Cell Lysis and Plasmid Isolation System

Abstract

The present disclosure provides devices, systems, and methods for isolating nucleic acids from cells, preferably in a continuous manner, using a reactor system.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 63 / 417,630, filed October 19, 2022, and U.S. Patent Application No. 63 / 468,322, filed May 23, 2023, each of which is incorporated herein in its entirety.

[0002] The present disclosure relates to a system for isolating plasmids from cells in a closed system that includes disposable materials. [Background technology]

[0003] The rapid growth of cell and gene therapy has created a need for systems that provide improved control of the lysis process and the ability to scale up the lysis process to process larger volumes of fluid. Furthermore, providing a system in a single-use format offers significant advantages in cost, versatility, and operation. The present disclosure provides solutions to these challenges. For example, in some embodiments, the single-use systems disclosed herein can achieve optimal mixing and residence times of multiple input fluids for cell lysis and plasmid isolation processes. Thus, the present disclosure provides solutions to these and other recognized and unrecognized challenges in the art. Summary of the Invention

[0004] The present disclosure provides a system for continuous in-line lysis of cells harboring plasmid DNA, comprising a single-use fluid path, the single-use fluid path comprising a first container containing a first fluid comprising cells harboring plasmid nucleic acid, fluidly connected to a first single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) fluid pathway, and an alkaline lysis buffer, fluidly connected to a second single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) fluid pathway. and a fourth container containing a neutralization buffer fluidically connected to the single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) flow path via a third pump through a fifth single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) fluid path, wherein the first and second fluids are mixed with a third single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) fluid path either directly or using a mixer. and a third fluid pathway is fluidly connected to at least one third container comprising a single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) tube that at least partially lyses the cells to release the plasmid nucleic acid; if more than one third container is present, each third container is fluidly connected in series with one or more further single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) fluid pathways and comprises an outlet fluidly connected to a fourth single-use fluid pathway; and said fourth and fifth single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) fluid pathways are combined, either directly or using a mixer, into a sixth single-use (in preferred embodiments, but also in some preferred embodiments, multiple-use and / or reusable) fluid pathway that contains a final product comprising the released plasmid and cell debris; and said first and second single-use (in preferred embodiments,However, in some preferred embodiments, the fluids in the (multiple-use and / or reusable) fluid paths are combined into a third fluid path fluidically connected to at least one third container, the fluids in the third container are maintained in the third container for a time sufficient to lyse the cells and release the plasmids from the cells with minimal degradation of the plasmids, and are discharged from a retention tube section (i.e., "reactor module") through a fourth fluid path, and the fluids in the fourth fluid path are combined with a neutralization buffer from the fourth container in a fifth fluid path (4a) either directly or using a mixer, and are discharged from a single-use flow path through a seventh fluid path. Other embodiments, including methods of using such systems, are also provided herein. [Brief explanation of the drawings]

[0005] [Figure 1] FIG. 1 is a diagram of a first exemplary system design. [Figure 2] FIG. 1 is a diagram of a second exemplary system design. [Figure 3] FIG. 1 is a diagram of an exemplary retention tube module (i.e., "reactor module"). [Figures 4A-4F] FIG. 1 is a diagram of an exemplary preferred "1-in-1-out" retention tube module (i.e., reactor module). [Figures 5A-5F] FIG. 1 is a diagram of an exemplary preferred "1 in 2 out" retention tube module (i.e., reactor module). [Figures 6A-6F] FIG. 1 is a diagram of an exemplary preferred "2-in 1-out" retention tube module (i.e., reactor module). [Figures 7A-7D] FIG. 1 is a diagram of an exemplary preferred mixing baffle and its placement. DETAILED DESCRIPTION OF THE INVENTION

