Single-use system and method for continuous homogenization or lysis - Patents.com

Abstract

The present invention relates to a continuous flow homogenization device and methods of using the device for lysis or disintegration of cells, tissues, and other biologics to create smaller particles and emulsions. The device includes a homogenization chamber in which the inlet material continuously enters the chamber and the homogenized material continuously flows out of the outlet. The homogenization chamber may include at least one impeller with or without homogenization enhancing media that rotates and / or reciprocates inside the chamber. The homogenization enhancing media may include rigid particles. All product contacting surfaces may be sterile and disposable. The complete homogenization process may be performed in a fully automated or manual manner with a controller managing the different inputs and outputs of the system components.

Description

[Technical field]

[0001] Related Applications This application claims priority from U.S. Provisional Application No. 63 / 296,984, filed January 6, 2022, the disclosure of which is incorporated by reference in its entirety.

[0002] The present invention relates to methods, devices and systems used for homogenizing organic and inorganic materials. [Background technology]

[0003] Lysis or homogenization of cells or tissues is used in many processes to recover useful products present inside the cells. For example, recombinant proteins produced by E. coli are contained intracellularly. The cells are lysed to release the recombinant proteins for further purification. Similarly, many viruses and viral vectors are produced inside cells (e.g., adeno-associated virus) and require lysis of the cells for their release. Because significant shear is produced by homogenizers, they can be used to reduce particle size and to produce emulsions and creams.

[0004] There are several notable methods that can be used for homogenization or lysis of cells from tissues or cell cultures. Some of these methods are based on mechanical lysis, while others are based on chemical lysis. Although chemical lysis can be used for small-scale research protocols, it is not the preferred method for biopharmaceutical manufacturing because the chemicals must be completely removed from the product after the lysis step. Regulatory agencies have restricted the use of detergents that are sometimes used to lyse cells because these chemicals are difficult to remove and may require several purification steps. This is one of the main reasons why commercial discontinuous or continuous homogenization systems are based on mechanical lysis. These systems are cleaned and reused because most of the product contacting parts are very expensive and the commercially available single use parts (e.g., tubing, bags) cannot withstand the high pressure or high shear generated by these homogenization systems. There are several other systems, including Dounce homogenizers, ultrasonic homogenizers, and bead mill homogenizers, that can be operated in a small-scale batch mode but cannot be scaled up.

[0005] Single-use technologies have gained significant momentum in recent years over reusable technologies for biomanufacturing because they eliminate the need for CIP / SIP (Clean-In-Place / Sterilization-In-Place) and the risk of cross-contamination between batches. They also reduce cycle time and capital investment between batches. There is a great need for single-use homogenization technologies since current homogenization technologies are not single-use. Summary of the Invention

[0006] Some embodiments of the present invention are directed to a homogenization device that includes a chamber including an inlet and an outlet, and at least one impeller within the chamber that is configured to (i) rotate or reciprocate, or (ii) rotate and reciprocate. The device is sterilized prior to use and is a single-use, disposable item.

[0007] In some embodiments, the chamber includes a homogenization enhancement medium. The homogenization enhancement medium may include incompressible or solid particles. The homogenization enhancement medium may include incompressible or solid particles with a size of 0.1 to 2000 microns. The homogenization enhancement medium may include non-porous particles. The homogenization enhancement medium may include porous particles. The homogenization enhancement medium may include particles with a rough surface. The homogenization enhancement medium may include particles with a smooth surface. The homogenization enhancement medium may include glass, metal, plastic, ceramic, or any other rigid particles.

[0008] In some embodiments, the chamber is made of plastic, hi some embodiments, the chamber is made of metal.

[0009] In some embodiments, the at least one impeller is connected to a shaft, the shaft being configured to oscillate.

[0010] In some embodiments, the impeller and shaft are made from plastic.

[0011] In some embodiments, the impeller and shaft are made from metal.

[0012] In some embodiments, all product contact surfaces are cleaned after processing a single batch.

[0013] In some embodiments, the device is sterilized and packaged prior to its use.

[0014] In some embodiments, the device is configured to homogenize a starting material. The starting material may be a cell culture. The starting material may be a microbial culture. The starting material may be organic. The starting material may be inorganic.

[0015] In some embodiments, the device is configured to lyse cells.

[0016] In some embodiments, the device is configured to mix materials.

[0017] In some embodiments, the device is configured to reduce the size of the starting material.

[0018] In some embodiments, the chamber includes a body defining an internal cavity, the inlet being at a lower portion of the internal cavity and the outlet being at an upper portion of the internal cavity.

[0019] In some embodiments, the device includes a filter between the outlet and the internal cavity.

[0020] In some embodiments, the device includes a shaft connected to at least one impeller.

[0021] In some embodiments, at least one impeller is single-blade connected to a shaft.

[0022] In some embodiments, a plurality of holes are defined in the single-blade impeller.

[0023] In some embodiments, the shaft extends from a top portion of the chamber body.

[0024] Some other embodiments of the present invention are directed to a system for homogenization of starting material, the system comprising a starting material container containing the starting material, a chamber including an inlet and an outlet, and a homogenization device including an impeller configured for rotational and / or reciprocating movement within the interior volume of the chamber, a first tubing line between the starting material container and the inlet of the chamber, a pump (P1) in the first tubing line, a first valve (V1) in the first tubing line, a recirculation container, a second tubing line between the recirculation container and the first tubing line, the second tubing line fluidly connecting the recirculation container and the inlet of the chamber, and a second tubing line. a second valve (V2) in the first tubing line, a third tubing line between the outlet of the chamber and the starting material container, a third valve (V3) in the third tubing line, a fourth tubing line between the recirculation container and the third tubing line, a fourth valve (V4) in the fourth tubing line, a fifth tubing line between the third tubing line and the first tubing line, and a fifth valve (V5) in the fifth tubing line; and a controller configured to direct the first through fifth valves to selectively open and close, to direct operation of the impeller, and to direct the pump to turn on and off.

