an electric field between the electrodes at a defined value, inducing a current through the electrodes, detecting a decrease in the induced current due to an increase in the resistance between the electrodes, identifying a plurality of values ​​of resistance between the electrodes over a period of time, predicting future values ​​of the resistance between the electrodes based on the identified plurality of values ​​of resistance, and controlling the DC power supply to increase the induced current based on the future values ​​of resistance to maintain the electric field between the electrodes at the defined value.

[0012] Further aspects and areas of applicability will become apparent from the description provided herein. It is understood that various aspects of the present disclosure may be implemented individually or in combination with one or more other aspects. It is also understood that the description and specific examples herein are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0013] The drawings described herein are only for purposes of illustrating selected embodiments rather than all possible implementations and are not intended to limit the scope of the present disclosure. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a block diagram of an electroporation device employing active compensation to maintain electric field strength, according to an example embodiment of the present disclosure. [Figure 2] 10 is a graph showing the voltage provided to the electrodes of an electroporation device when no active compensation is applied, the current induced through the electrodes, and the increase in resistance between the electrodes that reduces the electric field strength, according to another example embodiment. [Figure 3]10 is a graph showing the voltage provided to the electrodes of an electroporation device when no active compensation is applied, the current induced through the electrodes, and the increase in resistance between the electrodes that reduces the electric field strength, according to another example embodiment. [Figure 4] 10 is a graph illustrating the reduction in electric field strength for different electroporation processes without active compensation, according to another example embodiment. [Figure 5] 10 is a graph illustrating induced current through electrodes of an electroporation device employing active compensation according to another example embodiment. [Figure 6] 10 is a graph illustrating a predictive compensation technique employing "full data" linear projection according to another example embodiment. [Figure 7] 10 is a graph illustrating a predictive compensation technique employing "full data" linear projection according to another example embodiment. [Figure 8] 10 is a graph illustrating a prediction compensation technique employing a "moving window" linear projection according to another example embodiment. [Figure 9] 10 is a graph illustrating a prediction compensation technique employing a "moving window" linear projection according to another example embodiment. [Figure 10] 10 is a graph illustrating electric field strength when different predictive compensation techniques are employed, according to another example embodiment. [Figure 11] 10 is a graph illustrating biological performance results when different predictive compensation techniques are applied, according to another example embodiment. [Figure 12] FIG. 10 is a block diagram of an electroporation device including electrodes and a sensor that detects when fluid is present between the electrodes, according to another example embodiment. [Figure 13] FIG. 13 is a block diagram of fluid flow in the electroporation device of FIG. 12. [Figure 14] FIG. 2 is a block diagram of electrodes that can be employed in the electroporation device of FIG. 1 according to another example embodiment. [Figure 15] FIG. 2 is a block diagram of a power supply and control circuit that can be employed in the electroporation device of FIG. 1, according to another example embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0015] Corresponding reference numerals indicate corresponding (but not necessarily identical) parts and / or features throughout the several views of the drawings.

[0016] Example embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope to those skilled in the art. Numerous specific details are set forth, such as examples of specific components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, and that example embodiments may be embodied in many different forms and should not be construed as limiting the scope of the present disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0017] The terminology used herein is for the purpose of describing particular example embodiments and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" may be intended to encompass the plural forms as well, unless the context clearly dictates otherwise. The terms "comprises," "comprising," "including," and "having" are inclusive and thus specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The steps, processes, and operations of methods described herein should not be construed as necessarily requiring performance in the particular order discussed or illustrated, unless specifically identified as such. It should also be understood that additional or alternative steps may be employed.

[0018] Terms such as "first," "second," and "third" may be used herein to describe various elements, components, regions, layers, and / or sections, but these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms may only be used to distinguish one element, component, region, layer, or section from another region, layer, or section. Terms such as "first," "second," and other numerical terms, when used herein, do not imply a sequence or order unless clearly indicated by context. Thus, a first element, component, region, layer, or section described below could be referred to as a second element, component, region, layer, or section without departing from the teachings of example embodiments.

[0019] Spatial relative terms such as "in," "out," "below," "down," "belower," "up," "above," and the like may be used herein for ease of description to describe the relationship of one element or feature shown in the figures to another element or feature. Spatial relative terms may be intended to encompass different orientations of the device in use or operation in addition to the orientation shown in the figures. For example, if a device in the figures were turned upside down, elements described as being "below" or "below" other elements or features would then be oriented "above" the other elements or features. Thus, the example term "below" can encompass both an orientation of above and below. A device may be oriented differently (rotated 90 degrees or at another orientation), and the spatial relative terms used herein would be interpreted accordingly.

[0020] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0021] An apparatus for electroporating cells with a cargo according to an example embodiment of the present disclosure is shown in FIG. 1 and generally designated by reference numeral 100. As shown in FIG. 1, the apparatus 100 includes two electrodes 102, 104, a DC power supply 106 coupled across the electrodes, and a control circuit 108. The electrodes 102, 104 are spaced apart from one another to define a path (generally indicated by arrow F) for a fluid containing cells 112 and cargo 110 to flow therebetween. The electrodes have a resistance R between them. The control circuit 108 controls the DC power supply 106 to provide direct current (DC) electrical pulses at a voltage V to the electrodes 102, 104 to induce a current I through the electrodes 102, 104 and generate an electric field E between the electrodes 102 and 104, detect a decrease in the induced current I due to an increase in the resistance R between the electrodes, and control the DC power supply 106 to increase the induced current I to maintain the electric field E between the electrodes 102 and 104 at a defined value.