[0006] The present disclosure relates to a system for isolating plasmids from cells in a closed system including disposable materials (single-use in preferred embodiments, but also multiple-use and / or reusable in some preferred embodiments). The system provides a conduit for transporting plasmid-bearing cells to an area (retention tube module or reactor module) for lysing the cells and releasing the plasmids therefrom for a specified time period to provide a lysis mixture, followed by combining the lysis mixture with a neutralization buffer to stop the lysis reaction. Fluids containing cells and other materials move by pressure (or gravity) within the system from a starting point at a respective source (e.g., a cell source, a lysis buffer source, and / or a neutralization buffer source) through the conduits and / or retention tube modules to a product collection container and / or a waste container. The system includes at least one retention tube module in which cells are at least partially lysed in the presence of a lysis buffer for a time period suitable to release the plasmids from the cells without damaging the plasmids. In some embodiments, the system can include a source of cells (e.g., a cell container), a source of lysis buffer (e.g., a lysis container), a source of neutralization buffer (e.g., a neutralization buffer container), and a retention tube module for lysing the cells and releasing the plasmids therefrom. In preferred embodiments, the cell container is fluidly connected to a first conduit (also referred to as a fluid conduit) and at least a first pump, the lysis buffer container is fluidly connected to a second conduit and at least one second pump, and the neutralization buffer is fluidly connected to a third conduit and at least one third pump. In preferred embodiments, the first and second conduits merge into a fourth conduit that is fluidly connected to the retention tube module (e.g., a retention tube module inlet port). The retention tube module is fluidly connected to a fifth conduit that discharges the solution from the retention tube module (e.g., through a retention tube module outlet port). The fifth conduit can be fluidly connected to a third conduit containing a neutralization buffer (e.g., forming a sixth conduit).The alkaline plasmid mixture exiting the retention tube module is mixed with a neutralization buffer in the sixth conduit to reduce the alkalinity of the plasmid mixture. One or more static mixers can also be included in the fourth and sixth conduits. In preferred embodiments, a static mixer can be included in the fourth conduit between the junction of the first and second conduits and the retention tube module inlet port. In preferred embodiments, a static mixer can be included in the sixth conduit after the junction of the fifth and third conduits and the retention tube module inlet port. The system can also include a final product container and a waste container. System components are preferably constructed of disposable materials (e.g., plastic tubing, etc.). These single-use (in preferred embodiments, but also multiple-use and / or reusable in some preferred embodiments) systems can achieve optimal mixing and residence time for the multiple input fluids of the lysis process.

