Density gradient formation using sample particles

Abstract

A system for supplying a density gradient into a container is described. The system pumps a volume of sample particles and a density modifier into a mixing chamber that is in fluid communication with the proximal end of the probe. The mixing chamber mixes the sample particles and the density modifier together to form at least a partial density gradient. The system supplies the density gradient into the container through the distal end of the probe. The density gradient has a variation in density between the first and second ends, and at least a portion of the density gradient between the first and second ends includes the supplied volume of sample particles.

Description

Background Art

[0001] This application was filed as a PCT international application on February 13, 2024, claiming the benefit of priority to U.S. Provisional Patent Application No. 63 / 485,136, filed on February 15, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Particles including virus vectors such as adenovirus and adeno-associated virus (AAV), extracellular vesicles such as exosomes, and nucleic acids such as plasmid DNA can have various cell functions, structures, and mechanisms of action. For example, AAV can have different loading amounts, which makes AAVs different not only in molecular weight but also in density. In some cases, these types of particles can be separated using density gradients.

[0003] Typically, to separate particles using a density gradient, a sample of the particles is loaded into a container together with a density-modifying material to form a homogeneous solution having a uniform density. Then, centrifugation is performed on the container to form a density gradient. Finally, the particles move through the density gradient until they reach a density equal to their own. In addition to the time required for the particles to move through the density gradient until they reach a position matching their density, the time typically required to form the density gradient by centrifugation can take a lot of time to complete.

Summary of the Invention

Means for Solving the Problems

[0004] In general terms, this disclosure relates to separating particles by using a density gradient. In one possible configuration, the density gradient is formed without the use of centrifugation by automatically supplying the density gradient along with the supplied volume of sample particles to be integrated into it. In another possible configuration, the supplied volume of sample particles is integrated within the range of the density gradient. Various aspects are described in this disclosure, and this includes, but is not limited to, the following aspects:

[0005] One aspect relates to a system for supplying a density gradient into a container, the system comprising a processing network having a memory for storing instructions, the instructions which, when executed by the processing network, cause the processing network to pump sample particles and a density modifier into a mixing chamber that is in fluid communication with the proximal end of a probe, the mixing chamber to mix the sample particles and the density modifier together, and to supply a density gradient into the container through the distal end of the probe, the density gradient having a density variation between a first end and a second end, and at least a portion of the density gradient between the first end and the second end including the supplied volume of sample particles.

[0006] Another aspect relates to a method for supplying a density gradient into a container, the method comprising: pumping sample particles and a density modifier into a mixing chamber which is in fluid communication with the proximal end of a probe, the mixing chamber which mixes the sample particles and the density modifier together; and supplying a density gradient into the container through the distal end of the probe, the density gradient which varies between a first end and a second end, and at least a portion of the density gradient between the first end and the second end which includes the supplied volume of sample particles.

[0007] Various additional aspects will be described in the following explanation. These aspects may relate to individual features and combinations of features. Both the above general explanation and the following detailed explanation are illustrative and descriptive only and do not limit the broader concept of the invention. It should be understood that the embodiments disclosed herein are based on the broader concept of the invention. [Brief explanation of the drawing]

[0008] The following drawings, which form part of this application, illustrate the described art and are not intended to limit the scope of this disclosure in any way.

[0009] [Figure 1] Figure 1 schematically illustrates one embodiment of a system for generating a density gradient for centrifugal separation.

[0010] [Figure 2] Figure 2 illustrates an embodiment of the system in Figure 1 in which a mixer is housed inside the manifold and mixing chamber.

[0011] [Figure 3] Figure 3 is an isometric view of the probe, which has a distal end that is inserted into the container of the system in Figure 1 and a proximal end that is connected to the manifold and mixing chamber.

[0012] [Figure 4] Figure 4 illustrates an embodiment of the system in Figure 1 with the proximal end of the probe connected to the manifold and mixing chamber.

[0013] [Figure 5] Figure 5 schematically illustrates one embodiment of a method for generating a density gradient to separate particles, which can be carried out by the system in Figure 1.

[0014] [Figure 6]Figure 6 graphically illustrates an embodiment in which the supplied volume for each component along the radial length of the density gradient supplied by the system of Figure 1 is operated according to the method of Figure 5.

[0015] [Figure 7] Figure 7 schematically illustrates another embodiment of a method for generating a density gradient to separate particles, which can be carried out by the system in Figure 1.

[0016] [Figure 8] Figure 8 graphically illustrates another embodiment of the supplied volume for each component along the radial length of the density gradient supplied by the system of Figure 1, according to the operation of the method of Figure 7.

[0017] [Figure 9] Figure 9 schematically illustrates one embodiment of the computing component hardware of the system shown in Figure 1. [Modes for carrying out the invention]

[0018] Detailed explanation Figure 1 schematically illustrates one embodiment of a system 100 capable of generating density gradients for centrifugal separation. System 100 is a computer controlled to precisely supply gradients of any type, slope, or shape to the inside of a container 110. For example, system 100 can generate a linear density gradient having a density that increases progressively from top to bottom, and a stepped density gradient having at least two discrete steps of different densities.

