A liquid handling system, a method for calibrating a peristaltic pump in the system, a method for performing a mixing operation in the system, a reusable subsystem of the system, and a replaceable subsystem of the system.

Abstract

Embodiments provide a liquid handling system arranged to prepare and dispense small volumes of liquid formulations into output receptacles. The system can be used in a variety of small volume liquid formulation preparation applications, cell therapy being one example of such an application.

Description

Technical Field

[0001] The technical field of the present invention is to mix and dispense a small-volume liquid formulation with high precision for application in the preparation of liquid formulations for therapeutic agents and cell therapies, for example.

Background Art

[0002] Regenerative medicine and advanced cell therapies are new medical treatment techniques that manipulate living human-derived cells to create constructs or send immunogenic responses in a patient's body or stimulate repair responses. Some of these techniques can provide multiple doses to multiple patients from a single cell source (allogeneic cell products), but there is a growing recognition that it is safe and effective to process and provide cells from patients or matched donors. To produce a cell formulation (autologous formulation) specific to a patient or a matched donor, usually a small batch process is required. The batch process includes mixing of liquids to manipulate the concentration of cells and the composition of the carrier solution. Since these liquid formulations are used for medical treatment, high precision and a sterile environment are required. In current processes, generally manual intervention and checks are required (for example, manually calibrating a scale and weighing a bag containing liquid to ensure accuracy). Also, the accuracy may be limited by the tolerance of the equipment (such as a scale) used for checking.

[0003] When considering cell therapy, formulation, filling, and finishing are the final fluid steps performed on cell-based therapeutic products. Cells have a limited lifespan outside the body, and it is often impractical to immediately apply cell therapy to patients. Therefore, cell therapy products generally need to be stored for at least a short period. These formulations are typically cryopreserved. Cryoprotective media are commonly mixed with the cell product as part of the formulation process. Cells can become unstable and have a short survival time in cryoprotective media before freezing. The time they are exposed to the medium before freezing is limited. Missing this stability window of the cryoprotective can increase cell death. This is particularly undesirable in autologous therapies, where cell counts are generally low and every cell is considered precious.

[0004] As it is a formal pharmaceutical product, quality assurance scrutiny is rigorous throughout the entire process. Samples of the raw materials are used to direct formulation operations, such as dilution and the addition of cryopreservatives. Samples of the formulated raw materials are taken to verify the formulation process. Samples of the final product are taken to confirm the cell dosage and to complete the characterization of the active cell product. Such information is carefully reviewed before it can be administered to patients. Autologous cell formulation concerns very small batch formulations, where the input material is taken from a patient or a donor specially selected for the patient. The number of doses from such batches is generally quite small. Due to the many operations, time pressure, and small number of doses, these operations have been performed manually. To meet Good Manufacturing Practice (GMP) requirements for the medical product, a second person monitors all steps and records them in batch record documents. The batch records are reviewed as part of the product release activities.

[0005] Manual handling of liquids requires open processing where the cell product is exposed to the work environment, and maintaining sterility depends on the skill of the worker. Manual processing is also time-consuming. Because cells have a relatively short period during which they must be used to maintain their viability or safely frozen, manual processing to manufacture therapeutics can account for a significant portion of this time. Therefore, in order to provide a therapeutic treatment with maximum efficacy, this process shortens the remaining time in this window.

[0006] Another problem with manual processing is the human characteristic of making incremental, subtle differences to improve the process. It has been demonstrated that the same action can consistently be completely different depending on the person. This can, for example, affect cell products. For example, Manual mixing of cell products to maintain homogeneity in the mixture-suspension is highly inconsistent. In relation to sample separation and mixing, it is extremely difficult to separate a small amount of product sample that reflects the majority of a homogeneously mixed suspension. [Overview of the project] [Problems that the invention aims to solve]

[0007] While automated systems are emerging, a considerable amount of manual work is still required to achieve the accuracy and consistency necessary for medical applications.

[0008] There is a need for systems and automated methods for producing small quantities of highly concentrated and well-defined compositions for liquid formulation. [Means for solving the problem]

[0009] definition The following explanation uses the terms Qualification and Verification. The formal use of these terms in quality assurance should be understood.

[0010] Qualification is a planned and documented activity that ensures a process, apparatus, or cell product meets the specified requirements for that product. Qualification must be maintained through continuous measurement or verification.

[0011] Verification is the measurement of a process to determine whether it is functioning acceptablely well in order to meet the needs of a qualified process.

[0012] Calibration is the process of adjusting or checking measuring instruments (such as measuring devices and precision pumps) so that they can be used accurately and precisely.

[0013] According to one embodiment, a liquid handling system is provided, and the system is Reusable subsystems, Equipped with replaceable subsystems, Reusable subsystems are, Peristaltic pump and A valve assembly having multiple valves, Two or more bubble sensors, each positioned to detect bubbles in the fluid path, A system controller configured to receive input from a bubble sensor, control the operation of the peristaltic pump, and control the operation of a valve assembly, according to a programmed processing protocol. A case housing the peristaltic pump and valve operating assembly. It has, Single-use, replaceable subsystems are A fluid path manifold comprising one or more fixed-shape fluid paths, wherein at least one of the fluid paths is configured to engage with a valve assembly, thereby allowing the fluid path to be selectively opened and closed by the operation of the valve assembly, and the fixed shape of at least one fluid path is positioned near a bubble sensor when fixed to the housing, so that the bubble sensor can identify bubbles in the fluid path. A pump tube is configured to enable operational engagement between the peristaltic pump and the fluid path in order to generate fluid flow within the manifold by the operation of the peristaltic pump, Multiple liquid input ports are configured to connect to each liquid supply component in order to supply each liquid to one or more fluid pathways, To allow the gas to enter the fluid path, at least one gas inlet is connected to at least one of one or more fluid paths, It has one or more fluid pathways for dispensing fluid and at least one outlet port that communicates with the fluid, The replaceable subsystem provides a closed environment for mixing and dispensing liquid formulations. The controller is characterized by determining the volume of liquid in one of the fluid paths based on the operation of the peristaltic pump and input from at least one bubble sensor associated with each fluid path.

[0014] In one embodiment of the system, a combination of a fixed-shape fluid manifold and a bubble sensor arrangement allows for the measurement of a known volume in at least one region within the fluid path, and the controller utilizes this known volume measurement to calibrate the peristaltic pump.

[0015] In one embodiment, the peristaltic pump is automatically calibrated by a control device.

[0016] In some embodiments, the peristaltic pump is dynamically calibrated during the execution of one or more processing protocols.

[0017] In some embodiments, based on the bubble sensor input, the controller uses a known volume measurement to determine the volume of the dispensed product.

[0018] In some embodiments of the system, the bubbles in the fluid piping are used to separate small volumes of liquid, and the volume of each small volume of liquid is verified in at least two different regions within the flow path using bubble sensor data from each respective region.

[0019] In one embodiment of the system, the verified volume data includes dispensed volume data.

[0020] In an embodiment, the gas inlet can be an air inlet. In an embodiment, the air inlet includes a sterile filter.

[0021] In some embodiments, the system controller is further configured to control the system to determine a dosing formulation and the number of doses to dispense based on the particle count in the liquid sample to be processed, mix the determined formulation, and dispense the determined number of doses.

[0022] In one embodiment, the system controller includes an interpolation engine adapted to mathematically solve for the dose amount and formulation variables based on the target values and ranges of the formulation variables, the particle count, and the variable prioritization rules.

[0023] Another aspect provides a method of calibrating a peristaltic pump within a liquid handling system as described above, the method comprising introducing a volume of liquid into a flow path having a known volume between a first bubble sensor and a second bubble sensor; introducing a gas into the flow path such that a volume of liquid is preceded and followed by bubbles; operating the peristaltic pump to pass a volume of liquid through the flow path in the vicinity of the first bubble sensor so as to identify the transfer from liquid to bubbles and record the position of the peristaltic pump at the time of transfer; The steps include: operating a peristaltic pump to draw a certain volume of liquid through a known volume channel to a second bubble sensor, enabling the second sensor to identify the same transition from liquid to bubble, and recording the position of the peristaltic pump at the time of the transition; The steps include: calculating the volume of liquid that replaces each index of the peristaltic pump based on the recorded position and known flow path volume; It is equipped with.

[0024] It should be understood that to perform this calibration method, it is not necessary to know the actual volume of the liquid drawn through the fluid path. Since the calibration is based solely on identifying one end of a certain volume of liquid, knowledge of this volume is unnecessary.

[0025] Some embodiments provide a method for determining the volume between two bubble sensors. For example, determining the volume of a measuring loop between two bubble sensors. This method can also be used for calibrating a peristaltic pump. This method can be used to initially determine the volume of a measuring loop between two bubble sensors. This method can also be used to verify the volume of a measuring loop between two bubble sensors.