[0025] In some embodiments, the controller is configured to: (a) open the first valve and turn on the pump such that a cycle volume of starting material flows from the starting material container to the inlet and into the homogenization chamber; (b) close the first valve and open the fifth valve such that the cycle volume flows from the outlet to the inlet and into the homogenization chamber multiple times; (c) close the fifth valve and open the fourth valve such that the cycle volume is received in the recirculation container; and (d) repeat steps (a)-(c) until the starting material in the starting material container is depleted.

[0026] In some embodiments, the system further includes an air bubble sensor in the first tubing line, and the controller is configured to determine that the starting material in the starting material container is depleted based on measurements by the air bubble sensor.

[0027] In some embodiments, the controller is configured to: (a) open a first valve and turn on a pump so that starting material flows from the starting material container to the inlet and into the homogenization chamber; (b) open a third valve so that homogenized starting material flows from the outlet to the starting material container; and (c) keep the first and third valves open and the pump on until a desired homogenization is achieved based on measurements by optical sensors in the first and / or third tubing lines.

[0028] In some embodiments, the controller is configured to: (a) open the first valve, close the second valve, close the third valve, close the fifth valve, open the fourth valve, and turn on the pump such that starting material flows from the starting material container through the homogenization device to the recirculation container until the starting material in the starting material container is depleted; (b) close the first valve, close the fourth valve, and open the second and third valves such that starting material flows from the recirculation container through the homogenization device to the starting material container until the starting material in the recirculation container is depleted; and (c) repeat step (a) and / or step (b) multiple times until a desired homogenization is achieved based on measurements by optical sensors in the first and / or third tubing lines.

[0029] In some embodiments, the system further includes an air bubble sensor in the first tubing line, and the controller is configured to determine that the starting material in the starting material container and the recirculation container is depleted based on measurements by the air bubble sensor.

[0030] In some embodiments, the first through fifth tubing lines, the first through fifth valves, the air bubble sensor in the first tubing line (if provided), the pressure sensor in the first tubing line (if provided), the optical sensor in the first tubing line (if provided), the optical sensor in the third tubing line (if provided), the homogenization device, the starting material container, the recirculation container, and / or the pump are provided as a kit and are single-use disposable items.

[0031] Some other embodiments of the present invention are directed to a system for homogenization of a starting material. The system includes a starting material container containing the starting material, a chamber including an inlet and an outlet, and a homogenization device including an impeller configured for rotational and / or reciprocating movement within an internal volume of the chamber, a first tubing line between the starting material container and the inlet of the chamber, a pump (P1) in the first tubing line, a first valve (V1) in the first tubing line, a second tubing line between the outlet of the chamber and the starting material container, a second valve (V3) in the second tubing line, at least one optical sensor in the first tubing line and / or the second tubing line, and a controller, wherein the controller is (a) opening a first valve, turning on a pump, and rotating and / or reciprocating an impeller such that starting material flows from a starting material container to an inlet, into the homogenization chamber, and is at least partially homogenized; (b) opening a second valve such that the at least partially homogenized starting material flows from an outlet to the starting material container; and (c) keeping the first and second valves open and the pump on until a desired homogenization is achieved based on measurements by optical sensors in the first and / or second tubing lines.

[0032] Some other embodiments of the present invention are directed to a method for homogenizing organic and inorganic materials, the method including flowing a starting material from a starting material container through a homogenization device multiple times to homogenize the starting material, and collecting the homogenized material in the container.

[0033] In some embodiments, the flowing step includes: (a) pumping a cycle volume of starting material from a starting material container through a first tubing line to an inlet of a homogenization device; (b) closing a first valve (V1) in the first tubing line and opening a second valve (V5) in the second tubing line; (c) flowing the cycle volume from an outlet of the homogenization device through the second and first tubing lines to an inlet to the homogenization device multiple times while operating the homogenization device to homogenize the cycle volume; (d) closing the second valve (V5) and opening a third valve (V4) in the third tubing line such that the homogenized cycle volume enters a recirculation container; and (e) repeating steps (a)-(d) multiple times until determining that the starting material container is depleted.

[0034] In some embodiments, the method includes using an air bubble sensor in the first tubing line to determine when the starting material container is depleted.

[0035] In some embodiments, the flowing step includes: (a) pumping the starting material from a starting material container through a first tubing line to an inlet of a homogenization device; (b) flowing the starting material through the homogenization device while operating the homogenization device to at least partially homogenize the starting material; (c) flowing the at least partially homogenized starting material from an outlet of the homogenization device through a second tubing line to the starting material container to mix the at least partially homogenized starting material with the starting material; and (d) repeating steps (a)-(c) until the desired homogenization is achieved.

[0036] In some embodiments, the method includes using at least one optical sensor (OS1, OS2) in the first tubing line and / or the second tubing line to determine when the desired homogenization is achieved.