[0019] In some embodiments, system 100 includes features similar to those in the systems described in U.S. Provisional Patent Application No. 63 / 369,306, filed July 25, 2022, titled "AUTOMATIC DISPENSE OF DENSITY GRADIENTS", U.S. Provisional Patent Application No. 63 / 369,299, filed July 25, 2022, titled "NON-DESTRUCTIVE MEASUREMENT OF DENSITY GRADIENTS", and U.S. Provisional Patent Application No. 63 / 369,315, filed July 25, 2022, titled "REPLICATION OF DENSITY GRADIENTS", the disclosures of which are incorporated herein in their entirety by reference.

[0020] System 100 includes a reservoir 102, and each reservoir 102 holds a separate component for generating a density gradient inside a container 110. Each reservoir 102 is connected to a pump 104 for pumping the components held within the reservoir 102 into a manifold and mixing chamber 106. The pump 104 is programmed to pump components from the reservoir 102 at a predetermined volume and rate for mixing inside the manifold and mixing chamber 106.

[0021] In the embodiment shown in FIG. 1, system 100 includes four reservoirs such as a first reservoir 102a connected to a first pump 104a for pumping a first component into the manifold and mixing chamber 106, a second reservoir 102b connected to a second pump 104b for pumping a second component into the manifold and mixing chamber 106, a third reservoir 102c connected to a third pump 104c for pumping a third component into the manifold and mixing chamber 106, and a fourth reservoir 102d connected to a fourth pump 104d for pumping a fourth component into the manifold and mixing chamber 106. System 100 can include more than four reservoirs for holding more than four distinct components for generating a density gradient, or can include less than four reservoirs for holding less than four distinct components for generating a density gradient within container 110.

[0022] The components held within reservoir 102 are liquids that are pumped into the manifold and mixing chamber 106 for supplying a fluid flow within container 110. As an illustrative example, the first reservoir 102a can hold deionized (DI) water, the second reservoir 102b can hold a density modifier, the third reservoir 102c can hold a buffer solution, and the fourth reservoir 102d can hold sample particles. As an illustrative example, the sample particles can include viral vectors such as lentivirus, adenovirus, and adeno-associated virus (AAV), lipid nanoparticles carrying mRNA, extracellular vesicles such as exosomes, nucleic acids such as plasmid DNA, and other types of biological or synthetic nanoparticles.

[0023] All four components held in reservoirs 102a and 102d can be introduced into a single flow that proceeds through the manifold and mixing chamber 106. For example, DI water can be pumped from the first reservoir 102a into the manifold and mixing chamber 106 by the first pump 104a, the density modifier can be pumped from the second reservoir 102b into the manifold and mixing chamber 106 by the second pump 104b, the buffer solution can be pumped from the third reservoir 102c into the manifold and mixing chamber 106 by the third pump 104c, and the sample particles can be pumped from the fourth reservoir 102d into the manifold and mixing chamber 106 by the fourth pump 104d. Thus, various combinations of contents held in reservoirs 102a and 102d can be pumped into the manifold and mixing chamber 106.

[0024] In some embodiments, pumps 104a-104d include peristaltic pumps to provide a smooth pumped flow. In some further embodiments, pumps 104a-104d include syringe pumps which may be used when higher precision pumping is desired.

[0025] Figure 2 illustrates an embodiment of a mixer 200 housed inside the manifold and mixing chamber 106 of system 100. Referring here to Figures 1 and 2, various combinations of DI water, density modifier, buffer solution, and sample particles are introduced into a single flow that proceeds through the manifold and mixing chamber 106. Inside the manifold and mixing chamber 106, the mixer 200 includes mixing elements 202a and 202f, which mix the components together as the components pass through the mixing elements. The mixer 200 mixes the components together and, based on the relative concentrations of the components mixed by the mixer 200, generates a homogeneous flow of fluid for the probe 108 to supply a density gradient with spatial variations in density over the radial length of the container 110.

[0026] In some embodiments, the mixer 200 is a static mixer, and the mixing elements 202a-202f include staggered helical elements. In alternative embodiments, the manifold and mixing chamber 106 may include an alternative type of mixer, including a non-static mixer, and mixing elements.

[0027] In the embodiment provided in Figure 2, each helical element is set at 90° to adjacent helical elements to provide thorough mixing of the components over the length L of the mixer 200 inside the manifold and mixing chamber 106. Mixing elements 202a-202f mix the components together, eliminating pockets of low-density and / or high-density material. Mixing elements 202a-202f slice and rotate the components together multiple times, creating a substantially homogeneous flow for the probe 108 to supply a density gradient into the container 110. In an illustrative embodiment, the mixer 200 may include 12 mixing elements, the 12 mixing elements having an outer diameter OD of about 2.3 mm to about 2.4 mm, a total length L of about 27 mm to about 29 mm, and an individual missing element length-to-diameter ratio of about 1.