[0026] The step for determining the volume of the tube between the two bubble sensors is: a) A step of controlling the introduction of an initial volume of liquid into the tube, b) The step of operating a peristaltic pump to advance an initial volume of liquid so that the end of the liquid is identified near the first bubble sensor, c) A step of controlling the introduction of further liquid into the tube until liquid is detected by a second bubble sensor, d) A step of operating a peristaltic pump to transfer a total volume of liquid, consisting of an initial volume of liquid and an additional volume of liquid, to an external container. It holds.

[0027] Optionally, a further step may be taken: e) remove the external container holding the total volume of the liquid. The total volume can be measured to determine the volume of the tube between the two sensors from the outside. For example, the weight of the external container into which the liquid has been transferred can be measured before and after the transfer, and these weights and the known density (mass / ml) of the liquid can be used. 2 The volume of the liquid can be measured based on the difference between the two bubble sensors. The liquid volume corresponding to the tube volume can be stored in an external container and used for quality control purposes or external verification. Knowing the tube volume between the two bubble sensors allows for the calibration of other aspects of the system, including peristaltic pumps and tubing within the manifold.

[0028] Controlling the volume of liquid introduced into a tube can include measuring the volume of liquid introduced as it enters the tube.

[0029] Another embodiment provides a method for performing a mixing operation in the above-described liquid handling system, the method being: a) The step of operating a peristaltic pump to generate a fluid flow through the fluid path, b) The steps of operating one or more valves to control the selection and direction of fluid flow through a fluid path, c) A step of monitoring the fluid flow through the fluid path using a bubble sensor and determining the fluid volume based on fluid detection by at least one bubble sensor and the operation of a peristaltic pump, d) In response to the determination that a target volume of fluid has passed through the bubble sensor, the step of activating at least one valve to introduce bubbles into the fluid channel behind the target volume of fluid and to guide the fluid flow in the fluid channel, the step of guiding the flow of the target volume of fluid to a mixing reservoir, e) Repeat steps a) through d) for one or more additional fluids and additional target volumes, so that the fluids are mixed in the mixing reservoir, and It is equipped with.

[0030] The mixing method is, f) The procedure may further include the step of acting one or more valves to recirculate the mixed fluid so that it returns to the mixed reservoir from the mixing reservoir through a fluid path.

[0031] The mixing method is, g) The step may further include operating one or more valves to allow the mixed fluid to flow through one or more fluid paths to an outlet, and dispensing a target volume of the mixed fluid based on the operation of the pump and the flow of the mixed fluid detected by a bubble sensor:

[0032] The target volume to be dispensed can be the sample volume of the mixed fluid. For example, it may be a sample used to test the mixed composition before dispensing the production volume of the mixed fluid.

[0033] In another embodiment, a reusable subsystem of a liquid handling system is provided, the reusable subsystem is, Peristaltic pump and A valve assembly including multiple valves, Two or more bubble sensors, each positioned to detect bubbles in the fluid path, A system controller configured to receive input from a bubble sensor, control the operation of the peristaltic pump, and control the operation of a valve assembly, according to a programmed processing protocol. A case housing the peristaltic pump and valve operating assembly It has, The pump, valve assembly, and bubble sensor are arranged to engage with a fluid path manifold containing one or more fixed-shape fluid paths, thereby allowing the fluid paths to be selectively opened and closed by operation of the valve assembly, so that at least one fluid is located near each bubble sensor when fixed within the housing, and the pump engages with a pump tube configured to allow a movable engagement between the peristaltic pump and the fluid path, so that the operation of the peristaltic pump causes fluid flow in the manifold. The controller determines the volume of liquid in one of the fluid paths based on the operation of the peristaltic pump and input from at least one bubble sensor associated with each fluid path.

[0034] In another embodiment, a replaceable subsystem of a liquid handling system is provided, which is configured to engage with a reusable subsystem comprising a peristaltic pump, a valve assembly including a plurality of valves, two or more bubble sensors each positioned to detect bubbles in a fluid path, a system controller configured to receive input from the bubble sensors and control the operation of the peristaltic pump and the valve assembly according to a programmed processing protocol, and a case housing the peristaltic pump and the valve operating assembly. The replaceable subsystems are: A fluid path manifold comprising one or more fixed-shape fluid paths, wherein at least one of the fluid paths is configured to engage with a valve assembly, thereby allowing the fluid path to be selectively opened and closed by operation of the valve assembly, and the fixed shape of at least one fluid path is positioned near a bubble sensor when fixed within the housing, so that the bubble sensor can identify bubbles in the fluid path. A pump tube is configured to enable an operable engagement between the peristaltic pump and the fluid path so as to generate a fluid flow within the manifold by the operation of the peristaltic pump, Multiple liquid input ports are configured to connect to each liquid supply component in order to supply each liquid to one or more fluid pathways, To allow the gas to enter the fluid path, at least one gas inlet is connected to at least one of one or more fluid paths, One or more fluid pathways for dispensing fluid and at least one outlet port communicating with the fluid It has, The interchangeable subsystem provides a closed environment for mixing and dispensing liquid formulations. The controller is characterized by determining the volume of liquid in one of the fluid paths based on the operation of the peristaltic pump and input from at least one bubble sensor associated with each fluid path.

[0035] Next, embodiments incorporating all aspects of the present invention will be described illustratively with reference to the accompanying drawings. [Brief explanation of the drawing]

[0036] [Figure 1] Figure 1 is a typical block diagram of one embodiment of the system. [Figure 2] Figure 2 shows a basic example of a fluid transfer strategy that forms the basis of system operation. [Figure 3A] Figure 3A is a typical block diagram of a control system 300 for one embodiment of the system. [Figure 3B] Figure 3B is a typical block diagram of a control system 301 for one embodiment of the system. [Figure 4A] Figure 4A shows a basic representation of the equipment used in the pump calibration process. [Figure 4B] Figure 4B shows the steps of the pump calibration process. [Figure 5] Figure 5 is a flowchart of an example of a pump calibration process. [Figure 6] Figure 6 shows an example of one embodiment of the system. [Figure 7] Figure 7 is a schematic diagram of the fluid path for the single-use kit, associated valves, and bubble sensor, and also shows the peristaltic pump in the example in Figure 6. [Figure 8] Figure 8 shows the steps involved in calibrating the "known volume" loop using an external standard. [Figure 9] Figure 9 shows an example of additional equipment that may be used to calibrate a known volume loop against an external reference. [Figure 10]Figure 10 shows a table of tube calibration measurement results. [Modes for carrying out the invention]

[0037] The embodiment provides a liquid handling system configured for preparing small amounts of liquid formulations and dispensing them into output containers. This system can be used for a variety of small-volume liquid formulation preparation applications, one example being cell therapy.

[0038] Figure 1 is a typical block diagram of one embodiment of a liquid handling system, which comprises a reusable subsystem 100 and a replaceable subsystem 110. The reusable subsystem 100 includes a controller 10, a valve assembly 20, a peristaltic pump 30, and at least two bubble sensors 40. The valve assembly 20 has a plurality of valves, each configured to engage with a fluid path in a fluid path manifold 50 of the replaceable subsystem 110. The bubble sensors 40 are positioned along at least one fluid path to detect the presence or absence of liquid in the fluid path. These components are housed in a housing configured to operably accommodate the replaceable subsystem.

[0039] The interchangeable subsystem 110 provides a closed environment for mixing and dispensing liquid formulations. The interchangeable subsystem 110 includes a fluid path manifold 50 consisting of one or more fixed-shape fluid paths, and a flexible pump tube 60 operably engaged with a peristaltic pump, thereby causing the operation of the peristaltic pump to induce fluid flow in the fluid path. Multiple liquid input ports 45 are provided, each configured to connect to a respective liquid input source to supply its respective liquid to one or more fluid paths. For example, the liquid input source may be a liquid supply component such as a bag of input liquid with a tube connecting to the input port, or the liquid input source may be an external system. At least one outlet port 75, which is in fluid communication with one or more fluid paths, is also provided for dispensing fluid to one or more output containers or other systems. In one embodiment, a set of output containers can be incorporated into the interchangeable subsystem.

[0040] The interchangeable subsystem also includes a gas inlet 80 connected to at least one fluid path, allowing gas to enter the fluid path. In one embodiment, the gas inlet 80 includes a sterile filter, allowing filtered air to enter the fluid path. In other embodiments, the gas inlet may be configured to connect to a gas canister. The gas inlet allows gas to enter the fluid path, creating bubbles in the fluid path.