[0007] In preferred embodiments (see, e.g., Figures 1-2), the system includes at least one subsystem (or section) constructed of single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) materials (i.e., retention tube module (reactor module)) that effect lysis of plasmid-bearing cells such that the plasmid is released intact from the cells. Preferably, cell lysis is achieved without completely lysing the cells. In some embodiments, the system can include at least one subsystem (e.g., a skid, preferably an automated skid) for performing constant in-line lysis of cells, resulting in the intact release of the plasmid from the cells. In some embodiments, the system is designed to process batches on the order of 1000 L or more. In some embodiments, the flow rate of solutions through the system will be about 2-3 liters / minute. In some embodiments, the various flow paths (e.g., tubing) for product (e.g., a composition containing plasmid-bearing cells) and chemical (e.g., buffer) streams will be single-use (at least for individual systems) and will be replaced between batches. In some embodiments, the system includes a cell feed pump that transports cells from an upstream pool vessel. In some embodiments, the cell feed flow rate is controlled by input parameters to the control system and feedback from a flow meter immediately downstream of the cell feed pump. In some embodiments, the cell flow path can be combined with lysis buffer from a second pump fluidly connected to a lysis source (e.g., a reservoir). This flow then passes through an in-line mixing device to ensure robust mixing of the cells and lysis buffer. In preferred embodiments, the exposure time of the cells / plasmids to the lysis chemicals is only a few minutes and is adjustable depending on the number of cartridges used in the system. Analytical devices, such as pH or conductivity probes, can also be inserted downstream of the in-line mixing device. In some embodiments, a second flow meter can be included downstream of the lysis buffer pump.In some embodiments, the flow of lysis buffer can be controlled by proportional flow control from program inputs and feedback from a second flow meter based on feedback from a downstream analytical device. In preferred embodiments, the combined stream (e.g., cells and lysis buffer) then enters a retention tube section. In some embodiments, the retention tube section is specifically sized based on the combined stream flow rates and tubing dimensions to achieve a specific contact time between the lysis buffer and the cells. In preferred embodiments, the retention tube module is composed of individually packed "cartridges," each containing a specific length of tubing that may be the same or different between cartridges. In some embodiments, the retention tube section (e.g., cartridge, module) can be removable for ease of installation and to provide for tubing replacement after a particular run or between products. In some embodiments, the frame of the skid can hold each cartridge, allowing for the use of multiple cartridges. Following the retention tube section of the system, a neutralization buffer can be introduced from a third pump at the junction of the fluid conduit (in terms of flow direction) away from the retention tube module and the fluid conduit containing the neutralization buffer to generate the neutralization mixture. The neutralization mixture enters a static mixer to ensure proper neutralization and halt the lysis reaction. A separate analytical device, such as a pH probe or conductivity probe, can be inserted downstream of the second line mixing device. In some embodiments, a third flow meter can be included immediately downstream of the neutralization buffer pump. In some embodiments, the flow of the neutralization buffer can be controlled by proportional flow control from a program input and feedback from the third flow meter based on feedback from a downstream analytical device. In a preferred embodiment, the properly neutralized cell stream is discharged from the skid for downstream processing.

[0008] Therefore, the present disclosure provides a method for disposing of at least one single-use disposable tube (e.g., see FIGS. 1-2 (each " [ka] " represents a section / conduit comprised of single-use tubing). In various embodiments, such systems also include one or more fluid conduits (e.g., tubing, piping), housings or enclosures, valves, gaskets, one or more pumps (e.g., a pump module comprising a pump housing, a diaphragm, and a check valve), one or more vessels (e.g., a reservoir container, a cell source, a buffer source), and / or one or more pressure gauges fluidly connected to transport fluids comprising cells, lysis buffer, neutralization buffer, and / or a mixture thereof through the system. In some embodiments, the system can include at least one feed vessel and at least one disposable retentate vessel, and the at least one feed vessel and the at least one disposable retention tubing module are fluidly connected to other components of the system. In some embodiments, the feed vessel containing the plasmid-carrying cells can be a jacketed vessel comprising at least one heat transfer element.

[0009] In some embodiments, the feed vessel containing the plasmid-carrying cells, preferably comprising a disposable container (DC), can take the form of a standard bioreactor or fermentation vessel, as will be understood by those skilled in the art. In some embodiments, the feed vessel can be insulated, jacketed, and / or include one or more heat transfer systems. In some embodiments, the vessel can have a form disclosed in, for example, but not limited to, U.S. Pat. No. 8,658,419 (ABEC, Inc.), U.S. Pat. No. 9,228,165 B2 (ABEC, Inc.), and / or WO 2019 / 070648 A2 (ABEC, Inc.). In some embodiments, the disposable feed vessel can be a conical-bottom or tulip-bottom vessel and / or preferably has a capacity of at least 20, 100, 250, 500, 750, 1000, 1500, 2000, 2500, or 3000 liters (L). Other types of suitable feed vessels that can be used as disclosed herein are known in the art, as will be appreciated by those skilled in the art.