[0022] For example, the electrodes 102, 104 and associated components may form a tip. The electrodes 102, 104 and the fluid flowing between the electrodes 102, 104 may have a resistance R (e.g., the resistance of the tip in the presence of fluid). In such an example, the resistance R is equal to at least the resistance R of the fluid and the interface resistance Ri between the fluid and the surfaces of the electrodes 102, 104. As shown in FIG. 1, the electric field E represents the effective electric field generated across the fluid (e.g., between the electrodes 102 and 104) when the DC power supply 106 provides an electrical pulse at a voltage V (e.g., a regulated voltage). In the particular example of FIG. 1, the electric field E is equal to the voltage V across the electrodes 102, 104 (e.g., the voltage V minus any voltage drop) divided by the distance between the electrodes 102 and 104.

[0023] By increasing the induced current I through the electrodes 102, 104, transfection efficiency during electroporation can be maintained, and in some instances, improved. For example, during electroporation, resistance R may increase over time as a function of the number of electrical pulses provided by the DC power supply 106. In some instances, the increase in resistance R may be caused by an increase in interfacial resistance Ri. In such instances, the increase in resistance may be due to, for example, charged molecules, DNA, proteins, etc., attached to the surfaces of the electrodes 102, 104, causing electrode passivation, electrode degradation, etc. As a result of the increased resistance R, the induced current I may decrease, and the effective electric field E across the fluid flowing between the electrodes 102 and 104 may decrease. As a result, transfection efficiency may decrease over time, as further described below. However, if the induced current Ichip flowing through the chip can be increased, and in some instances maintained at a defined value through active compensation (e.g., as described further below), the effective electric field across the fluid during electroporation and transfection efficiency can be maintained, and in some instances improved.

[0024] Figures 2 and 3 show graphs 200, 300 illustrating electrical characteristics associated with the electrodes 102, 104 of Figure 1 during electroporation when active compensation is not employed. In the particular example of Figures 2 and 3, the electrical characteristics are associated with transfection of CHO-S cells with an expression plasmid containing DNA encoding an α-CTLA-4 / TxM-B fusion protein (e.g., cargo) as described in Example 1 herein.

[0025] In particular, FIG. 2 shows the resistance Rchip (line 202) associated with the tip and the electric field E (line 204) across the fluid of FIG. 1. FIG. 3 shows the induced current Ichip (line 302) flowing through the tip and the voltage Vchip (line 304) across the electrodes 102, 104. Testing has shown that the resistance Rchip (line 202) increases as the number of electrical pulses provided by the DC power supply 106 increases. This increase in resistance begins when fluid enters the path (e.g., chamber) between the electrodes 102 and 104. In the specific example of FIG. 2, the resistance Rchip begins to increase around the seventh pulse (e.g., when it begins to decrease), as shown by line 206. As the resistance increases over time, the induced current Ichip (line 302) flowing through the tip decreases and the voltage Vchip (line 304) increases, as shown in FIGS. 2 and 3. During this time, the electric field E (line 204) begins to decrease at line 206 and continues to decrease until the electroporation process ends at line 208.

[0026] 4 shows a graph 400 including lines 402, 404, 406, and 408 representing the electric fields of four experimental electroporation processes P1, P2, P3, and P4 for transfecting CHO-S cells with an expression plasmid containing DNA encoding an α-CTLA4 / TxM-B fusion protein (e.g., cargo) over different amounts of pulse, as described in Example 2 herein. In particular, line 402 (with a solid line configuration) represents the electric field during electroporation process P1, line 404 (with a dashed line configuration) represents the electric field during electroporation process P2, line 406 (with a dash-dotted line configuration) represents the electric field during electroporation process P3, and line 408 (with a dash-dotted line configuration) represents the electric field during electroporation process P4. See Example 2 herein.

[0027] 4 and Table 1 below, the effective electric field experiences a greater drop as the volume of fluid passing between electrodes 102 and 104 increases. The volume of fluid is related to the number of electrical pulses received during each electroporation process P1, P2, P3, P4. For example, electroporation process P1 processes 120 microliters (μL) of fluid (e.g., approximately 60 pulse duration) when process P1 terminates at line 410; electroporation process P2 processes 240 μL of fluid (e.g., approximately 83 pulse duration) when process P2 terminates at line 412; electroporation process P3 processes 400 μL of fluid (e.g., approximately 125 pulse duration) when process P3 terminates at line 414; and electroporation process P4 processes 600 μL of fluid (e.g., approximately 155 pulse duration) when process P4 terminates at line 416.

[0028] [Table 1]

[0029] As shown in Figure 4, the effective electric fields (lines 402, 404, 406, 408) associated with electroporation processes P1, P2, P3, and P4 begin to decrease at approximately pulse 20-25. At this time, the value of each effective electric field is approximately 1.31 kV / cm, as shown in Table 1 above. As shown in Figure 4 and Table 1 above, when electroporation processes P1, P2, P3, and P4 are completed, the electric field associated with process P1 (i.e., line 402) is 0.9 kV / cm, the electric field associated with process P2 (i.e., line 404) is 0.71 kV / cm, the electric field associated with process P3 (i.e., line 406) is 0.54 kV / cm, and the electric field associated with process P4 (i.e., line 408) is 0.46 kV / cm. Thus, as shown in Table 1 above, the electric field experienced during electroporation process P1 has a percent change of 31%, the electric field experienced during electroporation process P2 has a percent change of 46%, the electric field experienced during electroporation process P3 has a percent change of 58%, and the electric field experienced during electroporation process P4 has a percent change of 65%. Thus, the more fluid that passes between electrodes 102 and 104, the greater the drop in the effective electric field experienced.