[0041] In one embodiment of the system, the replaceable subsystem is a single-use kit having a manifold of tubing and peristaltic pump tubing accessible through a valve supported by a carrier frame that controls the kit shape when installed in the device (reusable subsystem). The valve and bubble sensors are positioned within the housing of the reusable subsystem so as to align with the tubing in the manifold that forms the fluid path. This allows for maintaining a constant volume of fluid contained in the tubing between the bubble sensors. In one configuration of the fluid path, a variation of 0.05 ml (1.6%) between replaceable subsystems has been demonstrated, compared to 3.2 ml.

[0042] The combination of peristaltic pump operation and valve operation can control the suction of liquid from the input source and the movement of fluid through the fluid path.

[0043] Fluid transfer is controlled by a peristaltic pump and the use of air to follow the fluid blocks through the piping. This strategy relies on the fluid piping having a sufficiently small diameter so that the surface tension of the fluid holds the fluid blocks together. This allows mixing and dispensing to be performed as a functionally closed process. The controller is programmed to control the operation to perform specific mixing and dispensing steps, as described below.

[0044] The controller may be implemented, for example, using a software-programmable microprocessor and memory, and may be programmed to perform mixing and dispensing processes as described below. Other embodiments may use programmable hardware components such as a programmable logic controller or field-programmable gate array that can be programmed to perform specific mixing and dispensing processes, and such embodiments may include support software that enables programming of the programmable hardware. Alternatively, embodiments may be implemented using dedicated circuitry or application-specific integrated circuits (ASICs) designed to control the execution of specific processes. It should be understood that embodiments in which a reusable subsystem includes a microprocessor and memory and allows customization of the mixing and dispensing processes using software are advantageous for research purposes as well as for individualized therapies where customization for individual patients may be required.

[0045] Embodiments of this system can be used to perform one or more of the following functions: Mix controlled volumes of liquids together. Separate the sample from the mixed cell suspension. Prepare the "dosage" of cells and carrier solution. Dispense a controlled amount into the output container.

[0046] The system's embodiments may include the following: A means for self-verifying the calibration of pump calibrations used to control all fluid transfers. Monitoring of fluid transfer in a way that provides verification of the transferred volume, independent of control functions. A means for determining and providing externally verifiable data regarding the volume of a tube between two sensors.

[0047] These features allow for the adjustment of fluids that contribute to the formulation and administration of the final drug.

[0048] As described in the background technology section, automated systems have been developed that automate at least several steps in the preparation of liquid formulations, completing operations within bags or tubes and creating a functionally closed process environment. This eliminates the risks associated with open processing. Automated systems can also create opportunities to address other process risks associated with manual processing, such as inconsistencies and errors due to operator variability.

[0049] In current automated processes, the accuracy of volume is more the responsibility of the equipment than the operator. Although the above example relates to cell therapy, these issues can also be applied to other types of therapeutic and non-therapeutic liquid formulations, especially the preparation of small-volume formulations.

[0050] Another advantage of automated processing is increased speed, including the speed of product freezing, which reduces the time required for cells to remain viable before freezing. Similarly, the time cells are mixed with cryoprotective agents before freezing is reduced, thereby decreasing cell death.

[0051] A further advantage of automation is precision. Embodiments of the system of this disclosure enable the precise handling of very small amounts of liquid formulations. In particular, the system incorporates several self-calibration and / or self-verification operations to assist in the precise handling of liquid formulations in order to automatically prepare therapeutic doses. These are described in more detail below.

[0052] Control of fluid transfer Figure 2 shows a basic example of a fluid transfer strategy that forms the basis of system operation.

[0053] The peristaltic pump 230 draws fluid from the first bag 210 through the control valve 240 and pushes it into the second bag 220, opening the control valve 245 to allow the fluid to enter the second bag 220. Once the target volume has been drawn from the first bag 210, the control valve 240 on the first bag closes, and valve 255 opens to allow air to be drawn in through the sterile filter 250. The pump continues to push the liquid into the second bag 220 using the air.

[0054] The accuracy of the fluid volume is controlled by precise control of the pump rotation and the known peristaltic pump dispensing volume per revolution. The combination of pump tubing and pump geometry allows for pump calibration in milliliters per revolution. Pump calibration can be performed automatically and dynamically, as will be explained in more detail below.

[0055] Each fluid control valve 240, 245 also has associated bubble sensors 215, 225.

[0056] The bubble sensors 215 and 225 detect the presence or absence of fluid in the tube by differential ultrasonic impedance across the tube. Other types of bubble sensors, such as optical sensors, can also be used.

[0057] The primary risk in the fluid transfer process using a peristaltic pump is whether the fluid is actually being transferred. A bubble sensor monitors the fluid in the tube as the transfer occurs. The bubble sensor sends a signal to the controller indicating the state of the fluid in the tube adjacent to the sensor, so the controller is informed of the passage of liquid and bubbles through the tube. The flow rate of the fluid that has moved through the tube after passing the bubble sensor can be calculated based on the fluid flow rate associated with the pump operation. The control system uses this information to determine the volume that has passed through each valve. Therefore, when fluid transfer occurs, the amount of fluid accumulated in the bag can be calculated from the pump's movement when the bubble sensor indicated the presence of fluid.

[0058] A peristaltic pump is a positive displacement pump having a rotor equipped with "rollers" or "wipers" that press against a flexible fluid tube, closing it. The rotational motion of the pump rotor moves the rollers along the tube, thereby moving the pinch point of the tube and advancing the fluid through the tube. Each rotation moves the same volume of fluid through the tube, and the rotation of the rotor is indexable such that the volume of fluid moved by a partial rotation is known and controlled based on the rotational position of the rotor. In particular, the volume of fluid moved is the same for each pump rotation or each rotor index advance. Thus, the volume moved by the pump can be known and controlled based on the pump index, thereby controlling the fluid flow rate with respect to the pump rotation speed. The volume of fluid can be calculated by knowing the fluid flow rate and monitoring the time it takes for the fluid to pass through a bubble sensor. More simply, instead of the fluid flow rate, the volume of fluid can be calculated from the pump index (or rotation) count required to move the volume of fluid that has passed through the bubble sensor. In this method, the controller increments a counter with each index increment of the pump rotation.

[0059] The count of fluid volume between the start and end of measurement, as detected by the bubble sensor, is directly proportional to the volume moved. This count value can be simply multiplied by the volume moved for each rotational index of the pump to determine the fluid volume. Therefore, the accuracy of the fluid volume calculation is independent of time. Furthermore, even when the pump rotational speed fluctuates, accurate fluid volume calculation is possible because only the rotational index count is required. Using the rotational index count to calculate the moved volume can be particularly advantageous in very small-volume mixing processes where the required fluid volume (e.g., added for mixing) may be less than one rotation of the pump.

[0060] Figure 3A is a typical block diagram of a control system 300 for one embodiment of the system, which is typical of processing modules that may be implemented with software and / or hardware components. The control system 300 includes a batch process and pump controller 310, which is the main process controller for a particular batch protocol; an accumulator module 320 that calculates and tracks the volume of liquid and other formulation parameters during the batch processing process; a valve controller 330 that acts on valves to control the flow of fluid during the batch processing process in response to instructions from the batch process controller 310; a calibrator 340 that automatically calibrates the peristaltic pump; a volume estimator 350 that estimates the volume of fluid based on a bubble sensor input 370; and a log 360 for recording information related to batch processing.

[0061] The accumulator 320 tracks the volume of liquid drawn from and dispensed from each reservoir or input source used in batch processing, for example, monitoring the volume of liquid drawn from one fluid bag and dispensed into a mixing reservoir (which may be a fluid bag or other container optionally engaged with components to facilitate mixing), and also tracking the volume within the mixing reservoir. The accumulator can also estimate the formulation concentration state at different stages of processing based on the accumulated volume data.

[0062] Returning to the example in Figure 2, fluid is drawn from the first bag 210, and when the pump draws in the target amount of fluid while the bubble sensor 240 is wet, the accumulator decreases the estimated volume in the first bag 210. Similarly, for the second bag 220, when the bubble sensor 245 for the second bag 220 is wet, the accumulator recognizes that fluid is being dispensed. The fluid is eventually dispensed into the second bag 220 along with air. Since the bubble sensor 245 recognizes air, the pump is still working, but no volume is accumulating. This method has been proven robust to volumes from 2 ml to 50 ml with a 6-sigma tolerance of 0.4 ml.

[0063] It should be understood that using this method, the volume of the same liquid is calculated based on bubble sensor data acquired in the example of Figure 2, during the draw-in from the first bag 210 and during the delivery to the second bag 220, and therefore, by comparing the calculated volumes from both parts of the system, internal verification / confirmation of this robust method for accurately calculating the delivered volume is possible. As a result, the first claim of the present invention is to perform independent verification of the volume of fluid transferred from both the source and destination containers, provided that bubble sensors are provided.