[0010] The disposable components of the system can be made of disposable (e.g., single-use), reusable (e.g., sterilizable), and / or replaceable / replaceable materials, which are commercially available to those skilled in the art. In preferred embodiments, the components of the system are made of disposable materials, preferably single-use disposable materials. In even more preferred embodiments, the components of the system, and at least the retention tube module, and optionally its components, can be made of disposable materials, preferably single-use disposable materials. Exemplary, non-limiting components that can be included in the system include Flexware® components (Mobius®), FlexReady Solution components (EMD Millipore Corporation), and / or Allegro™ components (Pall Corporation, Port Washington, NY). In some embodiments, the disposable fluid conduits can be made from disposable tubing. Preferably, the retention tube module includes at least one disposable flow path that includes one or more disposable fluid conduits. In some embodiments, disposable components can be and / or be used in conjunction with other disposable components, such as T-lines or valves (e.g., feed and / or retentate or permeate containers and / or conduit valves such as pinch or diaphragm valves). Typically, such components are compatible with other components of the system and are non-toxic, high-strength, hygienic, and / or reusable. In some embodiments, the system may also include one or more pumps, pressure sensors (e.g., including diaphragms), and / or similar components, which may or may not be fully or partially disposable and / or reusable.In preferred embodiments, the components of the system, individually and / or as one or more units thereof, can be sanitized (e.g., sterilized) prior to use using standard techniques, including, but not limited to, gamma radiation, ethylene oxide (ETO), bleach (e.g., Clorox), chlorine (e.g., NaOCl), peroxide, acid (e.g., peracetic acid), base (e.g., NaOH), formaldehyde (e.g., formalin solution), or heat, or suitable combinations thereof. The disposable components of the system and the disposable reactor vessel are made from disposable materials (e.g., plastic, thermoplastic elastomer, rubber, metal). Preferably, the disposable components and containers are made from plastic, thermoplastic elastomer, or a combination of low-density polyethylene and high-density polyethylene (LDPE and HDPE), or nylon.

[0011] In a preferred embodiment (see, e.g., FIGS. 1-2), the system moves reaction fluids from a source container through a reactor module (e.g., shown in preferred embodiments as component "3" in FIGS. 4-6) that houses cells bearing the plasmids, exposing the cells to an alkaline lysis buffer (e.g., preferably having a pH >13) for a specified time to generate a lysis mixture, and then combining the lysis mixture with a neutralization buffer to stop the lysis reaction and move the end product (e.g., plasmids and cellular debris) out of the system to a region of the system (i.e., reactor module (or "retention tube module" (e.g., shown in, e.g., FIGS. 3, 4) that lyses the cells and releases the plasmids therefrom). (see FIG. 5 ("1-in-1-out" configuration), and / or FIG. 5 ("1-in-2-out" configuration), and FIG. 6 ("2-in-1-out" configuration). The system includes at least one, and in some embodiments preferably one, serpentine path or conduit (single-use or multi-use), and components thereof, and / or combinations thereof. Cell-containing and other fluids move within the system by pressure (or gravity) from their starting points at their respective sources (e.g., cell source, lysis buffer source, and / or neutralization buffer source) through the conduits and / or retention tube modules to the product collection and / or waste containers.In a preferred embodiment, the system includes at least one cell container fluidly connected to a first fluid conduit and optionally a first pump, a lysis buffer container fluidly connected to a second conduit and optionally the first or second pump, a retention tube module including a retention module outlet port fluidly connected to a fourth conduit (optionally connected to a pump) connected to a fifth conduit and optionally a neutralization buffer container fluidly connected to a third pump, a sixth conduit optionally including a static mixer, and finally a waste container, wherein the first and second conduits merge through a retention tube module inlet port into a third conduit (optionally connected to a static mixer) that is fluidly connected to a retention tube module containing at least one section of disposable tubing pre-packaged in a rigid container (in embodiments where multiple sections are included, each section may contain single-use tubing of the same or different lengths and / or types). The fluid exiting the retention tube module contains the alkaline plasmid mixture and is mixed with a neutralizing buffer to reduce the alkalinity of the plasmid mixture. The system may also include a final product container and a waste container. The components of the system are most preferably constructed of disposable materials (e.g., plastic tubing, etc.). In most preferred embodiments, the components are single-use, although in some preferred embodiments, they can be multiple-use (e.g., reusable). These single-use systems can achieve optimal mixing and residence time for the multiple input fluids of the lysis process.