[0030] Furthermore, the yield associated with electroporation processes P1, P2, P3, and P4 may decrease as the volume of fluid passing between electrodes 102 and 104 increases (e.g., the number of electrical pulses increases). For example, as shown in Table 1 above, the titer of electroporation process P1 is 34.5 μg / mL at day 4, the titer of electroporation process P2 is 24.7 μg / mL at day 4, the titer of electroporation process P3 is 27.2 μg / mL at day 4, and the titer of electroporation process P4 is 22 μg / mL at day 4. Thus, as shown in FIG. 4 , the concentration of CHO-S cells transfected with α-CTLA4 / TxM-B protein decreases when the electric field associated with electroporation processes P1, P2, P3, and P4 decreases. Thus, transfection efficiency may decrease as the electric field E decreases.

[0031] However, transfection efficiency can be maintained at a desired level throughout the electroporation process by ensuring that the effective electric field across the fluid is sufficient. For example, referring to FIG. 1 , the control circuit 108 can monitor the induced current Ichip through the electrodes 102, 104 using the current sensor 114. In such an example, the control circuit 108 can monitor the current Ichip to detect a peak current value. If a decrease in the current Ichip is detected, indicating a decrease in the effective electric field E (e.g., if the current Ichip reaches a peak current value and then decreases), the control circuit 108 controls the DC power supply 106 to increase and maintain the current Ichip at a defined value, such as the peak current value, a desired value less than the peak current value, etc. Thus, in this particular example, the control circuit 108 controls the DC power supply 106 in response to detecting a decrease in the current provided by the DC power supply 106 to maintain the current Ichip at a defined value (e.g., initiate a compensation process).

[0032] In another example, the control circuit 108 may monitor the voltage Vchip across the electrodes 102, 104 using the voltage sensor 116 and, in response to the voltage Vchip being equal to or greater than a defined voltage threshold, control the DC power supply 106 to maintain the current Ichip at a defined value. For example, as described above, the voltage Vchip may begin to increase when a decrease in the effective electric field E occurs. In such an example, the control circuit 108 may detect when the voltage Vchip begins to increase by comparing the voltage to a defined voltage threshold. In response to the voltage Vchip reaching or exceeding the defined voltage threshold, the control circuit 108 may control the DC power supply 106 to maintain the current Ichip at a defined value, as described herein.

[0033] The induced current Ichip may be maintained by actively compensating for current losses. For example, the current Ichip may be maintained by adjusting the voltage Vps and / or the induced current Ichip provided by the DC power supply 106. The voltage Vps and / or the current Ichip may be adjusted one or more times during the electroporation process (e.g., randomly, periodically based on sensed parameters, etc.). In some examples, the voltage Vps and / or the current Ichip may be adjusted to a fixed constant value. In other examples, the voltage Vps and / or the current Ichip may be adjusted according to a desired positive / negative slope, a mathematical formula (e.g., a polynomial function), etc. For example, the DC power supply 106 may be controlled to provide a different (e.g., higher) current to compensate for current drop. In other examples, the DC power supply 106 may be controlled to provide a different voltage to compensate for current drop. In such examples, the voltage provided by the DC power supply 106 may be varied. For example, the control circuit 108 may control the DC power supply 106 to increase the voltage to a peak voltage value, to regulate the voltage to a different set point, and so on.

[0034] For example, given a chip voltage V supplied to the chip, the induced current I flowing through the chip in FIG. 1 is a function of the total chip resistance R. In such an example, Equation (1) below represents this relationship. As previously explained, the resistance R may increase as a function of the number of electrical pulses provided by the DC power supply 106, as shown in Equation (2) below. As a result of the increased resistance R, the current I decreases over time. Equation (1) Vchip=Ichip·Rchip Equation (2) Rchip = R (number of pulses)

[0035] The DC power supply 106 may be controlled based on defined parameters to compensate for a decreasing current I. For example, if the control circuit 108 detects a decrease in the current I, the control circuit 108 may control the DC power supply 106 to maintain the current I at a defined value, such as a peak current I, as shown in equation (3) below. In such an example, if it is desired that the DC power supply 106 provide a peak value of current I, equation (3) may be modified to equation (4) below. If the current I is maintained at a peak value (e.g., a constant), the voltage must increase as the resistance increases. In some examples, the control circuit 108 may control the DC power supply 106 to increase the voltage of the electrical pulse to a peak voltage value V or another suitable value. Equation (3) Vpeak=Ipeak·R0 Equation (4) Vpeak / R0=Ipeak=V(number of pulses) / R(number of pulses)

[0036] Therefore, to maintain the current Ichip at a peak constant value, the regulated voltage Vps provided by the DC power supply 106 may be increased to a set value Vset based on the resistance which varies as a function of the number of electrical pulses and a defined current level (e.g., peak current value), as shown in equation (5) below: Equation (5) Vset = Ipeak·R (number of pulses)

[0037] FIG. 5 shows a graph 500 illustrating the induced current Ichip of FIG. 1 over time. For example, graph 500 is divided into five stages: (1) the start of the electroporation process, (2) fluid flowing between electrodes 102 and 104, (3) decline, (4) current compensation, and (5) completion of the electroporation process. As shown in FIG. 5, the current Ichip increases to a peak value when fluid flows between electrodes 102 and 104. If the control circuit 108 detects a decline in the current Ichip (at the beginning of the decline stage), the voltage Vps provided by the DC power supply 106 increases to a set value Vset. As a result, as shown in FIG. 5, the current Ichip increases to and remains at its peak value (or another suitable defined value) during the compensation stage until the electroporation process is over. As previously explained, if the voltage Vps does not increase, the current Ichip decreases.

[0038] The induced current I may need to exceed a threshold value before compensation can begin. For example, as shown in FIG. 5, the induced current I may need to exceed a compensation threshold value before the control circuit 108 controls the DC power supply 106 to increase the current I. This may prevent the control circuit 108 from prematurely initiating the compensation phase when the induced current I peaks due to noise as the fluid enters the chamber.