[0028] Mixing chamber parameters such as flow rate, outer diameter OD, total length L, or the number of mixing elements on the mixer 200 are selected to avoid shear damage to the sample particles. In some embodiments, different mixers can be used interchangeably within the manifold and mixing chamber 106 based on the type of sample particles being mixed by the mixer 200. For example, different mixers having different sizes and / or designs and / or materials can be used interchangeably within the manifold and mixing chamber 106 to maximize mixing while minimizing shear / sample damage to sensitive sample particles such as lentiviruses.

[0029] Figure 3 is an isometric view of the probe 108, which has a distal end 112 inserted into the container 110 and a proximal end 114 that is in fluid communication with the manifold and mixing chamber 106. In some embodiments, the proximal end 114 of the probe 108 is directly connected to the manifold and mixing chamber 106. Alternatively, the proximal end 114 of the probe 108 can be indirectly connected to the manifold and mixing chamber 106 via tubing.

[0030] As shown in Figure 3, the distal end 112 is positioned toward the bottom of the internal volume 122 of the container 110 so that the probe 108 can supply a density gradient into the internal volume of the container. In embodiments where system 100 uses an underlay process to supply the density gradient, the probe 108 remains fixed in the same position while the density of the homogeneous flow supplied through the probe 108 gradually increases. Alternatively, in embodiments where system 100 uses an overlay process to supply the density gradient, the probe 108 can move upward while the density of the homogeneous flow supplied through the probe 108 gradually increases.

[0031] As shown in Figure 3, the container 110 is fixed and positioned relative to the probe 108 by a retainer 116 during the supply of the density gradient. In the embodiment of Figure 3, the retainer 116 includes a clamp for securely fastening the container 110 to the frame 118 of the system 100.

[0032] Figure 4 illustrates an embodiment with a proximal end 114 of a probe 108 connected to a manifold and mixing chamber 106. The manifold and mixing chamber 106 includes a manifold section 402 having inlet sections 404a and 404b, which respectively receive components pumped from reservoirs 102a and 102d by pumps 104a and 104d. The manifold and mixing chamber 106 further includes a mixing section 406, which houses a mixer 200 for mixing the components pumped from the reservoirs together before they reach the proximal end 114 of the probe 108.

[0033] The proximal end 114 of the probe 108 is secured by a set screw 408, which can be tightened or loosened around the proximal end 114 of the probe 108. The manifold and mixing chamber 106 are mounted on a motor-driven mechanism that precisely moves the probe 108 up and down to a desired position inside the container 110. In some embodiments, the probe 108 can be manually lowered to a desired position inside the container 110.

[0034] In some embodiments, the probe 108 includes a coating of a non-stick material. In some embodiments, the coating includes Teflon® and / or a similar type of material. In some embodiments, the coating is hydrophobic and / or non-wetting. The coating on the probe 108 prevents the density gradient supplied into the container 110 from sticking to or accumulating on the probe 108. This allows the probe 108 to be removed from the container 110 without unintentionally mixing any portion of the density gradient. The coating can also prevent the adsorption of sample particles and other components supplied through the probe 108 onto the probe 108.

[0035] In addition, the manifold and mixing chamber 106 and the probe 108 can be sterilized after each use of the system 100. Furthermore, the manifold and mixing chamber 106 and the probe 108 are free of endotoxins to protect the integrity of the sample particles and all components supplied through the manifold and mixing chamber 106 and the probe 108.

[0036] Referring to Figure 1, the system 100 may include a control panel 130 for receiving user input to generate a desired density gradient. In some embodiments, the control panel 130 may include a user interface 132, such as a touchscreen display, which may be used by the user to generate a desired density gradient, measure it, and store a profile of the density gradient. In further embodiments, the user interface 132 may include additional input devices, such as one or more physical buttons, which may be selected to control the operation of the system 100.

[0037] The separation of sample particles can be carried out by equilibrium zone centrifugation, which typically involves layering a sample of particles above a density gradient and then using centrifugal force to move the particles at different rates depending on their mass. As the particles move downward through the density gradient, zones containing particles of similar size are formed, with particles that settle more quickly moving ahead of those that settle more slowly. The zones into which the sample particles are layered limit the volume of sample that can be contained by the density gradient. In addition, the centrifugation time for such a technique is generally long, due to the time required for the particles of interest to settle within the density gradient zone and the additional time required to form the density gradient.

[0038] Alternative approaches for separating sample particles may include isodensity-density gradient ultracentrifugation (DGUC), which typically involves combining the sample particles with a density-forming material (such as cesium chloride (CsCl), potassium bromide, iodixanol, Nycodenz(R), or equivalent) to produce a homogeneous solution of a defined density.

[0039] The homogeneous solution is then loaded into a container for centrifugation, where centrifugal force creates a density gradient. After the density gradient is formed, the sample particles in the sample move along the density gradient to a position where the suspension density of the particles matches the density of the surrounding medium, so that the sample particles reach a stable equilibrium state. As used herein, a stable equilibrium state means that the sample particles are sufficiently separated to a point where they can be isolated for extraction, even if the sample particles do not become completely still.