[0037] In some embodiments, the flowing step includes the steps of: (a) opening a first valve (V1) in the first tubing line, opening a second valve (V4) in the second tubing line, closing a third valve (V5) in the third tubing line, closing a fourth valve (V2) in the fourth tubing line, and closing a fifth valve (V3) in the fifth tubing line; (b) pumping the starting material from a starting material container through the first tubing line to an inlet of the homogenization device; and (c) pumping the starting material through the first tubing line to an inlet of the homogenization device. (d) flowing the at least partially homogenized starting material from an outlet of the homogenization device through the fifth tubing line and the second tubing line to a recirculation vessel until determining that the starting material vessel is depleted; (e) closing a first valve (V1) in the first tubing line, a second valve (V4) in the second tubing line, and a third valve (V5) in the third tubing line. (f) pumping the at least partially homogenized starting material from the recirculation vessel through the fourth tubing line to an inlet of the homogenization device; (g) flowing the at least partially homogenized starting material into the homogenization device and operating the homogenization device to further homogenize the starting material; (h) flowing further homogenized starting material from an outlet of the homogenization device through the fifth tubing line to a starting material vessel until determining that the recirculation vessel is exhausted; and (i) repeating steps (a)-(h) while either stopping collecting the homogenized material in the recirculation vessel in step (d) or stopping collecting the homogenized material in the starting material vessel in step (h) until a desired homogenization is achieved.

[0038] In some embodiments, the method includes using an air bubble sensor in the first tubing line to determine when the starting material container or the recirculation container is depleted.

[0039] In some embodiments, the method includes using at least one optical sensor (OS1, OS2) in the first tube line and / or the sixth tube line to determine when the desired homogenization is achieved. [Brief description of the drawings]

[0040] [Figure 1] 1 is a schematic diagram of a homogenizer according to some embodiments. [Fig. 2A-2F] 11A-11D illustrate different impeller configurations, according to some embodiments. [Diagram 3] 1 is a cross-sectional view illustrating a reciprocating seal according to some embodiments. [Figure 4A] 1 is a graph illustrating experimental results. [Figure 4B] 1 is a graph illustrating experimental results. [Figure 4C] 1 is a graph illustrating experimental results. [Figure 4D] 1 is a graph illustrating experimental results. [Figure 5A-5B] 1 is a graph illustrating experimental results. [Figure 6] 1A-1D are side and front views that generally illustrate external features of a disposable homogenizer chamber, according to some embodiments. [Figure 7] 1A and 1B are back and cross-sectional views that generally illustrate internal features of a disposable homogenizer chamber, according to some embodiments. [Figure 8] 1 is a flow diagram of a system according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Although the embodiments described herein use a fluid with cells or particles as the starting material mixture, the devices and methods described herein are also applicable to mixtures containing any of liquids, solids, gases (e.g., air), and combinations thereof.

[0042] The devices and methods described herein use at least one inlet and at least one outlet (Figure 1). All product contact surfaces can be discarded after processing a batch. For biopharmaceutical manufacturing applications, all product contact surfaces are selected so that they are biocompatible and suitable for biopharmaceutical manufacturing (e.g., C-Flex, silicone, polypropylene, polyethylene, PVC, polycarbonate, 316L stainless steel). The complete homogenization chamber assembly and associated tubing are pre-sterilized (e.g., by steam, gamma irradiation, ethylene oxide, etc.) and packaged prior to its use. The inlet can be connected to the lower part of the chamber and the outlet can be connected to the upper part of the chamber (Figure 1). This configuration allows for settling of any dissolution or homogenization enhancement media before the dissolved product exits the chamber. In addition, this arrangement simplifies priming and removes any air bubbles from the outlet that may be introduced or generated during the process (Figure 7). A screen filter may be used at the outlet (e.g., 75 μm gasket sieve or filter in FIG. 7) and / or inlet to contain the homogenization enhancement media within the chamber. The screen filter is installed such that it is pressed against the outer body using a cap (bolted cap in FIG. 6) that is fastened using a screw or bolt. The pore size in the filter is selected such that it does not allow escape of homogenization enhancement media particles. The screen filter may be made of plastic or 316L stainless steel, or any other material suitable for cGMP manufacturing. The chamber is preferably cylindrical (FIGS. 6 and 7), but other shapes may also be used to improve mixing (e.g., right angle prism). The chamber may have features (e.g., baffles) to improve mixing and enhance shear. The outer casing of the homogenization chamber is made of plastic, but metals such as passivated 316L stainless steel may also be used to manufacture the chamber.

[0043] Non-compressible beads of various sizes and shapes made of materials with a specific gravity greater than 1.0 g / mL can be used as lysis or homogenization enhancement media. Beads are added to the homogenization chamber to enhance lysis as cells passing through the interstitial spaces between the beads are squeezed open and release intracellular material. In one embodiment, borosilicate or soda lime glass beads are used as homogenization enhancement media. In another embodiment, stainless steel (e.g., passivated 316L) beads are used. In another embodiment, ceramic beads (e.g., yttrium stabilized zirconium oxide beads) are used. The size of the beads or particles is generally 0.1-2000 microns and the shape is preferably spherical, spheroid, rod, cube, or rectangular, although other shapes can be used. In our results, a more dense medium (e.g., ceramic beads with a specific gravity of 6.0 g / cm3 compared to borosilicate glass beads with a specific gravity of 2.7 g / cm3) improved homogenization. Heavier media allowed for higher flow rates (10-50%) with similar homogenization results. The beads or particles may have smooth or rough exterior surfaces. Non-porous particles are generally preferred, although porous particles may also be used. Different types of homogenization enhancing media may be tested with the starting material to determine the optimal media for that type of starting material.