[0039] In some examples, the current Ichip may increase and / or maintain to a defined value (e.g., a peak current value) based on a reactive compensation technique. In such examples, the control circuit 108 of FIG. 1 may identify an increase in resistance Rchip and then control the DC power supply 106 to increase and / or maintain the induced current Ichip based on the identified increase in resistance between the electrodes 102 and 104. This process of identifying resistance changes and controlling the DC power supply 106 based on the resistance changes may be repeated as desired. For example, if it is desired to maintain the current Ichip at a peak current value, the peak current value may be determined based on the system voltage Vsys and the system resistance Rsys (e.g., the resistance Rchip and any parasitic resistances) as shown in equation (6) below. In such examples, if the resistance Rsys increases due to degradation (as described above), the voltage Vsys will increase and the current Ichip will remain at the peak current value. The resistance Rsys may be determined based on voltage and current measurements as shown in equation (7) below. Equation (6) Vsys=Ipeak·Rsys Equation (7) Rsys=Vmeas / Imeas

[0040] As previously described, the DC power supply 106 is controlled to maintain the current I at a peak current value. For example, the voltage V may be changed to maintain the current I. In such an example, a new system voltage V may be determined based on the changing resistance as shown in equation (8) below, using equations (6) and (7) above. The voltage V may change multiple times (e.g., V, V, etc.) as the resistance R continues to change over time. Equation (8) Vsys,new=Ipeak·(Vmeas / Imeas)

[0041] In some examples, before the control circuit 108 begins the current compensation stage, the current Ichip may have a decreasing trend, as shown in Figure 5. In such examples, the predicted voltage and current may be used for the first compensation value (instead of the measured voltage and current), as shown in equation (9) below: Equation (9) Vsys,new=Ipeak·(Vprojected / Iprojected)

[0042] Additionally, a feedback gain factor may be introduced to take into account the kinematics of the system. For example, the voltage difference ΔV between the new voltage value Vsys,new and the previous voltage value Vsys,previous may be determined using the following equation (10): In such an example, the DC power supply 106 may be controlled to provide a set voltage Vsys,set based on the voltage difference ΔV and the feedback gain factor G, as shown in the following equation (11): The feedback gain factor G may be a fixed value or may be a variable value, such as a value machine-learned from previous compensation results (based on online calculations, offline databases, etc.). Equation (10) ΔV=Vsys,new-Vsys,previous Equation (11) Vsys,set=G·ΔV

[0043] In other examples, the induced current Ichip may increase to and / or maintain at a defined value (e.g., a peak current value) based on a predictive compensation technique. In such examples, the control circuit 108 of FIG. 1 may predict a future value of the resistance Rchip between the electrodes 102 and 104 and then control the DC power supply 106 to increase to and / or maintain at a defined value (e.g., a peak current value) based on the predicted value of resistance. For example, the control circuit 108 may determine the resistance Rchip (e.g., varying resistance values) between the electrodes 102 and 104 multiple times and apply a linear projection between the determined resistances to predict the future value of the resistance. As described further below, the linear projection may be applied based on, for example, a "full data" linear projection or a "moving window" linear projection.

[0044] For example, FIG. 6 shows a graph 600 including measurements of the chip resistance R (line 602) and a linear projection (line 604) applied based on the "full data" linear projection. In this example, the chip resistance R is assumed to increase linearly as a function of the number of electrical pulses provided by the DC power supply 106 throughout the entire drop period. In this particular example, the control circuit 108 determines a peak current value after detecting a current drop over two consecutive pulses. At the peak current, the value of the chip resistance R is identified (at point 606 on line 602). For example, the value of the chip resistance R at the peak current may be determined based on current and voltage measurements, as previously described. This peak current value corresponds to the minimum value of the chip resistance R before it begins to increase during the drop. At the peak current (and minimum resistance), the pulse point value P is set equal to 0. After two consecutive pulses of current drop, the value of the chip resistance R is again identified (at point 608 on line 602). At this current, the pulse point value P is set equal to n.

[0045] Next, a linear projection (dashed line 604) may be applied (e.g., a linear fit) from the pulse point value P=0 (peak current) at point 606 to the pulse point value P=n (most recent pulse point) at point 608. This linear projection may be used to predict a future value of resistance (R_n+1) at the next pulse (e.g., pulse point value P=n+1) due to electrode degradation. This is shown as point 610 on the linear projection line 604. After the resistance (R_n+1) is predicted, the power supply 106 may be controlled to provide a new voltage Vsys,new based on the predicted resistance value and a defined value of the current Ichip (e.g., peak current value), as shown in equation (12) below: Equation (12) Vsys,new=Ipeak·R_n+1

[0046] The "full data" linear projection process may be repeated as desired (e.g., until the end of the electroporation process) to predict the resistance (R_n+2, R_n+3) at the next pulse and determine the voltage required to maintain the current Ichip at a defined value. For example, FIG. 7 illustrates a graph 700 showing multiple linear projections (e.g., dashed lines 702a-n) applied to predict resistance values ​​at future pulses. By using the "full data" linear projections illustrated in FIGS. 6 and 7, the applied linear projections, and thus the predicted resistance values, may be less susceptible to irregularities in voltage changes due to noise encountered in determining resistance values. However, using the "full data" linear projection method may not provide an accurate prediction if the chip resistance Rchip changes nonlinearly (e.g., nonlinear degradation).

[0047] 8 shows a graph 800 including measured chip resistance Rchip (line 802) and a linear projection (line 804) applied based on a "moving window" linear projection. In this example, the chip resistance Rchip is assumed to increase linearly (e.g., piecewise linearly) over local intervals. In the moving window projection method, the control circuit 108 of FIG. 1 determines a peak current value after detecting a current drop in two consecutive pulses. At the peak current, the value of the chip resistor Rchip is identified (e.g., measured, etc.). This resistance value on the line 802 is identified as point 806. At the peak current, a pulse point value P is set equal to 0. The value of the chip resistor Rchip is identified one or more times after the initial resistance value (e.g., after P=0). In the particular example of FIG. 8, the control circuit 108 identifies the value of the chip resistor Rchip at point 808 on the line 802 (corresponding to pulse point value P=n−1) and the value of the chip resistor Rchip at point 810 on the line 802 (corresponding to pulse point value P=n). In other examples, the control circuit 108 may identify more or fewer values ​​of the chip resistor.