[0012] The system consists of a serpentine path within a fluid conduit, e.g., fabricated components or tubing (single-use or multi-use) components, or a combination of the two. The system includes baffles designed to enhance mixing efficiency within the pathway. The system provides a specific set time for the reactant fluids to enter and exit the fluid conduit to process the reactant fluids into a more thoroughly mixed (preferably homogeneously mixed) solution. The specific set time can be determined by adjusting the length of the fluid conduit, the flow rate of the reactant fluids through the reactor module, the temperature, and / or other parameters. The design includes single or multiple inlets and / or outlets (at least one of each) leading to and / or from the reactor module, depending on the application and the number of fluids required to be added to the system. The design also includes single or multiple outlets that can be connected to another CSR® static mixer or further downstream processes. The inlets and / or outlets may be aligned with one another (e.g., horizontally relative to one another to provide balanced, geometrically uniform flow paths) or may be misaligned with one another (e.g., to provide downwardly sloping flow paths such that gravity drives the reaction fluids toward the outlets).

[0013] In a preferred embodiment, the fluid conduit (e.g., a tube) includes one or more mixing baffles therein. In the present disclosure, a mixing baffle is any structure provided by a fluid conduit that results in a change in the geometry of the fluid conduit, preferably the interior of the fluid conduit, which can provide or enable turbidity (or mixing) of the reaction fluid to improve mixing. The relatively consistent flow of the reaction fluid within the fluid conduit is interrupted by one or more mixing baffles, creating limited regions of turbulence and / or vortex flow of the fluid within the fluid conduit, which assists in lysing cells, thereby releasing plasmids therefrom, and / or further separating the released plasmids from cellular debris that may be attached to the plasmids. As the reaction fluid passes through reactor module 3, flowing through more than one mixing baffle further vortexes the cells and plasmids contained within the reaction fluid, further releasing the plasmids from cellular debris attached to the cells and / or plasmids. In some embodiments, the one or more mixing baffles can be formed by the geometry (or shape) of the interior channel of the fluid conduit. The geometric shape can be varied by any suitable means, but in preferred embodiments, it can be a protrusion (or extension) of material projecting from the inner surface of the fluid conduit toward the center and / or can be provided by a partial distortion of the normal shape of the fluid conduit (e.g., a bend such as a "kink" from circular). In some embodiments, one or more protrusions are positioned opposite one another with respect to the inner diameter of the fluid conduit (i.e., positioned perpendicular to one another). In some embodiments, one or more protrusions are not positioned opposite one another with respect to the inner diameter of the fluid conduit (i.e., not positioned perpendicular to one another). Some embodiments can include a combination of perpendicularly and non-perpendicularly positioned extensions. One or more mixing baffles can be positioned at any suitable location within the fluid conduit. In preferred embodiments, a first mixing baffle is positioned within about 25% of the length of the fluid conduit from an inlet and / or outlet of the fluid conduit (i.e., 25% of the distance of the length of the fluid conduit). In some such embodiments, a second mixing baffle is positioned within about 25% of the length of the fluid conduit from an opposite inlet and / or outlet.In some embodiments, multiple mixing baffles may be positioned along the length of the fluid conduit, for example, at a distance corresponding to about 25% of the length of the fluid conduit and / or a subsection thereof (e.g., within a horizontal section as shown in FIG. 6B). Other locations, as can be determined by one of ordinary skill in the art, may also be suitable. In some preferred embodiments, the mixing baffles extend partially toward the interior center point of the fluid conduit and include a triangular depression (e.g., a type of kink) that may extend the entire circumference of the tube or conduit or may be present less than the entire circumference (e.g., half the circumference). In preferred embodiments, as opposed to or in addition to one or more extensions, a first section of the fluid conduit (proximal to the inlet point) extends at an angle of about 90 degrees from its interior (and exterior) surface to reduce the diameter of the fluid conduit, and a second section extends from the first section to gradually expand the circumference of the fluid conduit (e.g., in a ramp shape, see the diagrams in FIGS. 6A and 6B). This type of mixing baffle provides a vortex-type mixing action, where fluid moves from an upwardly extending ramp section over a sharp edge (i.e., extending at an angle of approximately 90 degrees from the fluid conduit surface). Figure 6F provides an exemplary diagram of fluid flow over the ramp section of a vortex-type mixing baffle (arrows indicate fluid flow through a mixing baffle designed as in Figure 6A (see 6-3 in that figure)). In some embodiments, as shown in Figures 7A-7B, the fluid conduit can include multiple extensions (e.g., rectangular or triangular protrusions) that can take any suitable shape from the edge toward the center of the fluid conduit. In some embodiments, a mixture of different types of protrusions can extend from the inner wall of the fluid conduit (see, for example, Figure 7C). In some preferred embodiments, the mixing baffles can extend from the inner surface of the fluid conduit in a generally uniform distribution or in an alternating pattern, with each "first" row of one or more mixing baffles offset from the adjacent next row (see, for example, the pattern shown in Figure 7D).