[0044] A shaft with single or multiple impellers is placed inside the chamber (Figures 1, 2, and 7). Both the shaft and impellers are constructed of rigid materials (e.g., 316L stainless steel, polycarbonate, etc.). The impellers may be of many different shapes, including circular (Figure 2B), elliptical (Figure 2C), polygonal such as rectangular (Figures 2F and 7), or cross-shaped (Figures 2D and 2E). All these shapes may be with or without holes (Figure 2A) to enhance mixing. In addition to or instead of the impeller, a mesh or irregularly shaped wire (e.g., steel wool) may also be attached to the shaft. The edges of the impeller may be sharp (e.g., blade faces) to generate high shear at the blade edges. Sharp edges are generally not required when homogenization enhancement media is used. The plane of the impeller is parallel, perpendicular, or at an intermediate angle to the shaft. In one embodiment, the shaft is attached to the impeller at or near the center of the impeller. In another embodiment, the shaft is attached to the side of the impeller. The shaft and impeller may be manufactured as one piece (Figure 7). To drive the shaft, an alignment pin (Figure 7) may be used that fits into a slot in the shaft connected to the drive motor. The base may have threads (e.g., the threaded locking base of Figure 7) or other mechanisms to lock the outer body. Combinations of different shaped impellers and shafts of different motion may be used. The maximum width of the impeller is less than the inner diameter of the chamber, and the minimum width is preferably greater than 20% of the inner diameter of the chamber.

[0045] Rotational or reciprocating motion of the shaft, or a combination of rotational and reciprocating motion, are used to shear cells with or without homogenization enhancement media. The shaft can also be vibrated to generate mixing. The motion of the shaft can be driven from above or below. The reciprocating motion can be rotational (Figure 2A) or linear (up and down, Figure 2A, Figure 2B) or both (Figure 2A). The speed of motion can be adjusted by varying the rotational speed (0-50,000 RPM), the number of reciprocating motions (0-30,000 strokes per minute), or the vibration frequency (0-30000 vibrations per second). In our results, shafts or impellers driven from above or below (to provide rotational or reciprocating motion) yielded similar results. The stroke length during reciprocating motion can also be adjusted to enhance lysis. The rotational motion can be generated by a motor coupled either directly to the shaft or through a set of gears or belts and pulleys. A radial (Figure 7) or mechanical face seal is used to prevent leakage and maintain a seal between the external environment and the inside of the chamber. Two seals may be stacked to create a cavity in which pressure can be maintained by a supply of clean air (air channel in Figure 7). This air cavity maintains a positive pressure and creates an additional air barrier between the seal and the external environment. Typically, the external air pressure is maintained at 2-7 psi; however, higher pressures may also be used, depending on the seal type. A bearing may be installed within the disposable near the seal and held in place by a retaining clip (double row bearing in Figure 7).

[0046] The reciprocating motion to the shaft may be generated by a linear reciprocating motor or similar mechanism used in reciprocating saws. The shaft protruding from the chamber is clipped or affixed to a flexible tube or flexible bellows section (Figure 3). The other end of the flexible tube or flexible bellows section is clipped or affixed to a container (Figure 3). The shaft reciprocates within the flexible tube or flexible bellows section. This configuration creates a closed system (Figure 3) and also allows for limited rotation (e.g., -180 to +180 degrees) with the reciprocating motion. If both rotational and reciprocating motions are required, the motor may be attached to the arm of a reciprocating linear motor to provide both motions simultaneously. A sealed solution and shaft may not be required if the impeller is driven by an external magnetic force (e.g., levitation or magnetic drive). A closed system not only maintains sterility, but also prevents contamination or exposure of the environment from possible toxins, pathogens, and other harmful chemicals or agents.

[0047] In one embodiment of the present application, the starting material (e.g., cell culture) is introduced into the homogenization chamber through an inlet via a pump (e.g., a peristaltic pump) (Figure 7). The shaft is rotated and / or reciprocated at a set speed to generate mixing. The pump can be stopped for a short period of time as soon as the chamber is filled to ensure homogenization of the material from the start. Another way to achieve complete homogenization of the material from the start is to start the shaft movement before the introduction of the starting material. If pre-washing is required, a priming buffer can be introduced into the system before introducing the starting material.

[0048] The homogenized processed material exits through an outlet (Figure 7) and is collected in a bag or vessel. This process continues until all starting material has been pumped through the system. To improve homogenization, the homogenized material can be passed through the system multiple times from the collection bag to the starting material bag or recirculated. Flexible tubing and pinch valves are controlled by a controller or manually to create a flow path for recirculation or multiple passes. Once all starting material has been processed, residual material in the system can be dislodged with a buffer or liquid and the complete disposable flow path, including the chamber and associated tubing, can be sealed and safely discarded.

[0049] The homogenization of the material can be optimized by varying either the impeller motion (rpm, frequency), the size and volume of the homogenization enhancing medium, the chamber geometry, the impeller type, or the flow rate of the starting material. To make process development easier, the impeller motion is set at a fixed speed and the inlet flow rate is adjusted to achieve the desired homogenization. Since the residence time increases as the flow transporting the liquid mixture slows, a lower flow rate will generally provide a higher level of homogenization compared to a higher flow rate. In some cases, a higher flow rate may result in a higher level of homogenization due to increased shear. This can be tested by varying the flow rate during process development. Our results show that a larger impeller motion results in a higher level of homogenization, but a plateau can be reached after a certain speed (Figures 5A and 5B). Our results show that the total volume of beads is directly proportional to the level of homogenization. This can be used to scale up the process. For example, a process with 125 mL of homogenization enhancing media at a flow rate of 100 mL / min will produce similar results as a process with 1.25 L of homogenization enhancing media at a flow rate of 1 L / min. Impellers that generate high shear will result in improved homogenization. Overall, increasing shear by different means will result in higher levels of homogenization.