[0048] Next, a linear projection (line 804) may be applied (e.g., a linear fit) from pulse point value P=nm to pulse point value P=n (the most recent pulse point), where m is the data length of the segment (e.g., a defined number of electrical pulses over the local interval). In the example of FIG. 8 , m is equal to 3 (e.g., points 806, 808, 810), and point 810 is the most recent pulse point. Thus, in this particular example, a linear projection is linearly fit between point 806 (e.g., P=nm) and point 810 (e.g., P=n), as shown in FIG. 8 . This linear projection may then be used to predict a future value of resistance (R_n+1) at the next pulse (e.g., pulse point value P=n+1). This is shown as point 812 on linear projection line 804. After the resistance (R_n+1) is predicted, the power supply 106 may be controlled to provide a new voltage Vsys,new based on the predicted resistance value and a defined value of the current Ichip (e.g., a peak current value).

[0049] The "moving window" linear projection process may be repeated as desired (e.g., until the end of the electroporation process) to predict the resistance during the next pulse and determine the voltage required to maintain the current I at a defined value. For example, FIG. 9 shows a graph 900 illustrating multiple linear projections (e.g., dashed lines 902a-n) applied to predict resistance values ​​during future pulses. The data length (m) of each linear projection segment may be the same or different. By using the "moving window" linear projections shown in FIGS. 8 and 9, the control circuit 108 can respond with an aggressive compensation approach if the chip resistance R changes nonlinearly (e.g., nonlinear degradation). However, the use of the "moving window" linear projection method may introduce undesirable noise during the prediction process.

[0050] 1 may employ various components and / or methods when employing the predictive compensation techniques described above. For example, the "full data" and "moving window" linear projection techniques are based on linear regression for feedback control. In such examples, a simple gain factor G may be employed in conjunction with the feedback control.

[0051] In another example, the control circuit 108 may include a controller specific to the feedback control. For example, the control circuit 108 may include a proportional-integral-derivative (PID) controller, a proportional-integral (PI) controller, a proportional-derivative (PD) controller, and / or a proportional (P) controller. Furthermore, gain coefficients for the PID controller, the PI controller, the PD controller, and / or the P controller may be introduced to take into account the kinematics of the system. The feedback gain coefficients may be fixed values ​​or may be variable values.

[0052] In some examples, the feedback gain coefficients may be machine learned from previous compensation results (based on online calculations, offline databases, etc.), etc. For example, predictive compensation techniques may use machine learning methods based on known data points obtained in experiments, historical databases, etc. for the same cell line or same application (e.g., protein production) being transfected.

[0053] Tests have shown that the use of a "moving window" linear projection can produce better compensation results than other compensation techniques during the dip period. For example, FIG. 10 shows a graph 1000 including lines 1002, 1004, 1006, and 1008 representing normalized electric field values ​​over aligned time periods. In particular, line 1002 (with a dotted configuration) represents the electric field when a "moving window" linear projection is applied, line 1004 (with a dash-dot configuration) represents the electric field when a "full data" linear projection is applied, line 1006 (with a dashed configuration) represents the electric field when a response compensation technique is applied, and line 1008 (with a solid configuration) represents the electric field when no compensation technique is applied. After the 10-second mark (e.g., when the normalized electric field value peaks), a dip begins, and after the dip is detected, compensation using the "moving window" linear projection, "full data" linear projection, and response techniques begins. As shown in Figure 10, the electric field corresponding to the "moving window" linear projection technique (line 1002) is maintained at a normalized value of about 1 during the decay period, the electric field corresponding to the "full data" linear projection technique (line 1004) is maintained at a normalized value of about 0.95 during the decay period, and the electric field corresponding to the response compensation technique (line 1006) is maintained at a normalized value of about 0.85 during the decay period. Thus, in the example of Figure 10, the "moving window" linear projection technique provides better transfection efficiency than the "full data" linear projection and response compensation techniques.

[0054] Furthermore, electroporation processes using the "moving window" linear projection technique may be subject to greater yields than conventional electroporation processes. See Example 3 herein. For example, FIG. 11 shows a graph 1100 including lines 1102, 1104, 1106, 1108, and 1110 representing biological performance results when CHO-S cells are transfected with an α-CTLA4 / TxM-B fusion protein using different electroporation processes. In the example of FIG. 11, an expression plasmid containing DNA encoding the α-CTLA4 / TxM-B fusion protein was transfected into CHO-S cells using either an electroporation process applying the "moving window" linear projection technique as disclosed herein or a conventional electroporation process without compensation. In particular, lines 1102, 1104, and 1106 are associated with an electroporation process employing a "moving window" linear projection technique during compensation, line 1108 is associated with a Maxcyte® electroporation process without compensation, and line 1100 is a conventional electroporation process without compensation.

[0055] As shown, titration line 1102 (with dotted configuration) reaches 335.3 μg / mL when a 1.45 kV / cm electric field is applied to a 10 mL biphasic sample, titration line 1104 (with dashed configuration) reaches 306 μg / mL when a 1.45 kV / cm electric field is applied to a 10 mL sample, titration line 1106 (with dash-dot configuration) reaches 276.9 μg / mL when a 1.3 kV / cm electric field is applied to a 10 mL sample, titration line 1108 (with dash-dot configuration) reaches 214.2 μg / mL for a 100 mL sample, and titration line 1110 (with solid configuration) reaches 160.9 μg / mL for a 10 mL sample. Therefore, transfection efficiency can be much higher when compensation based on the "moving window" linear projection technique is applied (e.g., lines 1102, 1104, 1106) compared to when no compensation is applied (e.g., lines 1108, 1110).