[0064] When the final patient dose is delivered to the container, the accumulator readings of the source bag and the destination container independently determine the volume of liquid, rather than air, being aspirated and delivered, while the pump controls the dispensing volume. This is based on a flow rate or rotational index count, which is a function of the pump's rotation, and a bubble sensor output that senses the start and end of the volume of fluid moving through the sensor as the pump operates. A close agreement between these readings and the target dispensing volume systematically validates the fluid transfer step.

[0065] This method can be employed to measure the volume of input product for a process. A typical step is to extract the fluid from the input cell suspension bag and transfer it to a second bag configured for mixing the dispensed product. A volume accumulator, indicated by a bubble sensor, is drawn from the input bag and records the fluid received by the mixing bag. If the two accumulators do not match within a defined tolerance (typically 0.1 ml, but varies between 0.05 and 0.7 ml depending on the application), the control system returns the input material and repeats the transfer until the recorded volume falls within the tolerance.

[0066] Pump calibration verification The above techniques for calculating and verifying liquid volume rely on the reliability of the pump calibration, and in fact, it is common practice to include calibration protocols for drug dispensing processes using peristaltic pumps. A known issue with peristaltic pumps is that the flexible tubing engaged with the pump has an initial "warm-up period" during which the tubing properties, and consequently the pumping behavior, change until the tubing's flexibility stabilizes and the pumping behavior becomes consistent. For example, because the tubing is initially less flexible, the deformation rate and pumping volume change as the tubing's flexibility increases. In large batch processing or long-running operations, the pumping volume differences resulting from this initial phase may fall within acceptable limits for the specific application, or a specific initialization protocol may be used to "warm up" the system before calibrating the pump. However, neither is suitable for small-volume processing.

[0067] In small-batch autologous blood processing, accurate pump calibration is essential. The system embodiment includes a pump calibration function.

[0068] The self-verification function of this system embodiment is completed by means of verifying the pump calibration as part of the setup procedure. This calibration can also be performed periodically to verify or adjust the pump calibration.

[0069] Figure 4A is a basic representation of the apparatus used in the pump calibration process and shows part of apparatus 400, which includes a peristaltic pump mechanism 410 and bubble sensors 420, 430 associated with control valves (not shown) for opening and closing the fluid tube. The apparatus operates with a single-use kit consisting of a peristaltic pump tube 440 and a manifold (not shown) of the tube, accessed through valves supported by a carrier frame that controls the shape of the kit when installed in the apparatus. Thus, the volume of liquid contained in the tube between the bubble sensors can be kept constant in each single-use set, with a variation of 0.05 ml against a nominal volume of 3.2 ml. Therefore, the volume of the tube between the bubble sensors, i.e., the volume of fluid contained in the tube, can be known and used in calculations during processing. As described above, the combination of finely controllable pumping bubble sensors enables accurate calculation of the fluid volume in the system for the automation of the mixing protocol. The key to the accuracy of the liquid volume calculation is the precise calibration of the peristaltic pump. While peristaltic pumps have calibration data provided by the manufacturer, the accuracy of the pump calibration is crucial when using very small quantities of pumps, and it is desirable to be able to verify the pump calibration. Calibration protocols for peristaltic pumps can be included in the setup procedure, such as self-verification procedures to verify the pump calibration.

[0070] The process for calibrating the pump is shown in the flowcharts of Figures 4B and 5. The volume of the fluid tube between the two bubble sensors is known. This may include the pump engagement loop 440. The first step 510 of the measurement process is to draw a small block of fluid into the fluid path 440. The fluid block is pumped forward through the tube until the first bubble sensor 420 detects it (520), as shown as state 450 in Figure 4B. The position of the pump 410 is recorded when the first bubble sensor 420 detects the block of fluid in the tube 440 (530).

[0071] Next, pump 410 is reversed to pull the fluid block back through tube 440 until it is detected that the fluid block has exited the second bubble sensor 430 (550), as shown in Figure 4B as state 460 (540). The pump position is recorded at this second position (560). Based on the known tube volume between the two bubble sensors, the volume for each pump rotation (or index) can be calculated from the number of rotations (or partial rotations) required to move one end of the fluid from one bubble sensor to the next. This can be compared with the manufacturer's calibration data. This process is repeated (or performed) in the opposite direction, as illustrated as states 470 and 480 in Figure 4B, so that values ​​in both the forward and reverse directions can be compared with each other and with the manufacturer's calibration data. If the pump index volume calculated from the test measurements is within a preset tolerance (e.g., 1% of the manufacturer's calibration value), the pump calibration setting can be considered validated. This data can also be recorded in the process log. Calculated values ​​outside the acceptable range, or discrepancies between forward and reverse operation measurements, may result in calibration verification failures that may be repeated. Data regarding the number of verification attempts and their results can also be recorded.

[0072] The pump position is recorded with an accuracy of 0.002 ml or less. In this test method, since only one end of the fluid block needs to be detected by the bubble sensors 420 and 430, the volume of the fluid block can be excluded from the calculation of the volume of fluid moving with each index rotation of the peristaltic pump.

[0073] This process can be repeated (or performed) in the opposite direction, as illustrated as states 470 and 480 in Figure 4B. In the embodiment, the calibration method circulates a block of fluid back and forth to record measurements while monitoring for consistent measurements. The calculations rely on the bubble sensor function. If either the pump forward or pump reverse measurement does not match the instrument settings by more than a preset tolerance (e.g., 1%), an automated sequence can be executed to take additional samples of the pump calibration measurement. Statistical data obtained from the incremental samples are monitored to determine when sufficiently consistent results have been obtained (e.g., 6 × standard deviation < 0.2 ml). The calibration coefficient is automatically updated and reported in the process execution log. For example, a possible reason for the measurement to change and requiring the repeated calibration process is the "warm-up" phase of the pump's flexible tubing.

[0074] Once consistent readings are established, the forward and reverse pump calibrations can be estimated and compared to formally calibrated values ​​initially stored in system memory by the manufacturer or otherwise retained by the instrument. Not only the calibration verification results, but also data related to the calibration verification process (e.g., iterations and measurements) can be reported in the process execution log (to be recorded in memory along with other data from the batch process). If the results fall within a predefined tolerance range, the pump calibration is considered verified.

[0075] The embodiment may also perform dynamic recalibration or recheck of calibration during processing. It should be understood that, since the volume of the tube between each bubble sensor can be known or calculated based on the fixed shape of the manifold, the fluid block can be detected using any fluid block moving between two bubble sensors, and the pump rotation index calculated for the end of the fluid block passing through the next bubble sensor can be compared with the actual pump rotation index to verify pump operation. If a variation is detected, the amount of variation may be due to one or more changes in the system. For example, a change in ambient operating temperature may affect the fluid pressure, a change in the flexibility of the pump tube may change the pump efficiency, or an error or leak may occur in the system. The diagnostic function can determine whether the variation is due to drift within the operating tolerance or can be corrected by adjusting the calibration. If the diagnosis indicates a possible error, the process is stopped and the operator is warned about the error.

[0076] Verification of known volume A method for automatically calibrating or verifying peristaltic pumps within a functionally closed system utilizes the consistent volume created by the tubing of single-use kits controlled within a carrier frame sequentially controlled by geometric features on the process equipment. Calibrating the peristaltic pump requires measuring the fluid volume between two bubble sensors and ensuring that this volume remains known. Furthermore, since the tubing between the bubble sensors is part of a single-use subsystem, slight volume variations between kits (within manufacturing tolerances) are expected.

[0077] Qualifying pump calibration operations requires a means of measuring fluid volume within a controlled measuring loop, independent of variability in tubing, instruments, sensor function, or installation.

[0078] The volume determination step uses the same function to isolate the volume of fluid that the measuring system will use for calibration. Volume verification can be performed across multiple different single-use kits. Furthermore, different measuring instruments provide statistical reliability of the volume being measured (using resources deployed in the calibration itself) and therefore provide calibration determined from there.

[0079] This method uses automatic control of valves and pumps that interact with fluid detection sensors to isolate a controlled volume into a container that can be removed from the kit for accurate weight measurement. An example of the process is shown in Figure 8. In the first step A, the controlled volume of fluid 840 is transferred to the measuring loop 830. The peristaltic pump 810 can be operated to pull the fluid forward through the tube 830 until the fluid flows back to the first bubble sensor 820 in step B, triggering detection by the bubble detection sensor of the first bubble sensor 820. In this step, the peristaltic pump 810 is operated in reverse in step B.