[0050] The complete system can be operated manually, such that pump flow rates and impeller speeds are manually adjusted, or the system can be fully automated, with the user inputting parameters into a recipe screen that is connected to the controller, which receives and controls via various digital and analog inputs and outputs. These include pumps (speed, direction, start / stop, feedback, etc.), valves (open, close, feedback), blockage / pressure sensors, optical density and turbidity detectors, air bubble sensors, pH, conductivity, temperature sensors, weight load cells, accelerometers, speed, and leaks. Some of the sensors (e.g., blockage / pressure, accelerometer, leaks, microswitches) are used to improve the safety of the system. For example, if one or more of the tubes are blocked, the pressure rise detected by the blockage detector or pressure sensor will send a signal to the controller and the system will stop the pump and open the pinch valve to avoid high pressure and possible leaks. This information is provided to and from the controller as analog or digital signals. Commercially available automation systems from various vendors such as Siemens, Beckhoff, or Allen-Bradley are available as complete solutions including the controller, input / output digital or analog cards, and HMI. Examples are the Siemens SIMATIC S7-1200 kit, DeltaV automation, or the Beckhoff TwinCAT 3 automation platform.

[0051] The user inputs various process parameters into the process screens that are part of the HMI, which can be changed into the recipe that is executed each time the process is run. Process parameters include flow rates, recirculation times, pH, conductivity, turbidity, optical density, and absorbance values ​​to start or stop a step. Air bubble sensors are placed near the inlets to detect when the bioreactor or starting material container is empty.

[0052] Experiments were conducted to evaluate the effect of rotation type, speed, impeller geometry, and homogenization enhancement media. In several experiments, different types of incompressible beads (based on size distribution, shape, or material) were added to the homogenization chamber. Soda-lime glass, borosilicate glass, and ceramic beads ranged in size from 50 to 1500 microns.

[0053] A shaft with an impeller was placed inside the chamber. Combinations of differently shaped impellers and different shaft motions were used to compare the degree of lysis. Rotational or reciprocating motions of the shaft, or a combination of rotational and reciprocating motions, were tested to evaluate the amount of lysis with each combination. The speed of motion was adjusted by varying the RPM or the number of reciprocating motions. The stroke length during reciprocating motion was also adjusted to evaluate its effect on lysis performance. During the initial screen, lysis was performed in batch mode. Based on the lysis performance, the selected configuration was used to optimize the flow rate in continuous mode.

[0054] Several different configurations were tested to compare the degree of lysis and homogenization of Saccharomyces cerevisiae. After homogenization, the processed material was centrifuged at 10,000g for 3 minutes to pellet the insoluble matter. Absorbance measurements of the centrifuged supernatant at OD280 (to quantify relative intracellular protein release) and OD260 (to quantify relative intracellular DNA and RNA release) provided an effective method to quantify lysis. The amount of lysis was calculated as a percentage increase from the basal level. In addition, lysis was qualitatively verified by viewing the culture under a microscope.

[0055] The results from Figure 4A were obtained from a shaft configuration (as shown in Figure 2E) rotating at 3500 RPM with 24 impellers equidistantly spaced and mounted horizontally on a vertical shaft. The chamber shape was cylindrical with an inlet at the bottom and an outlet at the top. These results show minimal lysis (less than 20%) at all tested flow rates without homogenization enhancement media.

[0056] In another experiment (Figure 4B), a shaft fitted with a bellows mixer (Figure 2F, mounted parallel to the vertical shaft) was used in a cylindrical chamber and rotated at 200 RPM in the presence of 500 micron soda lime glass beads. These results show a 10-fold increase in the amount of protein released and near complete dissolution (as observed under a microscope) in less than 8 minutes. In a continuous flow experiment, the shaft was fitted with a bellows mixer with or without holes (Figures 6 and 7). These impellers were used in a cylindrical chamber and rotated at 500 RPM or 1000 RPM in the presence of 500 micron soda lime glass beads. After priming, the flow rate was maintained continuously at 50 mL / min. The results show that there is no significant increase in dissolution (as indicated by OD280 and OD260) with an increase in RPM above 500 (Figure 5A). A bellows mixer with or without holes was also tested. Our results from OD280 measurements (FIG. 5B) show that no significant homogenization differences existed between the two configurations at two different RPMs (500 and 1000).

[0057] To test reciprocating shaft mixing, a shaft with the configuration of FIG. 2B was placed in a cylindrical chamber and reciprocated at 2400 strokes per minute in the presence of 500 micron soda lime glass beads. The results (FIG. 4C) show nearly complete lysis in approximately one minute. The complete system was cleaned and the starting material was continuously fed into the homogenization chamber via a peristaltic pump set at flow rates of 50 and 100 mL / min. The results (FIG. 4D) show more than 10-fold lysis at a flow rate of 50 mL / min. When the homogenized sample was viewed under a microscope, the majority of the cells appeared to be lysed.