[0056] In some examples, a control circuit (e.g., control circuit 108 of FIG. 1) may monitor fluid flowing between the electrodes and, in response to detecting one or more parameters associated with the fluid, control an associated DC power supply to generate an electrical pulse and / or cease generating an electrical pulse. For example, FIG. 12 shows an apparatus 1200 for transfecting cells with a cargo, including electrodes 1202, 1204, 1210, and 1212 and a sensor 1206 coupled to electrodes 1210 and 1212. Electrodes 1202 and 1204 are generally similar to electrodes 102 and 104 of FIG. 1. Thus, electrodes 1202 and 1204 may be spaced apart from one another and may define a pathway 1208 for fluid containing cells and cargo to flow therebetween. Pathway 1208 may have a serpentine configuration or any other suitable configuration.

[0057] 12, electrodes 1210, 1212 are positioned at the inlet and outlet of a pathway 1208 (e.g., a fluid channel) opposite electrodes 1202, 1204. For example, as shown in FIG. 12, electrode 1210 (shown in dashed lines) is positioned at the inlet and outlet below electrodes 1202, 1204, and electrode 1212 (shown in solid lines) is positioned at the inlet and outlet above electrodes 1202, 1204. Electrodes 1210, 1212 may be employed to detect the flow of fluid into and out of fluid pathway 1208 between electrodes 1202 and 1204.

[0058] 12, the sensor 1206 may be in communication with a control circuit (not shown) or part thereof that controls a DC power supply (not shown) to compensate for the electric field drop, as described herein. The sensor 1206 may be a current sensor or an impedance sensor.

[0059] 12 , fluid passing through pathway 1208 is detected by sensing the current flowing through and / or the resistance across electrodes 1210, 1212 when a voltage is applied to electrodes 1210, 1212. For example, fluid flowing through pathway 1208 between electrodes 1202 and 1204 may change the current through electrodes 1210, 1212 and / or may change the resistance across electrodes 1210, 1212. In such an example, sensor 1206 may detect this change in current and / or resistance and send a signal to control circuitry indicating that the electroporation process has begun (e.g., fluid is flowing between electrodes 1210 and 1212), is completing, etc.

[0060] For example, FIG. 13 shows five stages of fluid flow and a graph of the sensed parameter (e.g., current or resistance) associated with electrodes 1210, 1212 over the five stages. In the first stage, fluid has not reached electrodes 1210, 1212, and the sensed parameter remains at its initial state (e.g., a steady-state value without fluid). In the second stage, fluid passes through electrodes 1210, 1212 at the inlet of path 1208, causing the sensed parameter to increase. In the third stage, fluid passes through electrodes 1210, 1212 at the inlet and outlet of path 1208, causing the sensed parameter to increase again. In the fourth stage, fluid passes through electrodes 1210, 1212 at the outlet of path 1208, causing the sensed parameter to decrease. In the fifth stage, fluid has passed through electrodes 1210, 1212, causing the sensed parameter to decrease again (e.g., return to a steady-state value). Thus, the control circuitry may control the DC power supply to generate electrical pulses based on an increasing parameter (e.g., when fluid is present in stages 2-4) and stop generating electrical pulses based on a steady-state parameter (e.g., at stage 5). Additionally, if it is known when the electric field begins to drop relative to when fluid is present, the control circuitry may begin controlling the DC power supply to compensate for the electric field drop when the chamber is filled with fluid (e.g., stage 3). This allows the control circuitry to begin the compensation process earlier compared to other techniques based on detecting a drop in current provided by the DC power supply, for example.

[0061] Figure 14 illustrates another device 1400 that can monitor fluid flowing between electrodes and control an associated DC power supply to generate electrical pulses and / or cease generating electrical pulses in response to detecting one or more parameters associated with the fluid. For example, as shown in Figure 14, device 1400 includes fluid paths 1420, 1422, 1424, a check valve 1426 in fluid path 1422, sensors 1406a-d in fluid communication with paths 1420, 1422, 1424, and electrodes 1202, 1204 of Figure 12 that define path 1208. As shown, fluid path 1208 (between electrodes 1202 and 1204) is coupled between fluid paths 1420 and 1424, and fluid path 1422 (e.g., a lateral path) extends from fluid path 1420. In the example of FIG. 13, the sensors 1406a-d may be in communication with, or part of, a control circuit that controls a DC power supply (not shown) to compensate for the electric field drop, as described herein.

[0062] When the electroporation process is initiated, fluid begins to flow through pathways 1208, 1420, 1422, and 1424, as indicated by the dashed arrows. In particular, fluid flows through pathway 1420 (e.g., the inflow pathway), enters pathway 1208, and exits pathway 1424 (e.g., the outflow pathway). In such an example, one or both sensors 1406a-b may sense fluid entering pathway 1208 (e.g., the tip) and send a signal to the control circuit. This allows the control circuit to detect the presence of fluid, indicating that the electroporation process has begun. Accordingly, the control circuit may control the DC power supply to generate an electrical pulse based on the presence of fluid. The control circuit may then begin controlling the DC power supply to compensate for the drop in the electric field in response to any one of sensors 1406a-c detecting the presence of fluid. This may initiate the compensation process at an earlier point in time compared to other techniques based on detecting a drop in the current provided by the DC power supply, for example.

[0063] Additionally, the control circuitry may stop the electroporation process based on a parameter associated with the fluid. For example, sensor 1406c may detect a drop in fluid and / or an absence of fluid, indicating that the fluid has passed through pathway 1208 (e.g., the tip) and the electroporation process has ended. In such an example, sensor 1406c may send a signal to the control circuitry, which may control the DC power supply to stop generating electrical pulses (e.g., stopping the electroporation process) and / or control the fluid pump to turn off.