[0080] In step C, additional fluid 840 is transferred to the measurement loop until the second fluid sensor 825 is triggered. It should be understood that the volume of fluid between these sensors represents the “known fluid volume” as described above. In step D, the pump is operated forward and the fluid representing the measurement loop volume is transferred to the external container 850. The medium used to push the liquid volume through the fluid tube may be filtered air as described above. In step E, the external container is isolated from the rest of the fluid assembly. For example, a valve can be used to isolate the external container 850. Optionally, the external container can be removed for calibration of the loop fluid measurement. To enable removal of the external container, a separation point can be provided at a point that is predictable in terms of mass. By measuring the weight of the external container before and after fluid addition, it becomes possible to calculate the tube volume based on mass. For example, if the weight of the liquid is known to be X g / ml, the volume can be easily calculated from the difference in weight of the empty and filled containers.

[0081] Figure 9 shows an example of an additional device that can be used to calibrate a known volume loop against an external reference. In this example, the external container is connected to the liquid handling system using a Luer connector 910 between the external container and the system being measured. Other options are also possible. The advantage of the Luer connector is that it is a standard and commonly used connector for medical devices and is highly reliable. It can also be detached and reconnected. In another embodiment, the external container is part of a single-use kit, connected via tubing, and detached once full by cutting the tubing.

[0082] Figure 10 shows an example of a table of measurement values ​​for the calibration loop using the method described above. These results show a 5% 6-sigma measurement variability for the loop volume.

[0083] The advantage of the described system embodiment is that processing accuracy can be verified within the system without requiring manual intervention. This is made possible by the closed system, the fixed shape of the fluid path within the fluid path manifold, and the use of bubble sensors at multiple locations along the fluid path. The use of bubble sensors and the gaps between them means that the liquid volume can be estimated in multiple regions within the system based on the output of the bubble sensors. Thus, the same block of liquid can have its volume estimated multiple times in different regions of the fluid path, and if a comparison of these multiple estimates shows agreement, this indicates an accurate measurement. In this way, the volume of the liquid block dispensed into the output container or bag is already verified within the system, thus reducing reliance on manual methods such as weighing.

[0084] Formulation, filling, and finishing are the final fluid steps performed for cell-based therapeutic formulations. The systems described herein have the advantage of enabling strategies and methods for process self-validation by automated equipment that completes the formulation, filling, and finishing operations.

[0085] An example of a system embodiment is shown in Figure 6, and a schematic diagram of the main functional components is shown in Figure 7. In the apparatus of this embodiment, the reusable subsystem 100 includes a mixer and hanger for supporting fluid bags above a case housing a control system, a peristaltic pump, a valve assembly, and a bubble sensor. The mixer can mechanically massage the bags to facilitate mixing of the liquid held in the bags. In some embodiments, tubing may also be supplied either directly from the system or via one or more input bags to another system, such as a centrifuge. In the embodiment shown in Figure 6, the single-use subsystem is mounted within the case. Each bag can be connected to the fluid input path of a single-use kit manifold, and multiple small bags can be connected to the output of the fluid path to dispense a formulation of the processing volume into them.

[0086] Embodiments may also include a recirculation function that circulates the fluid through the manifold's fluid pathway, optionally combining it with additional input fluid during this circulation and returning it to the mixing bag. This circulation may be performed multiple times, either to combine with additional input fluid or as a mixing step. This provides the ability to create a homogeneous suspension of fluid by combining bulk mixing in the bag with detailed circulation of the suspension through single-use kit tubing. A sample can then be taken from the recirculated flow, which has a very representative example of the bulk of the homogeneous suspension.

[0087] Figure 7 is a schematic diagram of the fluid pathway, associated valves, and bubble sensor of the single-use kit, and also shows the peristaltic pump.

[0088] In the embodiments shown in Figures 6 and 7, the input bag is on the right and the dilution reagent is on the left to facilitate rinsing of the input bag. During reagent dispensing, when valve F is open, liquid from the reagent bag is dispensed through the bubble sensor, and when valve D is open, the action of the pump allows this liquid to move through the fluid path to the mixing bag. The valves can be selectively operated to allow input from each input source to enter the fluid flow path, and the operation of the pump controls how much liquid is drawn into the system from each input source. The operation of the pump controls the volume of liquid drawn from each input source, and since the amount pumped up with each rotation of the pump is calibrated as described above, the volume is determined based on the known fluid path shape and the operation of the pump.

[0089] The pump also operates to deliver the input liquid to the mixing bag. Due to the narrow dimensions of the fluid channel and the surface tension of the liquid, the liquid can be pushed through the fluid channel using air, and the input liquid passes through the fluid channel as a block. An air inlet with a sterile filter is provided, and when valve A is open, the pump's operation draws air into the fluid path. This air is used to push the block of liquid around the fluid path. The system can be programmed to control many different changes in the movement of liquid in selectively controlled volumes between reservoirs to mix a precise formulation.

[0090] Gaps or bubbles can also be used to divide the dispensable volume of liquid within a fluid pathway. For example, when dispensing multiple samples of a mixed formulation, air valve A can be activated to create bubbles in the fluid piping between each sample volume. The volume of each sample is calculated as it is drawn from the mixing bag and then verified as the block of liquid passes through the bubble sensor. In this way, the system provides a self-verification function.

[0091] To consider the advantages of the disclosed system, it is worthwhile to examine alternatives to the prior art. The key step in formulating filling and finishing is the volume of fluid being transferred. Commercial prior art systems available on the market at the time of filing rely on weighing of suspended bags or mass measurement using trays on a platform scale to determine the volume of fluid transferred.

[0092] The response from the weighing system depends on the offset or tare adjustment and the scale factor. Tare is controlled by determining when it should occur in the process and avoiding it. The scale factor has little variation, but because there is no secondary information, there is a risk of errors each time the system is used. Therefore, verification of all weighing equipment should be part of every batch operation.

[0093] Each transfer can be verified by installing weighing systems on the source and destination containers. Substantial scaling errors or other interferences from one of the weighing devices will be evident in the discrepancy between the two measurements. Another problem with weighing batch inputs and outputs is interference, which is a systemic issue in these systems. Because sealed systems are connected by tubes that move with the filling and emptying of bags, the achievable weighing accuracy is limited, potentially exacerbating reliability issues associated with weighing (such as unexplained changes occurring between the two weighing devices).

[0094] In contrast, the embodiments of the system described herein are built upon the inventors' experience with the use of peristaltic pump tubes and control of pump rotation, and advantageously provide an internal verification system.

[0095] The inventor's system relies on the known behavior of the pump tube, which is influenced by the material and shape of the pump tube, as well as the shape of the pump roller and the pressure applied to the pump tube. While there are many potential variables, such systems are widely used for precise fluid delivery in large-scale filling and finishing systems. Pump tubes are manufactured to a high standard for this purpose.

[0096] Nevertheless, using peristaltic pumps as the primary fluid volume control exposes the process to the following risks: 1. The pump tubing was either incorrectly calibrated or damaged. 2. Instead of just liquid, air or bubbles are accidentally pumped into the fluid piping.

[0097] In embodiments of this system, ultrasonic bubble sensors are used. These devices detect the presence or absence of fluid in a tube by ultrasonic acoustic impedance. A threshold is set to detect bubbles in the tube. Such devices are commonly used in intravenous infusion pumps to protect patients from receiving tiny gaseous bubbles instead of liquid. In this application, since only tiny bubbles in the fluid flow are of interest, the threshold of the bubble sensor can be fairly coarse. Air gaps are intentionally used to divide specific portions of liquid in the fluid path.

[0098] The ability to utilize bubbles to separate specific volumes of liquid, and the ability to verify these specific volumes within the system, enables highly accurate mixing and tracking of the concentration of each component in the mixed formulation. This data is recorded during the mixing process and can be used for quality control and qualification of the output product. Because volume and concentration can be accurately verified internally, the burden of manual verification is reduced. This has the advantage of improving processing and treatment times. Especially for delicate samples, reducing manual steps and shortening preparation time can lead to significant benefits in quality and treatment outcomes.

[0099] In some embodiments, consideration of additional interference sources, such as temperature control, is not included. However, temperature measurement is a good example of an in-process measurement that needs careful consideration to ensure robust qualification. The only practical way to qualify temperature measurement is to always calibrate the measurement system from time to time and demonstrate that the frequency of calibration is sufficient to maintain acceptable accuracy. In the case of a manually calibrated system, this is a tedious manual process that can be prone to errors.

[0100] The advantage of the system described herein is its internal verification function. This reveals the following: In-process pump tube calibration verification - Pump tube calibration compares the measured volume with the controlled volume within the kit assembly. Input Product Volume Measurement - The volume of product transferred from the input bag is measured and used for batch data. The sample volume is recorded independently and removed from the product pool. Product volume pool - manages input liquid, diluent, and cryobuffer as batch data. The volume dispensed into the delivery bag is measured independently of the dispensing controller and recorded for review. Low correlations are recorded and highlighted for further investigation.