[0014] In preferred embodiments of reactor module 3 shown in Figures 4 ("1-in-1-out" configuration), 5 ("1-in-2-out" configuration), the fluid conduits (e.g., tubes) therein include at least one (preferably two) mixing baffles (e.g., depressions in the conduits or tubes) therein, and Figure 6 preferably includes more than two mixing baffles along the length of the fluid conduits or tubes. Exemplary, but preferred, mixing baffles are shown in Figures 4A, 4B, 5A, 5B, 6A, and 7.

[0015] The preferred embodiment shown in Figure 4 (Figure 4A) is a "one-in, one-out" design, where a single fluid conduit introduces fluid into the reactor module ("fluid inlet point") and a single fluid conduit discharges fluid from the reactor module ("fluid outlet point"). Figures 4B and 4C show recesses positioned within the fluid conduit, with a first mixing baffle ("A") positioned approximately midway through the fluid conduit and a second mixing baffle ("B") positioned between the first mixing baffle ("A") and the fluid outlet on the opposite side of the retention module from the fluid inlet.

[0016] Another preferred embodiment, shown in Figure 5 (Figure 5A), is a "one-in, two-out" design, in which a single fluid conduit introduces fluid into reactor module 3 (the "fluid entry point") and at least two fluid conduits discharge fluid from the reactor module (the "fluid exit point"). Figures 5B and 5C show depressions disposed within the fluid conduits, with a first mixing baffle ("A") positioned approximately midway along the fluid conduit and a second mixing baffle ("B") positioned between the first mixing baffle ("A") and the fluid exit on the opposite side of the retention module from the fluid inlet. The presence of two fluid exit points in this preferred embodiment provides another point within the system where components can mix, thus effectively providing a third mixing baffle.

[0017] Another preferred embodiment, shown in FIG. 6, illustrates a "two-in, one-out" design in which two fluid conduits introduce reaction fluids into reactor module 3 ("fluid entry points") and the reaction fluids exit through one outlet ("fluid exit point"). FIG. 6A illustrates a preferred design for the mixing baffle arrangement in this embodiment. FIG. 6B illustrates an embodiment in which multiple alternating sections of fluid conduits are arranged within the reactor module. Each alternating section may or may not include at least one mixing baffle (1, FIG. 6A). Each mixing baffle ("A") is located approximately midway through the section of fluid conduit in which it resides. This section is connected to the next section that does not include a mixing baffle. This arrangement is repeated throughout the reactor module. In the illustrated example, seven alternating sections are included, with sections 1, 3, 5, and 7 including mixing baffles, while sections 2, 4, and 6 do not. Thus, this embodiment provides multiple mixing baffles distributed throughout the fluid conduits. The presence of two fluid exit points in this preferred embodiment provides another point within the system where components can mix, thereby effectively providing a third mixing baffle.