[0064] In some examples, the check valve 1426 can function as an occlusion detector to detect a fluid blockage in one of the paths 1208, 1420, 1424. For example, if a fluid blockage is present, pressure can build up inside the check valve 1426. If the pressure exceeds a defined activation threshold of the check valve 1426 (e.g., 20-40 psi), the check valve 1426 can open, allowing fluid to flow through the path 1422 and past a sensor 1406d positioned outside the valve 1426. In such examples, the sensor 1406d detects fluid (indicating a blockage in one of the paths 1208, 1420, 1424) and sends a signal to the control circuit to generate an electrical pulse and / or stop the fluid pump. In other examples, the check valve 1426 can send a signal to the control circuit based on the state of the valve 1426 (e.g., open or closed) to indicate whether a blockage is present. In such an example, sensor 1406d may not be employed.

[0065] The sensors 1406a-d may be any suitable sensor. For example, any one of the sensors 1406a-d may be an ultrasonic sensor, an optical sensor (e.g., an infrared sensor), or the like. In such examples, the fluid may be detected by the fluid, waves reflected by movement through the fluid, and / or light. The optical sensor may provide and / or detect visible or non-visible light. In some examples, one or more light sources, such as LEDs, lasers, infrared light, etc., may be employed in conjunction with and / or be part of the sensors 1406a-d. Furthermore, in the particular example of FIG. 13, the four sensors 1406a-d are positioned external to the electrodes 1202, 1204. In other examples, any one of the sensors 1406a-d may be positioned along the pathway 1208 (e.g., between the electrodes 1202 and 1204), if desired. Additionally, in some instances, only one sensor (eg, sensor 1406a, sensor 1406b, etc.) may be utilized, more than four sensors may be utilized, and so forth.

[0066] The electrodes disclosed herein can be any suitable type of conductive electrode. For example, any one of electrodes 102, 104, 1202, 1204, 1210, 1212 can include a mesh plate, a solid plate, or the like, having porosity that allows fluid containing cells and cargo to pass through.

[0067] Additionally, the power supplies and control circuits disclosed herein may include a variety of suitable components. For example, FIG. 15 shows an apparatus 1500 including a DC power supply 106 and the control circuit 108 of FIG. 1. In the example of FIG. 15, the DC power supply 106 includes a power converter 1502 and a pulse circuit 1504. The power converter 1502 may include a DC-DC power conversion circuit and / or an AC-DC power conversion circuit having any suitable converter topology. For example, the power converter 1502 may receive DC or AC power and output a regulated DC voltage to the pulse circuit 1504. In some examples, the power supply 106 may include an AC-DC rectifier and a DC-DC buck, boost, etc., conversion circuit coupled to the AC-DC rectifier.

[0068] Pulse circuit 1504 of Figure 15 receives the regulated DC voltage from power converter 1502 and converts the DC voltage into DC electrical pulses. The electrical pulses are then provided to the electrodes (e.g., electrodes 102, 104 of Figure 1, electrodes 1202, 1204 of Figure 12, etc.) as described herein. For example, pulse circuit 1504 may include one or more switching devices that interpret the DC voltage from power converter 1502 and create the electrical pulses.

[0069] In the example of FIG. 15 , the control circuit 108 includes a controller 1506 and a pulse generator 1508 coupled to the controller 1506. As shown, the controller 1506 receives a feedback signal representing an output parameter (e.g., output voltage) of the power converter 1502 and provides one or more control signals to the power converter 1502 for controlling one or more switching devices within the power converter 1502 based on the feedback signal. In some examples, the controller 1506 controls the switching devices of the power converter based on the voltage Vchip sensed by the voltage sensor 116 of FIG. 1 and / or the induced current Ichip sensed by the current sensor 114 of FIG. 1 to adjust the output voltage of the power converter at different set points. Accordingly, the controller 1506 may control the amplitude of the electrical pulses. In such examples, the controller 1506 may control the output of the power converter 1502 to compensate for the electric field drop, as described herein.

[0070] The controller 1506 provides signals to the pulse generator 1508, which provides one or more control signals to the pulse circuit 1504 for controlling the switch devices to generate electrical pulses based on the signals received from the controller 1506. In some examples, the pulse generator 1508 may adjust the frequency, pulse width, duty cycle, etc. of the electrical pulses based on the signals received from the controller 1506. [Example]

[0071] Example 1 (Figs. 2 and 3) Cell preparation: Prior to the experiment, Gibco™ CHO-S™ cells (ThermoFisher Cat. A11557-01) were grown in CD-CHO medium (ThermoFisher Cat. 10743029) + 8 mM L-glutamine. CHO-S cells were cultured and harvested at a concentration of less than 2e6 cells / mL. On day 0 of the experiment, 5% homemade sodium-based electroporation buffer was present at 37°C. On day 1 of the experiment, 1 mM sodium butyrate was added to bring the 5% homemade sodium-based electroporation buffer to 32°C. On days 4 and 8 of the experiment, homemade sodium-based electroporation buffer was fed. During the electroporation process, viability and VCD (viable cell density) follow similar trend lines.

[0072] Cells were harvested, washed with homemade sodium-based electroporation buffer (sodium concentration 8 mM / L, conductivity = 10-14 mS / cm, osmolality = 265-300 mOSM / L, pH 7.2-7.4), and resuspended in homemade sodium-based buffer at a cell density of 100e6 cells / mL as shown in Table 2 below for homemade sodium-based electroporation buffer.