[0058] Figure 8 shows the overall flow schematic of the system. The flow rate, impeller speed or RPM, and other important parameters are input by the user through the HMI screen. V1-V5 are pinch valves and are closed at the beginning of the process. The homogenization chamber is fully assembled with the homogenization medium. Pressure or occlusion sensor PS1 is used to monitor the pressure in the system, and if the pressure exceeds the safe pressure limit (set in the HMI), the pump will stop and all valves will be in the closed state. At the beginning of the process, the starting material from the bag or vessel (e.g., cell culture, microbial culture, mixture of liquids, mixture of liquids and solids, mixture of gas and solids, mixture of gas and liquids, etc.) enters the homogenization chamber by flowing (through pump P1) through a tube that passes through air bubble sensor BS1, open pinch valve V1, optical sensor OS1, and pressure sensor PS1. The impeller in the homogenization chamber starts when liquid enters the system (when the air bubble sensor BS1 detects liquid plus the time it takes for the starting material to travel the set length of tubing at the set flow rate). The homogenized material exits through the outlet tubing via the optical sensor OS2 and open pinch valve V4 into the recirculation bag. Homogenization efficiency can be monitored by the optical sensor (e.g., measuring OD280 for changes in protein content or OD260 for changes in DNA content). This process is continuous until the air bubble sensor BS1 detects a steady air flow due to the absence of starting material in the starting material bag. At this point, the homogenized material in the recirculation bag can be used for subsequent processes. Buffers can be used to prime the system at the beginning of the process and to expel any remaining material inside the homogenization chamber at the end of the process.

[0059] There may be multiple tubing lines in the system. For example, there may be a first tubing line between the starting material container and the inlet of the homogenization chamber, a second tubing line between the recirculation container and the inlet of the homogenization chamber, a third tubing line between the outlet of the homogenization chamber and the starting material chamber, a fourth tubing line between the recirculation container and the third tubing line, and / or a fifth tubing line between the third tubing line and the first tubing line. As used herein, the term "tubing line" generally means a fluid conduit and may include one or more (flexible) tubes, fittings, manifolds, and the like. As seen in FIG. 8, components such as pumps, sensors, and valves may be present in the various tubing lines.

[0060] To improve homogenization, in addition to increasing the impeller speed or decreasing the flow rate (inlet flow rate) of pump P1, three strategies can be used: In all cases, all valves are closed at the start of the process.

[0061] In the first strategy, the starting material flows or passes through the homogenization chamber multiple times before sending it to a recirculation bag. The following steps are performed:

[0062] Step 1: A set volume of starting material (cycle volume) is drawn by pump P1 through open valve V1 and forced into the inlet of the homogenization chamber. Homogenized material leaving the chamber at the outlet re-enters the inlet through open valve V5 (valve V1 closed) until the desired cycle is completed. Valves V2, V3, and V4 are closed during this step.

[0063] Step 2: In that phase, valve V1 is open, valve V5 is closed, and valve V4 is open so that material equal to the cycle volume passes to the recirculation bag.

[0064] Step 3: Go to step 1 and the process continues until air bubble sensor BS1 detects a steady air flow indicating the end of the starting material. At this point, the homogenized material in the recirculation bag can be used for subsequent processes.

[0065] In the second strategy, the homogenized material is continuously pumped back into the starting material bag until the starting material is homogenized to the desired degree. In this case, the final material is collected in the same bag or vessel as the starting material. The starting material is drawn in by pump P1 through open valve V1 and forced into the inlet of the homogenization chamber. The material exiting the chamber at the outlet passes through open valve V3 into the starting material and mixes with it. The process is stopped when the desired homogenization is achieved. At this point, the homogenized material in the original starting material bag can be used for subsequent processes. Valves V2 and V4, if present, are closed in this strategy. Valves V2 and V4 and the recirculation vessel (and associated tubing lines) can be omitted from the system in this strategy.

[0066] In the third strategy, the following steps are implemented:

[0067] Step 1: The homogenized material is collected in a recirculation bag as in the sequence described above where recirculation is not performed.

[0068] Step 2: Once the starting material has completely exited the original bag, the homogenized material collected in the recirculation bag is pumped back to the homogenization chamber by pump P1 via open valves V2 and V3 and closed valves V1, V4, and V5. This process continues until the recirculation bag is empty (stable air at air bubble sensor BS1).

[0069] Step 3: Step 1 is repeated for the desired number of cycles. The process is stopped either after step 1 and the final processed material has been collected from the recirculation bag, or after step 2 and the final processed material has been collected from the starting material bag.

[0070] A recirculation process as described above may also be incorporated to improve homogenization efficiency.

[0071] The present invention is described herein with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. In the drawings, the relative sizes of regions or features may be exaggerated for clarity. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein, but rather these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0072] It should be understood that terms such as first, second, etc. may be used herein to describe various elements, components, regions, layers, and / or sections, and that these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another region, layer, or section. Thus, a first element, component, region, layer, or section discussed below may be named a second element, component, region, layer, or section without departing from the teachings of the present invention.

[0073] Spatially related terms such as "below," "below," "lower," "above," "above" and the like may be used herein for ease of description to describe the relationship of one element or feature to another element or feature as illustrated in the figures. It should be understood that the spatially related terms are intended to encompass different orientations of the device during use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, an element described as "below" or "below" the other element or feature would be oriented "above" the other element or feature. Thus, the term "below" can encompass both an above and below orientation. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially related descriptors used herein interpreted accordingly.

[0074] As used herein, the singular forms "a", "an" and "the" are intended to include the plural unless expressly stated otherwise. It is further understood that the terms "include", "comprise", "including", "including" and / or "comprising" as used herein specify the presence of the stated features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. When an element is referred to as being "connected" or "coupled" to another element, it is understood that it may be directly connected or coupled to the other element or that intervening elements may be present. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. When the terms "about" or "substantially equal" are used herein, the intended meaning is that the value is plus or minus 5% of the specified value.

[0075] It should be noted that any one or more aspects or features described with respect to one embodiment may be incorporated into a different embodiment, but are not specifically described in that regard. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. Applicant reserves the right to modify any original application claim, including the right to amend any original application claim to depend on and / or incorporate any feature of any other claim, even if not originally claimed in such manner, or to file any new claim accordingly. These and other objects and / or aspects of the present invention are described in detail in the specification set forth herein.