[0040] The time required to form a density gradient depends primarily on the g-force applied during centrifugation, which is a significant limiting factor for large-volume workflows because the g-force decreases with increasing volume. For example, increasing the volume of the density gradient for large-scale workflows can reduce throughput because, due to lower centrifugation rates, more time is required to form the density gradient.

[0041] When separating sample particles using the DGUC technique, additional factors can also contribute to a decrease in throughput. For example, temperature can affect the time required to form a density gradient. This is because using lower temperatures to protect the integrity of the sample particles can add time to form the density gradient because the diffusivity is slowed. Also, the type of density modifier used, in particular the molecular weight and / or diffusivity and viscosity of the density modifier, can affect the time required to form a density gradient.

[0042] Figure 5 schematically illustrates one embodiment of method 500 for generating a density gradient to separate sample particles. System 100 can implement method 500 to reach equilibrium and significantly reduce the centrifugation time for separating particles with high resolution. Method 500 is advantageous for workflows with a large number of particles. Method 500 is also advantageous for small-scale workflows, as centrifugation is performed at a lower speed without significantly increasing the time required to reach a stable equilibrium, and small-scale workflows are implemented to separate particles that are unstable under high g forces.

[0043] As shown in Figure 5, method 500 includes an action 502 to lower the distal end 112 of the probe 108 near the bottom of the internal volume 122 of the container 110. An embodiment of this arrangement is shown in Figures 1 and 3.

[0044] Method 500 includes an operation 504 to supply a density gradient into container 110. The density gradient is supplied as a homogeneous mixture of a density modifier pumped from a second reservoir 102b, a buffer solution pumped from a third reservoir 102c, and sample particles pumped from a fourth reservoir 102d, so that sample particles are supplied directly into the density gradient. This differs from equilibrium-area centrifugation and isodense DGUC described above.

[0045] In some embodiments, operation 504 includes performing an underlay process, in which the distal end 112 of the probe 108 remains positioned near the bottom of the internal volume 122 of the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually increases. Alternatively, operation 504 may include performing an overlay process, in which the distal end 112 of the probe 108 moves upward within the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually decreases.

[0046] In some embodiments, the density gradient supplied in operation 504 is a continuous gradient in which the density gradually decreases as it moves along the radial length of the container 110. In such cases, the continuous gradient can be either linear or logarithmic. In alternative embodiments, the density gradient supplied in operation 504 is a stepped gradient with a defined interface between different layers having different densities.

[0047] Each position along the radial length of the density gradient has a density based on the relative concentration of the component pumped from the reservoir 102. For example, increasing the amount of density modifier mixed by the manifold and mixing chamber 106 increases the density of a particular portion of the density gradient, while decreasing the amount of density modifier mixed by the manifold and mixing chamber 106 decreases the density of a particular portion of the density gradient.

[0048] Next, method 500 includes an operation 506 to determine whether the density gradient is complete. If the density gradient is not complete (i.e., "No" in operation 506), method 500 continues to supply the density gradient in operation 504. If the density gradient is complete (i.e., "Yes" in operation 506), method 500 can proceed, in at least some embodiments, to an operation 508 to supply a volume having a density lighter than the lightest density of the density gradient on top of the density gradient.

[0049] In some embodiments, the volume supplied to the upper part of the density gradient mainly consists of DI water. Operation 508 can be performed, in particular, to eliminate any air pockets inside the sealed tube that remain when container 110 is a sealed tube by removing probe 108. Otherwise, air pockets can cause weakening within the sealed tube during centrifugation, especially at high g forces. Also, by supplying the upper volume mainly consisting of DI water, probe 108 is cleaned and ready to supply a second density gradient into another container. In other embodiments, such as when container 110 is an open upper tube, operation 508 is optional.

[0050] Next, method 500 includes an operation 510 to remove the probe 108 from the container 110. Operation 510 may include slowly removing the probe 108 so as not to disturb the density gradient. As discussed above, the probe 108 may include a coating to prevent the density gradient from sticking to the probe 108 during its removal.

[0051] Next, Method 500 includes an operation 512 to place the container 110 inside a centrifuge for centrifugation so that the sample particles are separated within a density gradient generated by Method 500. In some embodiments, operation 512 may include a user manually placing the container 110 inside the centrifuge and, optionally, allowing the user to operate the centrifuge and perform centrifugation. In further embodiments, operation 512 is automated. For example, a mechanical actuator such as a robotic arm may be used to automatically place the container 110 inside the centrifuge, and the centrifuge then automatically performs centrifugation of the container 110. In some cases, ultracentrifugation is performed to analyze the sample particles as they are separated within a density gradient formed by Method 500.

[0052] In method 500, pumps 104a and 104d are programmed to control the flow of each liquid component into the manifold and mixing chamber 106, respectively, so as to have a given volume to be supplied to generate a density gradient with spatial density variation over the radial length of container 110. This allows system 100 to precisely control the concentration of each liquid component in each portion of the density gradient supplied by probe 108.