[0101] The system embodiment can also provide reports such as batch closeouts, including volume matching reports, based on data recorded during the execution of the mixing and dispensing processes.

[0102] Automated formulation and dosing Due to the self-calibration and validation advantages of the system described above, this system is suitable for automating at least a critical part of the dosage adjustment process for cell therapy. Potentially, the entire dosage adjustment process can be automated using the embodiments of the disclosed system.

[0103] For context, the following paragraph outlines an example of the process for coordinating autologous cell therapy. As a first step, cells are collected from the patient (or, in the case of allogeneic cell therapy, from a patient-compatible donor). These cells are preserved alive and cultured (also called extended) in a culture medium for, for example, several hours to about 10 days. During this time, target cell-specific treatments may be performed before or during the culture period. The cultured cells, along with at least a portion of the culture medium, are transferred to a centrifuge where target cells and "good" cells can be separated from dead cells, non-target cells, and other waste.

[0104] For example, a backflow centrifuge can be used to separate target cells from other cells and waste particles, and the culture medium can be diluted and flowed through a separate carrier medium. For instance, due to variations in size and weight between target cells and other components (dead cells, non-target cells, cell debris, and other waste particles), the target cells will gather where the forces acting on these cells (fluid flow and centrifugal force) balance out in the centrifuge chamber. The backflow centrifuge works to separate the target cells and concentrate them in a fluidized bed. These target cells (or good cells) form the basis of cell therapy, but the resulting cell count is unknown. Cell counts vary from patient to patient due to many variables. Variables that affect the cell count include the patient's biological factors and variables acting on the culture process, such as ambient temperature and pressure, the concentration of culture medium components, and the surrounding atmosphere. The resulting cell count can also be unpredictable. For example, the cell count may be very low, such as only 100,000, 1 million, tens of millions, or hundreds of millions of cells.

[0105] Once target cells are separated from waste, they typically need to be prepared into doses for administration to a patient. Because cell culture is time-consuming and target cell isolation requires specialized equipment, this preparation is usually performed in a laboratory or other facility away from the patient. For transport and delivery to the patient, the cells need to be preserved. This is typically done by freezing them in a mixture containing cryoprotective agents and preparing them in individually administerable doses. Dosage parameters required for treatment can be defined. These parameters include the dose volume and dosage formulation (e.g., carrier medium, proportion and composition of cryoprotective agents), and the minimum number of doses required for treatment. However, the exact formulation composition can vary depending on the cell concentration. The precise volume required for formulation usually depends on the cell count in the dose. For example, the carrier solution consists of a mixture containing cryoprotective agents. The cells for the dose are suspended in the carrier solution. The volume of the carrier solution can be manipulated to dilute the concentration of the cells to be administered. Examples include the minimum cell count per dose, the range of associated cryoprotective volumes based on the cell count, and the range of other formulation components. These components in the formulation may vary depending on the therapeutic objective. For example, the dosage formulation for stem cell therapy for cancer treatment will differ depending on the type of cancer being treated.

[0106] The number of feasible doses depends on the number of cells obtained for use in these doses. Therefore, the exact dosage and number of doses cannot be predetermined and must be determined after the cells have been separated and counted. Cell counting has traditionally been performed by manually or automatically counting cells in a small amount of liquid (e.g., 0.1 ml) and calculating the cell count per ml. For example, a small sample of the input sample (often called a QC sample) is extracted, and the cell count is counted in this sample. (This can be done manually or using an external cell counting system.) Since the system can record or measure the volume of the input sample, the total number of cells can be calculated by simply multiplying the total number of cells per ml by a predetermined volume of suspension.

[0107] Once the cell count is calculated, the potential number of doses that can be produced from the total number of cells is calculated. Since the number of doses must be an integer, given a target or desired cell count for each dose, there is a high probability that some cells will remain. For example, from the cell count, it is determined that 60 million cells can be obtained for use in therapeutic administration. The parameters stored in the system are a target cell count of 40 million cells per dose, with an acceptable range of 25 million to 50 million cells per dose. From the available 60 million cells, only 40 million cells can be administered in one dose, leaving 20 million cells, which is below the minimum dose threshold of 25 million. In such a case, the options are: Prepare one dose using 140 million cells, and discard the remaining 20 million cells. 2. For example, administer 30 million cells twice, or administer 35 million cells once and 25 million cells twice. 3. Increase the cell count per dose to 50 million to maximize the number of cells used and minimize the number of cells discarded to 10 million. 4. To use all cells, increase the cell count per dose to 60 million. This may be undesirable as the dose is outside the acceptable range.

[0108] Option 2 maximizes cell utilization and maintains the dose within the specified range. Option 3 is preferable to Option 1 because it minimizes the number of cells discarded while remaining within the specified range. Furthermore, since this option involves only a single dose, this treatment may be acceptable even if this dose is at the maximum acceptable cell count, or even higher, as in Option 4. Clearly, determining the number of doses and the cell count per dose is related to evaluating various trade-offs and constraints for determining the dose.

[0109] Traditionally, clinicians determine these dosage compromises. Once the therapeutic dosage is determined, further calculations are needed to determine the formulation of each dosage. Here again, these calculations may involve trade-offs, compromises, and constraints. Currently, decisions regarding dosage formulations and the calculation of the volume of components needed to formulate the dosages are performed manually.

[0110] Clinicians can use computers as calculation tools, for example, by using spreadsheet software to assist with calculations. However, this process is still essentially manual and heavily relies on the clinician's experience and knowledge. Because many variables are involved, the calculations are time-consuming. It should also be understood that manual processing can lead to errors and inconsistencies. The manual process of calculating dosages is also time-consuming, which can result in a longer time for cells to survive before the dosage is frozen.

[0111] One embodiment of the disclosed system is configured to enable automatic calculation of dosage formulations based on cell counts. An example of a control system 301 configured to perform dosage calculations is shown in Figure 3B, which, like Figure 3A, includes a batch process and pump controller 310, which is the primary process controller for a particular batch protocol; an accumulator module 320, which calculates and tracks liquid volume and other formulation parameters during the batch processing process; a valve controller 330, which acts valves to control the fluid flow during the batch processing process in response to instructions from the batch process controller 310; a calibrator 340, which automatically calibrates the peristaltic pump; a volume estimator 350, which estimates the fluid volume based on a bubble sensor input 370; and a log 360, which records information related to the batch processing.

[0112] The accumulator 320 tracks the volume of liquid drawn from and delivered to each reservoir or input source used in batch processing, for example, monitoring the volume of liquid drawn from one fluid bag and delivered to a mixing reservoir (which may be a fluid bag or any other container that engages with a component that facilitates mixing), and also tracking the volume within the mixing reservoir. The accumulator can also estimate the formulation concentration state at different stages of processing based on the accumulated volume data.

[0113] In this embodiment, the controller 301 further includes an interpolation engine 380 that can operate in real time to calculate dosing parameters based on the number of cells injected. In this embodiment, the controller stores formulas and algorithms for dosing calculation, variables for dosing calculation, and variable attributes and values ​​such as ranges or boundary conditions for each variable, as well as variable prioritization data. The variables and associated attributes are typically based on therapeutic requirements and may include information such as the following: Dosage-related – has a range or boundary condition that defines the minimum and maximum dose. The administered cell count may include one or more ranges specifying upper and lower limits, or thresholds.

[0114] Details of the carrier formulation component variables and tissue cryoprotection (Cryoprotect) include constraints, concentration ranges, and cell concentration dependence.

[0115] Each variable can have a target value and an acceptable range defined. The interpolation engine uses these defined target values ​​and ranges to mathematically solve for the variables and determine the dosage formulation for a given cell count. Constraints include thresholds for viable treatment, such as the minimum cell count per dose and the minimum number of doses. If there are insufficient cells available to provide a viable treatment, the formulation may be stopped, and a warning may be issued to the clinician.

[0116] The system also remembers prioritization rules that can be changed between batches. For example, the prioritization might be: 1. the maximum number of doses per cell count, 2. the maximum cell count within a given range, 3. rounding down to an integer dose, and 4. whether it is possible to exceed the maximum or minimum range to avoid discarding excess cells. The ranking of such prioritization rules is variable and can also be predefined by the clinician based on the cell therapy. This prioritization is used by the interpolation engine to mathematically solve the variables and determine the dose formulation.

[0117] It should be understood that the target values ​​and ranges for each variable, as well as the prioritization rules, can be selected for each batch before physical processing begins. These parameters, ranges, and priorities may differ between batches. In some embodiments, historical data 390 may be stored, which includes previous batch data with previously used parameters, ranges, and prioritizations. In this embodiment, a clinician or technician can select previously used settings based on the batch type. In some embodiments, the settings may be stored as a batch type profile. For example, stored batch profiles can be retrieved via a user interface, either by searching a picklist or by searching from data records.