[0076] Unless otherwise specified, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. It is further understood that terms such as those defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of the relevant art, and not to be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0077] The foregoing is illustrative of the present invention and should not be construed as limiting thereof. Although several example embodiments of the present invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the example embodiments without substantially departing from the teachings and advantages of the present invention. Accordingly, all such modifications are intended to be included within the scope of the present invention as defined in the claims. The present invention is defined by the following claims, with equivalents of the claims included therein.

Claims

1. a chamber having an inlet and an outlet; at least one impeller within said chamber configured to (i) rotate or reciprocate, or (ii) rotate and reciprocate; Equipped with The homogenization device is sterile prior to use and is a single-use, disposable item.

2. The device of claim 1 , wherein the chamber contains a homogenization enhancing medium.

3. The device of claim 2 , wherein the homogenization enhancing medium comprises incompressible or solid particles.

4. The device of claim 2, wherein the homogenization enhancing medium comprises incompressible or solid particles having a size between 0.1 and 2000 microns.

5. The device of claim 2 , wherein the homogenization enhancing medium comprises non-porous particles.

6. The device of claim 2 , wherein the homogenization enhancing medium comprises porous particles.

7. The device of claim 2 , wherein the homogenization enhancing medium comprises particles having a rough surface.

8. The device of claim 2 , wherein the homogenization enhancing medium comprises particles having smooth surfaces.

9. The device of claim 2 , wherein the homogenization enhancing medium comprises glass or metal particles.

10. The device of claim 1 , wherein the chamber is made of plastic.

11. The device of claim 1 , wherein the chamber is made of metal.

12. The device of claim 1 , wherein the at least one impeller is connected to a shaft, the shaft being configured to oscillate.

13. The device of claim 12 , wherein the impeller and shaft are made from plastic.

14. The device of claim 12 , wherein the impeller and shaft are made from metal.

15. 10. The device of claim 1, wherein all product contact surfaces are discarded after processing a single batch.

16. The device of claim 1 , wherein the device is sterilized and packaged prior to its use.

17. The device of claim 1 , wherein the device is configured to homogenize a starting material.

18. The device of claim 17 , wherein the starting material is a cell culture.

19. The device of claim 17 , wherein the starting material is a microbial culture.

20. The device of claim 17 , wherein the starting material is organic.

21. The device of claim 17 , wherein the starting material is inorganic.

22. The device of claim 1 , wherein the device is configured to lyse a cell.

23. The device of claim 1 , wherein the device is configured to mix materials.

24. The device of claim 1 , wherein the device is configured to reduce a size of the starting material.

25. The device of claim 1 , wherein the chamber comprises a body defining an internal cavity, the inlet being in a lower portion of the internal cavity and the outlet being in an upper portion of the internal cavity.

26. 26. The device of claim 25, further comprising a filter between the outlet and the internal cavity.

27. 26. The device of claim 25, further comprising a shaft connected to the at least one impeller.

28. 30. The device of claim 27, wherein the at least one impeller is single-blade connected to the shaft.

29. 30. The device of claim 28, wherein a plurality of holes are defined in the single-blade impeller.

30. The device of claim 27 , wherein the shaft extends from a top portion of the chamber body.

31. 1. A system for homogenization of a starting material, comprising: A starting material container containing the starting material; a homogenization device comprising a chamber including an inlet and an outlet, and an impeller configured for rotational and / or reciprocating movement within an interior volume of the chamber; a first tubing line between the starting material container and the inlet of the chamber; a pump (P1) in said first tubing line; a first valve (V1) in the first tubing line; A recirculation vessel; a second tubing line between the recirculation vessel and the first tubing line, the second tubing line fluidly connecting the recirculation vessel and the inlet of the chamber; a second valve (V2) in the second tubing line; a third tubing line between the outlet of the chamber and the starting material container; a third valve (V3) in the third tubing line; a fourth piping line between the recirculation vessel and the third piping line; a fourth valve (V4) in the fourth tubing line; a fifth tubing line between the third tubing line and the first tubing line; a fifth valve (V5) in the fifth piping line; a controller configured to command the first through fifth valves to selectively open and close, to command operation of the impeller, and to command the pump to turn on and off; A system comprising:

32. The controller includes: (a) opening the first valve and turning on the pump so that a cycle volume of starting material flows from the starting material container to the inlet and into the homogenization chamber; (b) closing the first valve and opening the fifth valve such that the cycle volume flows from the outlet to the inlet and into the homogenization chamber multiple times; (c) closing the fifth valve and opening the fourth valve such that the cycle volume is received in the recirculation vessel; (d) repeating steps (a)-(c) until the starting material in said starting material container is depleted; 32. The system of claim 31 configured to:

33. 33. The system of claim 32, further comprising an air bubble sensor in the first tubing line, the controller being configured to determine when starting material in the starting material container is depleted based on measurements by the air bubble sensor.

34. The controller includes: (a) opening the first valve and turning on the pump so that starting material flows from the starting material container to the inlet and into the homogenization chamber; (b) opening the third valve so that the homogenized starting material flows from the outlet to the starting material container; (c) keeping the first and third valves open and the pump on until a desired homogenization is achieved based on measurements by optical sensors in the first tubing line and / or the third tubing line; 32. The system of claim 31 configured to:

35. The controller includes: (a) opening the first valve, closing the second valve, closing the third valve, closing the fifth valve, opening the fourth valve, and turning on the pump such that the starting material flows from the starting material container through the homogenization device to the recirculation container until the starting material in the starting material container is depleted; (b) closing the first valve, closing the fourth valve, and opening the second valve and the third valve so that the starting material flows from the recirculation vessel through the homogenization device to the starting material vessel until the starting material in the recirculation vessel is depleted; (c) repeating step (a) and / or step (b) a number of times until a desired homogenization is achieved based on measurements by optical sensors in the first tube line and / or the third tube line.