[0073] [Table 2]

[0074] α-CTLA-4 / TxM-B Fusion Protein (N-844-2) DNA Preparation: N-844-2 is an anti-CTLA-4 / TxM molecule in which an anti-CTLA-4scFv is attached to an IL-15RαSu moiety. The preparation and disclosure of the anti-CTLA-4scFv / IL-15RαSu molecule are disclosed in International Publication No. WO 2018 / 075989, which is incorporated herein by reference in its entirety. The DNA sequence of N-844-2 was grafted into a mammalian expression plasmid. Then, a DNA plasmid containing the N-844-2 sequence was prepared in milligram quantities using an Endo Free DNA Giga Kit (Qiagen Catalog No. 12391). The expression plasmid containing DNA encoding the α-CTLA4 / TxM-B fusion protein was diluted in water and mixed with the cell suspension immediately prior to the electroporation process.

[0075] Electroporation: The cell and DNA mixed suspension was placed in a 50 mL falcon tube and pumped into the electroporation chip using a peristaltic pump and tubing. The pulse waveform was 40 V, the pulse width was 450 μs, the pulse interval was 2.7 seconds, and the flow rate was 2 mL / min. The voltage V(p) and current I(p) were recorded throughout the experiment as a function of each pulse (p), and the resistance R(p) was calculated as R(p) = V(p) / I(op). R0 = min(R(p)) is when the fluid completely fills the electroporation chamber, which is set as the initial electrode-fluid interface resistance Ri in Figure 1. The effective electric field E = V(p) * R0 / (R(p) * d), where d was the distance between the electrode pairs. Figures 2 and 3: V, I, R, and E were then plotted as a function of pulse length p.

[0076] Example 2 (Figure 4) Cell preparation and α-CTLA-4 / TxM-B fusion protein (N-844-2) DNA preparation were the same as in Example 1.

[0077] Electroporation: The cell and DNA mixture suspension was placed in a 1.5 mL tube and pumped into the electroporation chip using a peristaltic pump and tubing. Four different total volumes of cell and DNA mixture were tested in this experiment: 120 μL, 240 μL, 400 μL, and 600 μL. The pulse waveform was 38 V, the pulse width was 450 μs, the pulse interval was 0.27 s, and the flow rate was 1 mL / min. The voltage V(p) and current I(p) were recorded throughout the experiment as a function of each pulse (p), and the resistance R(p) was calculated as R(p) = V(p) / I(op). R0 = min(R(p)) is when the fluid completely fills the electroporation chamber, which is the initial electrode-fluid interface resistance R in Figure 1. i The effective electric field is set as E=V(p) * R0 / (R(p) * The effective electric field E was then plotted as a function of pulse length p (Figure 4).

[0078] Example 3 (Figure 11) Cell preparation and α-CTLA-4 / TxM-B fusion protein (N-844-2) DNA preparation were the same as in Example 1.

[0079] Electroporation: The cell and DNA mixture suspension was placed in a 50 mL tube and pumped into the electroporation chip using a peristaltic pump and tubing. Five different electroporation conditions were compared using the same cell preparation as described above. The five different conditions are shown in Table 3 below.

[0080] [Table 3]

[0081] Figure 11 shows that using the compensation method improves protein production titer results. Titers were measured using a Protein A biosensor with Octet Red 96e with a standard curve prepared using N-601 (a monoclonal IgG1 antibody).

[0082] The above description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or limiting of the present disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, are interchangeable where applicable, and can be used in selected embodiments even if not specifically shown or described. Individual elements or features of a particular embodiment can also be modified in many ways. Such modifications should not be considered a departure from the present disclosure, and all such modifications are intended to be encompassed within the scope of the present disclosure.

Claims

1. 1. A device for electroporating cells with cargo, comprising: two electrodes spaced apart from one another and defining a pathway for fluid containing the cells and the cargo to flow therebetween, the electrodes having a resistance between them when fluid flows through the pathway; a DC power source coupled across the electrodes; a control circuit configured to control the DC power supply to provide a plurality of electrical pulses at a voltage to the electrodes to generate an electric field between the electrodes at a first defined value; induce a current through the electrodes; detect a decrease in the induced current due to an increase in the resistance between the electrodes; identify at least a first value and a second value of the resistance between the electrodes over a period of time; apply a linear projection between the first value of resistance and the second value of resistance; predict a future value of the resistance between the electrodes based on the linear projection; and increase the induced current to a second defined value based on the future value of the resistance to control the DC power supply to maintain the electric field between the electrodes at the first defined value; An apparatus comprising:

2. The apparatus of claim 1 , wherein the control circuitry is configured to apply the linear projection based on a data length of a defined number of electrical pulses.

3. 2. The apparatus of claim 1, wherein the control circuit is configured to identify the first value of the resistance when the induced current is at a second defined value.

4. 4. The apparatus of claim 3, wherein the second defined value of the induced current is a peak value of the induced current.

5. The apparatus of claim 1 , wherein the second defined value of the induced current is a peak value of the induced current.

6. 10. The apparatus of claim 1, wherein the control circuitry is configured to control the DC power source to increase the voltage of the plurality of electrical pulses to increase the induced current.

7. 1. A method of electroporating a cell with a cargo, comprising: flowing a fluid containing the cells and the cargo through a pathway defined by two electrodes spaced apart from one another, the electrodes having a resistance therebetween as the fluid flows through the pathway; controlling a DC power source to provide a plurality of electrical pulses at a voltage to the electrodes, thereby inducing a current through the electrodes that generates an electric field between the electrodes at a defined value; detecting a decrease in the induced current due to an increase in the resistance between the electrodes; determining at least a first value and a second value of the resistance between the electrodes over a period of time; applying a linear projection between the first value of the resistance and the second value of the resistance; predicting a future value of the resistance between the electrodes based on the linear projection; controlling the DC power supply to increase the induced current based on the future value of the resistance to maintain the electric field between the electrodes at the defined value; A method comprising:

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