[0118] Flaving the interpolation engine 380 integrated with the controller 301 of the fluid processing system has the advantage that the calculation of the dosage formulation is rapid, and once the formulation is solved, the controller can automatically prepare the dosage according to the determined formulation using the method described above.

[0119] In one embodiment, the cell count is manually entered by a clinician or technician. In this embodiment, a sample of enriched cells is extracted, calculated, and the cell count per ml is entered into the system. Based on this value, the controller is configured to determine the total cell count based on the total volume of enriched cells and the cell count. Then, using this value and the batch dose parameters, range, and priority, the optimal dose formulation is mathematically determined. The optimal dose formulation is solved by an interpolation engine using an iterative process. This iterative process is broadly characterized by the following steps. A. Determine the number of administrations and the integer value of the residual amount based on the total cell count and the target cell count per dose (or target cell concentration and dose). B. If there is no residue, does the integer value of the total number of doses meet the minimum number of doses requirement? If the minimum number of doses is met, proceed to determining the dosage formula (Step E). If the minimum number of doses is exceeded and additional doses are prioritized, proceed to determining the dosage formula (Step E). If the minimum number of doses or the target number of doses is exceeded and priority is given to increasing the dose concentration, recalculate the dose concentration (Step C). C. If there is residue, or if the minimum dose is exceeded and it is desirable to increase the dose concentration, recalculate the dose based on the total cell count so that the cell count per dose increases. If the dose and cell concentration are within acceptable limits, proceed to determining the dose calculation formula (step E). D. If the minimum target dose is not met in step A and there are no residues, the system may determine, based on the threshold requirements for treatment, that a viable treatment cannot be formulated and may output a warning message to the clinician or technician. Processing may be stopped at this step so that the clinician can input further instructions. For example, it may output instructions to return the cells to growth medium or to perform further tests. If the minimum treatable threshold is met, the system may continue to determine the most preferable dosage option based on prioritization data and available cell counts. Once the viable treatment doses and cell counts per dose are determined, the formulation of the dose is determined (step E). E. Formulating dosages based on cell count per dose: The amount of cryoprotective agent used in the dosage formulation is based on the cell count per dose. The interpolation engine iteratively solves the variable values ​​based on formulas and prioritization rules, adjusting the dosage formulation to match the cell count and treatment. This includes varying the dosage within a defined range, as well as adjusting the relative concentrations of the formulation components.

[0120] It should be understood that the process described above may actually involve iterative calculations at each step, and / or the steps may be repeated iteratively until the optimal formulation is determined. Once the formulation is determined (i.e., the ratio of components to be mixed for each dose, and the dosage), the system automatically performs the steps of mixing the formulation and dispensing individual doses for freezing using the process described above. This automation can significantly reduce the time it takes for cells to be removed from the culture medium or growth medium and frozen in individual doses.

[0121] Automatic calculation and subsequent dosage preparation are made possible by the automatic calibration and verification of the volume within the system, which means that the measurement of the components to be mixed into the formulation is accurate and reliable, and it should be understood that the dosage dispensed is also accurate.

[0122] In some embodiments of this system, samples (QC samples) are automatically dispensed for manual cell counting. Embodiments of this system are also envisioned to be used for automated mixing and dispensing of doses where the cell count at the input concentration of cells or other particles is already known (e.g., determined from another process outside the liquid formulation system) before the concentrated cells or particles in suspension are introduced into the system. The steps of volume measurement, dose calculation, mixing, and dispensing of the concentrate can be carried out as described above.

[0123] Embodiments of this system can also be configured to integrate with a system capable of automatic determination of cell counts (or particle counts). Cell count data can be input to the formulation process via an inter-system machine communication interface, which may be a wired or wireless interface. Cell counts can be based on detected characteristics of the cell concentration in the suspension. For example, the optical characteristics (density, turbidity, spectrum) of the cell concentration in the suspension can be measured using an optical sensor. Other sensors, such as electrical sensors, may also be used. The cell concentration is estimated based on these characteristics and the cell type.

[0124] Determining the particle count in a suspension is a common quality control measurement performed on particle-based therapeutic products because it is necessary to guide the next step in the process. Such particle counts are typically achieved by instrumental or manual optical methods used to measure particles in a small sample of a diluted suspension in a known total volume. The particle count in the total volume of the suspension is then extrapolated based on the small sample count. The problem is that this sampling and particle count operation requires waiting for the information to be obtained before proceeding with the primary processing. Furthermore, these methods for particle counting are susceptible to many influences that cause variability in measurement results, resulting in count variability ranging from ±20%. Combining indirect observation of the entire particle population with density sensors and the accumulation of validation data for the same product and process environment may provide sufficient reliability to complete the process without intra-step sampling.

[0125] Examples of counterflow centrifugation systems that may include functionality for automated cell count estimation are disclosed in the applicant's earlier patent publications WO2019 / 140491 and WO2018 / 204992, and such systems may be connected to the disclosed systems via centrifugation piping (see Figure 7) to enable the integration of the functions of the two systems. In this example, in the counterflow centrifugation process, the fluidized bed of particles generated in the rotating chamber is characterized by particle attributes that affect the Stokes sedimentation behavior in the fluid medium supporting the particles, namely nominal external dimensions or diameter, bulk density and external morphology. Attributes of the fluid medium include density and viscosity (both temperature-sensitive), and secondary properties such as thixotropy and shear sensitivity of viscosity. Despite the complexity of such interactions, consistent fluidized bed behavior is obtained, where the particle density in the fluidized bed is consistent when measured as particle count per unit volume (e.g., particle count per ml), by reproducing the process with nearly consistent input materials and operating conditions. While the density of particles in the suspension is substantially similar across separate batches processed using consistent input materials and operating conditions, it should be understood that the volume of the fluidized bed can vary significantly between batches, reflecting changes in particle count.

[0126] Embodiments of the centrifugation system in such cases may include particle count estimation. In these embodiments, the counterflow centrifugation controller is further configured to determine the particle count based on the volume of the recovered concentrate and to determine the particle density estimation of the concentrate based on particle characteristics and operating parameters. The particle density estimation may be based on empirical data, e.g., historical data from previous batch processing with input materials and operating conditions correlated with the current processing batch. For example, such data may be stored externally and input to the controller along with processing procedure data and parameters for the specific process being performed. Alternatively, the controller may be configured to monitor the execution of the process and take in data characterizing the particle density of each process performed (e.g., density sensor output, particle count estimation, or validated particle count data). In such embodiments, the controller may identify one or more correlated previous / historical processing events and store and retrieve such data in a database or other data repository to compare with the current processing event in order to find suspension characteristics to be used for particle density estimation. Once the particle density is determined, the total cell count is calculated from the density and measured volume. This estimated cell density or cell count can be output to the control device 301 for use in the dosing formulation process. In some embodiments, quality control (QC) samples may also be output for verification purposes. However, for processes (e.g., batch types) where the system has a record of accurate automated cell count estimation, the extraction of QC samples can be omitted to maximize the cell count available for formulation into dosing.

[0127] The automated processing enabled by this system can significantly reduce the time it takes for cells to be removed from the culture medium (or in the body) and frozen for therapeutic administration. Furthermore, the ability to integrate with external systems such as centrifugation systems can increase the number of autonomous processing steps, potentially further improving processing speed and consistency. Reduced processing time can have a significant impact on cell viability and therapeutic outcomes.

[0128] Integrating autonomous closed-loop systems can also reduce the risk of exposure to pollutants and environmental conditions. Thus, it's not just speed that's improved.

[0129] Those skilled in the art will understand that many modifications can be made without departing from the spirit and aspects of the present invention.

[0130] When prior art documents are referenced in this specification, it should be understood that this does not constitute an acknowledgment that such documents form part of the general knowledge in the relevant art in Australia or any other country.