32. The system of claim 31 configured to:

36. 36. The system of claim 35, further comprising an air bubble sensor in the first tubing line, the controller being configured to determine that starting material in the starting material container and the recirculation container is depleted based on measurements by the air bubble sensor.

37. 32. The system of claim 31 , wherein the first through fifth tubing lines, the first through fifth valves, the air bubble sensor in the first tubing line (if provided), the pressure sensor in the first tubing line (if provided), the optical sensor in the first tubing line (if provided), the optical sensor in the third tubing line (if provided), the homogenization device, the starting material container, the recirculation container, and / or the pump are provided as a kit and are single-use disposable items.

38. 1. A system for homogenization of a starting material, comprising: A starting material container containing the starting material; a homogenization device comprising a chamber including an inlet and an outlet, and an impeller configured for rotational and / or reciprocating movement within an interior volume of the chamber; a first tubing line between the starting material container and the inlet of the chamber; a pump (P1) in said first tubing line; a first valve (V1) in the first tubing line; a second tubing line between the outlet of the chamber and the starting material container; a second valve (V3) in the second tubing line; at least one optical sensor in the first tubing line and / or the second tubing line; Controller and The controller comprises: (a) opening the first valve, turning on the pump, and rotating and / or reciprocating the impeller such that starting material flows from the starting material container to the inlet, into the homogenization chamber, and is at least partially homogenized; (b) opening the second valve so that the at least partially homogenized starting material flows from the outlet to the starting material container; (c) keeping the first and second valves open and the pump on until a desired homogenization is achieved based on measurements by the optical sensors in the first tubing line and / or the second tubing line; A system that is configured to:

39. 1. A method for homogenizing organic and inorganic materials, comprising: flowing the starting material from a starting material container through a homogenization device multiple times to homogenize the starting material; collecting the homogenized material in a container; The method includes:

40. The flowing step includes: (a) pumping a cycle volume of the starting material from the starting material container through a first tubing line to an inlet of the homogenization device; (b) closing a first valve (V1) in the first tubing line and opening a second valve (V5) in the second tubing line; (c) operating the homogenization device to homogenize the cycle volume while flowing the cycle volume from an outlet of the homogenization device through the second and first tubing lines to the inlet and through the homogenization device a plurality of times; (d) closing the second valve (V5) and opening a third valve (V4) in a third piping line so that the homogenized cycle volume enters a recirculation vessel; (e) repeating steps (a)-(d) a plurality of times until determining that the starting material container is depleted; 40. The method of claim 39, comprising:

41. 41. The method of claim 40, comprising using an air bubble sensor in the first tubing line to determine when the starting material container is depleted.

42. The flowing step includes: (a) pumping the starting material from the starting material container through a first tubing line to an inlet of the homogenization device; (b) flowing the starting material through the homogenization device while operating the homogenization device to at least partially homogenize the starting material; (c) flowing the at least partially homogenized starting material from an outlet of the homogenization device through a second tubing line to the starting material container to mix the at least partially homogenized starting material with the starting material; (d) repeating steps (a)-(c) until the desired homogenization is achieved.

40. The method of claim 39, comprising:

43. 43. The method of claim 42, further comprising the step of using at least one optical sensor (OS1, OS2) in the first tube line and / or the second tube line to determine when the desired homogenization is achieved.

44. The flowing step includes: (a) opening a first valve (V1) in a first tubing line, opening a second valve (V4) in a second tubing line, closing a third valve (V5) in a third tubing line, closing a fourth valve (V2) in a fourth tubing line, and closing a fifth valve (V3) in a fifth tubing line; (b) pumping the starting material from the starting material container through the first tubing line to an inlet of the homogenization device; (c) flowing the starting material through the homogenization device and operating the homogenization device to at least partially homogenize the starting material; (d) flowing the at least partially homogenized starting material from an outlet of the homogenization device through a sixth tubing line and the second tubing line to a recirculation vessel until determining that the starting material vessel is depleted; (e) closing the first valve (V1) in the first tubing line, closing the second valve (V4) in the second tubing line, closing the third valve (V5) in the third tubing line, opening the fourth valve (V2) in the fourth tubing line, and opening the fifth valve (V3) in the fifth tubing line; (f) pumping the at least partially homogenized starting material from the recirculation vessel through the fourth piping line to the inlet of the homogenization device; (g) flowing the at least partially homogenized starting material through the homogenization device and operating the homogenization device to further homogenize the starting material; (h) flowing the further homogenized starting material from the outlet of the homogenization device through the sixth tubing line and the fifth tubing line to the starting material container until determining that the recirculation container is exhausted; (i) repeating steps (a) through (h) while either stopping collecting homogenized material in said recirculation vessel in step (d) or stopping collecting homogenized material in said starting material vessel in step (h) until a desired homogenization is achieved; 40. The method of claim 39, comprising:

45. 45. The method of claim 44, including using an air bubble sensor in the first tubing line to determine when the starting material container or the recirculation container is depleted.

46. 45. The method of claim 44, further comprising the step of using at least one optical sensor (OS1, OS2) in the first tube line and / or the sixth tube line to determine when the desired homogenization is achieved.

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