[0053] Figure 6 graphically illustrates an embodiment of the supplied volume (Y-axis) for each component along the radial length (Y-axis) of the density gradient 600 supplied by the system 100, according to the operation of Method 500. In this embodiment, the density gradient 600 is a continuous gradient. Each portion along the radial length of the density gradient 600 includes a combination of density modifier pumped from the second reservoir 102b, a buffer solution pumped from the third reservoir 102c, and sample particles pumped from the fourth reservoir 102d, so that the sample particles are directly supplied into the density gradient 600. Thus, in this embodiment, the density gradient 600 includes the supplied volume of sample particles along the entire radial length of the density gradient 600.

[0054] As shown in Figure 6, the upper portion of the density gradient 600 (i.e., the left side of Figure 6) contains the minimum supply volume of density modifier so that it has the lowest density within the density gradient 600, and the lower portion of the density gradient 600 (i.e., the right side of Figure 6) contains the maximum supply volume of density modifier so that it has the highest density within the density gradient 600, and thus the density of the density gradient 600 increases progressively. In some embodiments, the density gradient 600 has a density range of about 1.0 g / mL to 1.8 g / mL. This range can accommodate viral vectors whose densities may range from about 1.3 g / mL to 1.5 g / mL, such as those for adenoviruses and adeno-associated viruses (AAVs).

[0055] In the illustrative embodiment shown in Figure 6, the density gradient 600 has a total volume of approximately 39 mL, containing approximately 21 mL of sample particles. A larger volume of sample particles can be introduced into the density gradient 600 by reducing the slope of the supply volume of the density modifier component, or by using a higher concentration stock solution of the density modifier.

[0056] Since the density gradient is formed during operation 504 in method 500, the centrifugation time typically required to form the density gradient is significantly reduced, or even eliminated, which shortens the overall time for separating the sample particles. For example, when starting with a homogeneous solution of the density gradient material to be mixed with the sample particles (e.g., isodense DGUC), two equilibrium states are reached at different times; namely, the first equilibrium state is reached when the density gradient is stable, followed by the second equilibrium state when the movement of the sample particles is stable along the radial length of the density gradient. The sample equilibrium state is not fully reached when the density gradient is first formed. Instead, it may take several more hours to reach the sample equilibrium state.

[0057] Method 500 eliminates the first step in this two-step process, as centrifugation for forming a density gradient is significantly reduced or even eliminated, and centrifugation is performed only over the second step (e.g., the sample equilibrium state). Method 500 allows sample particles to move to their respective equilibrium positions without the need to first form a density gradient. An isodense DGUC process for separating sample particles, which takes about 20 hours, can be shortened to less than 5 hours by Method 500.

[0058] Feeding particles from a sample within a continuous density gradient is not easily achievable using conventional density gradient formation techniques, which are primarily manual processes. This is because it would be impossible to control sample introduction and density gradient formation with the required level of precision manually.

[0059] Figure 7 schematically illustrates another embodiment of Method 700 for generating a density gradient to separate sample particles. Method 700 can be performed by System 100 to further reduce the centrifugation time for separating particles. Method 700 is particularly advantageous for early development, analytical, and other small-volume workflows where rapid time is critical for various samples.

[0060] Method 700 includes an action 702 to lower the distal end 112 of the probe 108 near the bottom of the internal volume 122 of the container 110. Action 702 is substantially similar to action 502 in Method 500 described above.

[0061] Next, method 700 includes operation 704 of supplying a first portion of the density gradient into container 110. In operation 704, the first portion includes a homogeneous mixture of DI water pumped from a first reservoir 102a, a density modifier pumped from a second reservoir 102b, and a buffer solution pumped from a third reservoir 102c. Sample particles are not supplied into the first portion of the density gradient.

[0062] In some embodiments, operation 704 includes performing an underlay process, in which the distal end 112 of the probe 108 remains positioned near the bottom of the internal volume 122 of the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually increases. Alternatively, operation 704 may include performing an overlay process, in which the distal end 112 of the probe 108 moves upward within the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually decreases.

[0063] Figure 8 graphically illustrates the exemplary supplied volumes of each component within each portion of the density gradient 800 supplied by the system 100 according to the operation of method 700. As shown in the embodiment provided in Figure 8, the first portion 802 of the density gradient 800 includes a combination of DI water, a density modifier, and a buffer solution. The first portion 802 does not contain sample particles, which have a supplied volume of 0. In the first portion 802, the supplied volume of DI water gradually decreases while the supplied volume of the density modifier gradually increases, which progressively increases the density of the first portion 802 of the density gradient 800. This illustrates an underlay process.

[0064] In the embodiment provided in Figure 8, the density gradient 800 is a continuous gradient in which the density gradually decreases as it moves along the radial length of the container 110. In an alternative embodiment, the density gradient 800 supplied by method 700 may be a stepped gradient with distinct interfaces between different portions having different densities.

[0065] Referring to Figure 7, method 700 then includes an operation 706 to determine whether the first portion of the density gradient is complete. If the first portion of the density gradient is not complete (i.e., "No" in operation 706), method 700 continues to supply the first portion of the density gradient in operation 704. If the first portion of the density gradient is complete (i.e., "Yes" in operation 706), method 700 proceeds to operation 708 to supply the second portion of the density gradient into container 110.