[0131] In the claims and the above description of the invention, unless otherwise required by the context, explicit wording, or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” are used in a comprehensive sense, that is, to identify the presence of the described features, but not to exclude the presence or addition of further features in various embodiments of the invention. [Prior art documents] [Patent Documents]

[0132] [Patent Document 1] International Publication No. WO2019 / 140491 [Patent Document 2] International Publication No. WO2018 / 204992

Claims

1. A liquid handling system, Reusable subsystems, Interchangeable subsystems and Equipped with, The aforementioned reusable subsystem is Peristaltic pump and A valve assembly including multiple valves, Two or more bubble sensors, each positioned to detect bubbles in the fluid path, A system controller configured to receive input from the bubble sensor, control the operation of the peristaltic pump, and control the operation of the valve assembly according to a programmed processing protocol, A case housing the peristaltic pump and the valve assembly It has, The single-use, replaceable subsystem is A fluid path manifold comprising one or more fixed-shape fluid paths, wherein at least one of the fluid paths is configured to engage with the valve assembly, thereby allowing the fluid path to be selectively opened and closed by operation of the valve assembly, and at least one of the fixed-shape fluid paths is positioned in close proximity to the bubble sensor when fixed to the case, so that the bubble sensor can identify bubbles in the fluid path; A pump tube is configured to enable an operable engagement between the peristaltic pump and the fluid path so as to generate fluid flow within the fluid path manifold by the operation of the peristaltic pump, Multiple liquid input ports configured to be connected to each liquid supply component in order to distribute each liquid to one or more fluid paths, To enable the gas to enter the fluid path, at least one gas inlet is connected to at least one of the one or more fluid paths, To dispense the fluid, at least one outlet port is connected to one or more fluid pathways and Having The interchangeable subsystem provides a closed environment for mixing and dispensing liquid formulations. A liquid handling system in which the system controller determines the volume of liquid in one of the fluid paths based on the operation of the peristaltic pump and input from at least one bubble sensor associated with each of the fluid paths.

2. The liquid handling system according to claim 1, wherein the combination of a fixed-shape fluid path manifold and the arrangement of a bubble sensor enables the measurement of a known volume in at least one region within the fluid path, and the system controller utilizes the measurement of the known volume to calibrate the peristaltic pump.

3. The liquid handling system according to claim 2, wherein the peristaltic pump is automatically calibrated by the system controller.

4. The liquid handling system according to claim 3, wherein the peristaltic pump is dynamically calibrated during the execution of one or more processing protocols.

5. A liquid handling system according to any one of claims 2 to 4, wherein the system controller utilizes a measurement of a known volume to confirm the volume of the dispensed product based on the input of a bubble sensor.

6. A liquid handling system according to any one of claims 1 to 4, wherein bubbles in a fluid piping are used to separate small volumes of liquid, and the volume of each small volume of liquid is verified in at least two different regions within the fluid path using bubble sensor data from each region.

7. The liquid handling system according to claim 1, wherein bubbles in a fluid piping are used to separate small volumes of liquid, the volume of each small volume of liquid is verified in at least two different regions in the fluid path using bubble sensor data from each region, and the verified volume data includes dispensed volume data.

8. The liquid handling system according to claim 7, wherein the gas inlet is an air inlet.

9. The liquid handling system according to claim 8, wherein the air inlet includes a sterile filter.

10. The liquid handling system according to any one of claims 1 to 4, wherein the system controller is further configured to determine a dosage formulation and the number of doses to be dispensed based on a particle count in the liquid sample to be processed, and to control the system to mix the determined formulation and dispense the determined number of doses.

11. The liquid handling system according to claim 1, wherein the system controller is further configured to determine a dosage formulation and the number of doses to be dispensed based on a particle count in a liquid sample to be processed, to mix the determined formulation and to dispense the determined number of doses, and the system controller includes an interpolation engine adapted to mathematically solve for dosage and formulation variables based on target values ​​and ranges of formulation variables, particle count, and prioritization rules for variables.

12. A method for calibrating a peristaltic pump in a liquid handling system according to claim 1, The steps include introducing a certain volume of liquid into a flow path having a known volume between a first bubble sensor and a second bubble sensor, The steps include introducing a gas into a flow path such that the aforementioned volume of liquid is preceded and followed by bubbles, The steps include operating a peristaltic pump to pass the aforementioned volume of liquid through a channel near the first bubble sensor so that the transition from liquid to bubbles can be identified, and recording the position of the peristaltic pump at the time of the transition, The steps include operating the peristaltic pump to draw the aforementioned volume of liquid through a known volume channel to the second bubble sensor so that the second bubble sensor can identify the same transition from liquid to bubble, and recording the position of the peristaltic pump at the time of the transition, The steps include: calculating the volume of liquid to replace each index of the peristaltic pump based on the recorded position and known flow path volume; A method for providing this.

13. A method for calibrating a peristaltic pump according to claim 12, further comprising a method for determining the volume of a tube between two bubble sensors, the method is: a) A step of controlling the introduction of an initial volume of liquid into the tube, b) The step of operating the peristaltic pump to advance the initial volume of liquid so that the end of the fluid is identified near the first bubble sensor, c) A step of controlling the introduction of further liquid into the tube until liquid is detected by the second bubble sensor, d) The step of operating the peristaltic pump to transfer the total volume of liquid, consisting of the initial volume of liquid and any further volumes of liquid, to an external container. A method of having.

14. A method for performing a mixing operation in a liquid handling system according to claim 1, wherein the method is: a) The step of operating the peristaltic pump in order to generate a fluid flow through the fluid path, b) The step of operating one or more valves to control the selection and direction of the fluid flow through the fluid path, c) A step of monitoring the fluid flow through the fluid path using the bubble sensor, and determining the volume of the fluid based on fluid detection by at least one bubble sensor and the operation of the peristaltic pump, d) In response to the determination that the target volume of the fluid has passed through the bubble sensor, the steps include activating at least one valve to direct the flow of the fluid in the fluid path, including introducing bubbles into the fluid path behind the target volume of the fluid and directing the flow of the target volume of fluid towards the mixing reservoir, e) Repeat steps a) through d) for one or more additional fluids and additional target volumes, thereby mixing the fluids in the mixing reservoir and providing a mixed fluid. A method for providing this.

15. f) A method for performing the mixing operation according to claim 14, further comprising the step of activating one or more valves to recirculate the mixed fluid from the mixing reservoir back to the mixing reservoir through the fluid path.

16. g) A method for performing a mixing operation according to claim 14, further comprising the steps of allowing the mixed fluid to flow through one or more fluid paths to an outlet, and operating one or more valves to dispense a target volume of the mixed fluid based on the operation of the peristaltic pump and the flow of the mixed fluid detected by a bubble sensor.

17. A method for performing the mixing operation according to claim 16, wherein the target volume dispensed is the sample volume of the mixed fluid.

18. A reusable subsystem of a liquid handling system, wherein the reusable subsystem is Peristaltic pump and A valve assembly including multiple valves, Two or more bubble sensors, each positioned to detect bubbles in the fluid path, A system controller configured to receive input from the bubble sensor, control the operation of the peristaltic pump, and control the operation of the valve assembly according to a programmed processing protocol, A case housing the peristaltic pump and valve assembly Equipped with, The peristaltic pump, valve assembly, and bubble sensor are arranged to engage with a fluid path manifold including one or more fixed-shape fluid paths, thereby allowing the fluid paths to be selectively opened and closed by the operation of the valve assembly, and at least one fluid is positioned close to each of the bubble sensors when fixed within the case, so that the fluid paths can be selectively opened and closed by the operation of the valve assembly, and the bubble sensor can identify bubbles in the fluid paths, and the peristaltic pump engages with a pump tube configured to allow a movable engagement between the peristaltic pump and the fluid paths so that the operation of the peristaltic pump causes fluid flow into the fluid path manifold, A reusable subsystem in which the system controller determines the volume of liquid in one of the fluid paths based on the operation of the peristaltic pump and input from at least one bubble sensor associated with each fluid path.

19. A replaceable subsystem of a liquid handling system, the replaceable subsystem is configured to engage with a reusable subsystem comprising a peristaltic pump, a valve assembly including a plurality of valves, two or more bubble sensors, each arranged to detect bubbles in a fluid path, a system controller configured to receive input from the bubble sensors according to a programmed processing protocol, control the operation of the peristaltic pump, and control the operation of the valve assembly, and a case housing the peristaltic pump and the valve assembly, the replaceable subsystem is configured to engage with a reusable subsystem, A fluid path manifold comprising one or more fixed-shape fluid paths, wherein at least one of the fluid paths is configured to engage with the valve assembly, thereby allowing the fluid path to be selectively opened and closed by operation of the valve assembly, and the at least one fixed-shape fluid path is positioned near the bubble sensor when the manifold is fixed within the case, so that the bubble sensor can identify bubbles in the fluid path. A pump tube is configured to enable a movable engagement between the peristaltic pump and the fluid path, so as to generate fluid flow within the fluid path manifold by the operation of the peristaltic pump, Multiple liquid input ports configured to connect to respective liquid supply components for distributing each liquid into one or more fluid pathways, To enable the gas to enter the fluid path, at least one gas inlet is connected to at least one of the one or more fluid paths, To dispense the fluid, at least one outlet port is connected to one or more fluid pathways and Equipped with, The interchangeable subsystem provides a closed environment for mixing and dispensing liquid formulations. A replaceable subsystem in which the system controller determines the volume of liquid in one of the fluid paths based on the operation of the peristaltic pump and input from at least one bubble sensor associated with each fluid path.

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