[0066] In operation 708, the second portion includes a homogeneous mixture of sample particles pumped from the fourth reservoir 102d, a density modifier pumped from the second reservoir 102b, and a buffer solution pumped from the third reservoir 102c. In some embodiments, operation 708 includes performing an underlay process, in which the distal end 112 of the probe 108 remains positioned near the bottom of the internal volume 122 of the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually increases. Alternatively, operation 708 may include performing an overlay process, in which the distal end 112 of the probe 108 moves upward within the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually decreases.

[0067] As shown in the embodiment provided in Figure 8, the second portion 804 of the density gradient 800 includes a combination of sample particles, a density modifier, and a buffer solution. In this embodiment, the second portion 804 does not include a supplied volume of DI water, and the supplied volume of DI water is 0. Instead, the supplied volume of sample particles replaces the supplied volume of DI water in the second portion 804. In an alternative embodiment, the second portion 804 may include a supplied volume of DI water, in which case the sample particles in the second portion 804 would have a supplied volume less than the supplied volume illustrated in the embodiment of Figure 8 in order to accommodate the progressively increasing density of the second portion 804.

[0068] In the second section 804, the volume of sample particles supplied gradually decreases while the volume of density modifier supplied gradually increases, thereby gradually increasing the density of the second section 804. This represents an underlay process. As shown in Figure 8, the sample particles are supplied directly into the second section of the density gradient, which differs from equilibrium-region centrifugation and isodense DGUC.

[0069] Referring to Figure 7, method 700 then includes an operation 710 to determine whether the second portion of the density gradient is complete. If the second portion is not complete (i.e., "No" in operation 710), method 700 continues to supply the second portion of the density gradient in operation 708. If the second portion of the density gradient is complete (i.e., "Yes" in operation 710), method 700 proceeds to operation 712 to supply the third portion of the density gradient into container 110.

[0070] In operation 712, the third portion includes a homogeneous mixture of DI water pumped from the first reservoir 102a, a density modifier pumped from the second reservoir 102b, and a buffer solution pumped from the third reservoir 102c. Sample particles are not supplied into the third portion of the density gradient.

[0071] In some embodiments, operation 712 includes performing an underlay process, in which the distal end 112 of the probe 108 remains positioned near the bottom of the internal volume 122 of the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually increases. Alternatively, operation 712 may include performing an overlay process, in which the distal end 112 of the probe 108 moves upward in the container 110 while the density of the homogeneous flow supplied through the probe 108 gradually decreases.

[0072] As shown in Figure 8, the third portion 806 within the density gradient 800 contains a combination of DI water, a density modifier, and a buffer solution. The third portion 806 does not contain sample particles, which have a supplied volume of 0. In the third portion 806, the supplied volume of DI water gradually decreases while the supplied volume of the density modifier gradually increases, which progressively increases the density of the third portion 806 of the density gradient 800. This illustrates an underlay process.

[0073] Method 700 includes an operation 714 to determine whether a third portion of the density gradient is complete. If the third portion is not complete (i.e., “No” in operation 714), Method 700 continues to supply the third portion of the density gradient in operation 712. If the third portion of the density gradient is complete (i.e., “Yes” in operation 714), Method 700 may proceed to an operation 716 to supply the upper volume, followed by an operation 718 to remove the probe 108 from the container 110, followed by an operation 720 to place the container 110 inside a centrifuge for centrifugation to separate the sample particles within the density gradient by Method 700. Operations 716–720 can be substantially analogous to operations 508–512 of Method 500, as described above.

[0074] As shown in Figure 8, the density gradient 800 generated by method 700 includes the supplied volume of sample particles within the range of the density gradient 800. For example, the density gradient 800 includes the supplied volume of sample particles only within a second portion 804 sandwiched between a first portion 802 and a third portion 806 that do not include the supplied volume of sample particles. In further embodiments, the density gradient 800 may include multiple portions that include the supplied volume of sample particles. These multiple portions that include the supplied volume of sample particles may be discontinuous with respect to each other such that the portions that include the supplied volume of sample particles are separated by portions that do not include the supplied volume of sample particles.

[0075] Supplying sample particles within discrete locations of a continuous density gradient is not readily possible using conventional density gradient techniques, which are primarily manual processes, because it would be impossible to manually control sample particle supply and density gradient formation with the required level of precision. Advantageously, Method 700 allows the density gradient 800 to have a shorter centrifugation time to separate the sample particles, since the sample particles can be supplied closer to locations where their expected density lies along the radial length of the density gradient 800. This reduces the distance the particles must travel to reach their equilibrium positions, and therefore further shortens the centrifugation time.

[0076] In some embodiments, the system 100 uses a predictive model to automatically determine, based on the type of sample particles to be separated, the relationship between the highest and lowest densities in the gradient (such as the radial length and / or slope of the supplied volume of the component across the radial length), whether the gradient is linear, logarithmic, continuous, or stepwise, and, when it is desired not to uniformly disperse the sample particles within the density gradient, the length of the container 110 or the location for supplying the sample particles within the density gradient as a function of the estimated density of the sample particles.

[0077] Furthermore, to maximize the efficiency of the centrifuge, the density gradient supplied according to the operation of Method 500 and Method 700 can be supplied to precisely match a specified known gradient composition or to match an experimental gradient that has been shown to be successful. For example, the size and / or geometry of the container, the geometry of the centrifuge rotor, temperature, centrifugation speed (rpm / rcf), and other parameters can determine the optimal density gradient profile for separating sample particles. Predictive models can also consider these parameters when supplying a density gradient that matches the optimal density gradient profile.

[0078] The volume of sample particles introduced into the density gradient 800 generated by Method 700 is less than the volume of sample particles introduced into the density gradient 600 generated by Method 500. In this example, approximately 5.7 mL of sample is introduced into the density gradient 800, which has a total volume of 39 mL. Therefore, Method 700 can be particularly advantageous for early development, analytical, and other small-volume workflows where rapid time is important for various samples.

[0079] By selectively feeding sample particles directly into these continuous density gradients, density gradients of 600 and 800 can be generated, significantly increasing the throughput for separating sample particles by reducing the overall centrifugation time. This maximizes throughput and efficiency when separating large sample volumes, while minimizing the time required for efficiently separating relatively small sample volumes.

[0080] In addition, since centrifugation is eliminated or significantly reduced for density gradient formation by methods 500 and 700, these methods can utilize a wider variety of density modifier materials, such as sucrose, which would otherwise be impractical for use in equilibrium-zone centrifugation and isodensity DGUC. For example, methods 500 and 700 can generate a continuous density gradient using a density modifier with a lower density (e.g., sucrose), which would require much longer centrifugation times and / or much greater centrifugal forces in equilibrium-zone centrifugation and isodensity DGUC techniques, which are impractical.

[0081] In another example, the use of iodixanol as a density modifier can sometimes be impractical in conventional particle separation techniques that use density gradients. Unlike cesium chloride (CsCl), iodixanol has a relatively high molecular weight and is also viscous. At high speeds, iodixanol forms a very steep gradient, which can limit the resolution between sample species. At low speeds, the density gradient formed by using iodixanol will form very slowly. Methods 500 and 700 overcome these challenges associated with using iodixanol as a density modifier, as the centrifugation required to form the density gradient is significantly reduced or even eliminated, so that the slope of the density gradient formed by methods 500 and 700 can be controlled without compromising the centrifugation time.

[0082] Figure 9 schematically illustrates one embodiment of the computing hardware of System 100 for implementing aspects of the present disclosure. As shown in Figure 9, System 100 includes one or more processing devices 902, a memory storage device 904, and a system bus 906 that connects the memory storage device 904 to one or more processing devices 902. One or more processing devices 902 may include a central processing unit (CPU). In some cases, one or more processing devices 902 are part of a processing network having memory for storing instructions, which, when executed by the processing network, cause the processing network to implement various aspects, features, and functionalities described herein.

[0083] As shown in Figure 9, the memory storage device 904 may include random access memory ("RAM") 908 and read-only memory ("ROM") 910. Basic input logic and basic output logic, which have basic routines useful for transferring information between elements within the system 100 during startup, etc., may be stored in the ROM 910.

[0084] System 100 may also include a mass storage device 912, which includes an operating system 914 and can store software instructions and data 916. The mass storage device 912 is connected to a processing device 902 via a system bus 906. The mass storage device 912 and its associated computer-readable data storage medium provide a non-volatile, non-transient storage device for System 100.

[0085] The description of computer-readable data storage media contained herein refers to the mass storage device 912, but it should be understood by those skilled in the art that the computer-readable data storage medium may be any available non-transient physical device or product from which the system 100 can read data and / or instructions. The computer-readable storage medium may consist entirely of non-transient media. The mass storage device 912 is an embodiment of the computer-readable storage device.

[0086] Computer-readable data storage media include volatile and non-volatile, removable and non-removable media implemented in any method or technique for storing information such as computer-readable software instructions, data structures, program modules, or other data. Exemplary types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technologies, or any other media that may be used to store information and may be accessed by such devices.

[0087] System 100 can operate in a networked environment using logical connections to other devices via network 920. System 100 connects to the communication network 920 through a network interface unit 918 connected to a system bus 906. The network interface unit 918 can also connect to additional types of communication networks and devices, including those via Bluetooth®, Wi-Fi, and cellular telecommunications networks, including 4G and 5G networks. The network interface unit 918 can connect System 100 to additional networks, systems, and devices. System 100 also includes an input / output unit 922 for receiving and processing inputs and outputs from peripheral devices.

[0088] The mass storage device 912 and RAM 908 can store software instructions and data. The software instructions may include an operating system 914, which is suitable for controlling the operation of system 100. The mass storage device 912 and / or RAM 908 can also store software instructions and data 916, which, when executed by the processing device 902, provide the functionality of system 100 discussed herein.

[0089] The various embodiments described above are provided for illustrative purposes only and should not be construed as limiting in any way. Various modifications can be made to the embodiments described above without departing from the true spirit and scope of this disclosure.

Claims

1. A system for supplying a density gradient within a container, wherein the system is A processing network having a memory for storing instructions, wherein when an instruction is executed by the processing network, the processing network receives The method involves pumping sample particles and a density modifier into a mixing chamber that is in fluid communication with the proximal end of the probe, wherein the mixing chamber mixes the sample particles and the density modifier together. The method involves supplying the density gradient into the container through the distal end of the probe, wherein the density gradient varies between the first end and the second end, and at least a portion of the density gradient between the first end and the second end includes the supplied volume of the sample particles. A system equipped with a processing circuit network to perform the following actions.

2. The system according to claim 1, wherein the density gradient is a continuous gradient in which the density increases between the first end and the second end.

3. The system according to claim 1, wherein the density gradient is a stepped gradient having an interface of different densities between the first end and the second end.

4. When the aforementioned instruction is executed by the processing network, the processing network further: The system according to claim 1, wherein the density gradient is supplied such that the supplied volume of the sample is included throughout the entire density gradient.

5. When the aforementioned instruction is executed by the processing network, the processing network further: The system according to claim 1, wherein the density gradient is supplied such that the volume to be supplied of the sample particles is included in a portion of the density gradient sandwiched between portions of the sample particles that do not include the volume to be supplied.

6. When the aforementioned instruction is executed by the processing network, the processing network further: The first portion of the density gradient is supplied, wherein the first portion includes the supplied volume of the density modifier without the sample particles. The provision of a second portion of the density gradient, wherein the second portion includes the volume of the density modifier and the sample particles to be supplied. The means of supplying the third portion of the density gradient, wherein the third portion includes the supplied volume of the density modifier without the sample particles. The system according to claim 5, which causes the following to be performed.

7. When the aforementioned instruction is executed by the processing network, the processing network further: The first portion of the density gradient is supplied by increasing the volume of the density modifier to be supplied and decreasing the volume of deionized water to be supplied, thereby increasing the density of the first portion along the first portion. The second portion of the density gradient is supplied by increasing the volume of the density modifier to be supplied and decreasing the volume of the sample particles to be supplied, thereby increasing the density of the second portion along the second portion. The third portion of the density gradient is supplied by increasing the volume of the density modifier to be supplied and decreasing the volume of the deionized water to be supplied, thereby increasing the density of the third portion along the third portion. The system according to claim 6, which causes the following to be performed.

8. When the aforementioned instruction is executed by the processing network, the processing network further: Before supplying the density gradient, the distal end of the probe is lowered toward the bottom of the container, After supplying the density gradient, the probe is removed from the container. The system according to claim 1, which causes the following to be performed.

9. The system according to claim 1, wherein the density of the density gradient is in the range of 1.0 g / mL to 1.8 g / mL between the first end and the second end.

10. A method for supplying a density gradient within a container, wherein the method is The method involves pumping sample particles and a density modifier into a mixing chamber that is in fluid communication with the proximal end of the probe, wherein the mixing chamber mixes the sample particles and the density modifier together. The method involves supplying the density gradient into the container through the distal end of the probe, wherein the density gradient varies between the first end and the second end, and at least a portion of the density gradient between the first end and the second end includes the supplied volume of the sample particles. Methods that include...

11. The method according to claim 10, wherein the density gradient is a continuous gradient in which the density increases between the first end and the second end.

12. The method according to claim 10, wherein the density gradient is a stepped gradient having an interface of different densities between the first end and the second end.

13. The method of claim 10, further comprising supplying the density gradient such that the supplied volume of the sample particles is included throughout the entirety of the density gradient.

14. The method according to claim 10, further comprising supplying the density gradient such that the volume to be supplied of the sample particles is included in a portion of the density gradient sandwiched between portions of the sample particles that do not include the volume to be supplied.

15. The first portion of the density gradient is supplied, wherein the first portion includes the supplied volume of the density modifier without the sample particles. The provision of a second portion of the density gradient, wherein the second portion includes the volume of the density modifier and the sample particles to be supplied. The means of supplying the third portion of the density gradient, wherein the third portion includes the supplied volume of the density modifier without the sample particles. The method according to claim 14, further comprising:

16. The first portion of the density gradient is supplied by increasing the volume of the density modifier to be supplied and decreasing the volume of deionized water to be supplied, thereby increasing the density of the first portion along the first portion. The second portion of the density gradient is supplied by increasing the volume of the density modifier to be supplied and decreasing the volume of the sample particles to be supplied, thereby increasing the density of the second portion along the second portion. The third portion of the density gradient is supplied by increasing the volume of the density modifier to be supplied and decreasing the volume of the deionized water to be supplied, thereby increasing the density of the third portion along the third portion. The method according to claim 15, further comprising:

17. Before supplying the density gradient, the distal end of the probe is lowered toward the bottom of the container, After supplying the density gradient, the probe is removed from the container. The method according to claim 10, further comprising:

18. The method according to claim 10, wherein the density of the density gradient is in the range of 1.0 g / mL to 1.8 g / mL between the first end and the second end.

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