[0018] In a preferred embodiment, the present disclosure provides a system (shown in FIG. 1) for continuous in-line lysis of cells carrying plasmid DNA, comprising a single-use fluid path, said fluid path comprising: a first container (1) containing a first fluid comprising cells carrying plasmid nucleic acid, fluidly connected to a first single-use fluid path (1a) via a first pump (P1); a second container (2) containing a second fluid, an alkaline lysis buffer (lysis buffer inlet), fluidly connected to a second single-use fluid path (2a) via a second pump (P2); and a fourth container (4) containing a neutralization buffer fluidically connected to a single-use flow path through a fifth single-use fluid path (4a) via a third pump (P3), wherein the first and second fluids are combined, either directly or using a mixer, into a third single-use fluid path (3a), which in turn is connected to at least one third container (3) (the third container being a retention tube cartridge) comprising disposable (e.g., preferably single-use) tubing that at least partially lyses cells to release plasmid nucleic acid. and if there is more than one third container (3b), each third container is fluidly connected to one another in series by one or more further single-use fluid paths (3b), and has an outlet fluidly connected to a fourth single-use fluid path (3c), and said fourth (3c) and fifth (4a) single-use fluid paths are combined, either directly or using a mixer, into a sixth single-use fluid path (5) containing the final product (released plasmids and cell debris) and the fluid (cells) in said first single-use fluid path (1a) and said second single-use fluid path (1b) The fluid in the main pathway (2a) (alkaline lysis buffer) is combined into a third fluid pathway (3a) fluidly connected to at least one third container, said third fluid pathway (3a) being fluidly connected to at least one third container comprising a disposable retention tube (retention tube cartridge), and the fluid in said third container (3 / 3b) being maintained in the third container (retention tube section) for a time sufficient to lyse the cells and release the plasmids from the cells with minimal degradation of the plasmids;The system provides a system in which fluid from the retention tube section is discharged through a fourth fluid path (3c), and the fluid in the fourth fluid path is combined with neutralization buffer from a fourth container (4) either directly in a fifth fluid path (4a) or using a mixer, and discharged through a seventh fluid path (6) (product outlet) to a single-use flow path. In some preferred embodiments, the retention tube section (3 / 3b) includes multiple sub-sections fluidically connected to each other, each sub-section containing disposable tubing prepackaged in a rigid container, and each sub-section may contain single-use tubing of the same or different lengths and / or types therein. In preferred embodiments, the retention tube section (3 / 3b) includes four such sub-sections. In some embodiments, the flow of cells, lysis buffer, and / or neutralization buffer through the system is controlled by a process controller, such as a programmable logic controller (PLC) or distributed control system (DCS), and a human-machine interface (HMI) is used to input process recipe parameters, monitor the process, and collect process data. In some embodiments, at least one in-line analytical device controls the pH and / or conductivity of at least one fluid, buffer, and / or stream. In some embodiments, the flow of cells, lysis buffer, and / or neutralization buffer is controlled via feedback from an in-line analytical device, such as, but not limited to, pH and conductivity. In some embodiments, the alkaline lysis buffer has a pH less than 13 (i.e., pH<13, e.g., preferably about 7 or greater, e.g., about 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, or 12.5), and optionally, the alkaline lysis buffer is about 0.1-0.2 N NaOH. In some embodiments, the sufficient time is about 2 to about 10 minutes. In some embodiments, the lysis reactor is fluidly connected to a neutralization reactor comprising a fluid path downstream of the lysis reactor, and a neutralization buffer is added to and mixed with the flowing lysis stream. In some preferred embodiments, the retention tube section comprises disposable tubes pre-packaged in a rigid container. In some preferred embodiments,The fourth fluid path comprises a static mixer. In some preferred embodiments, the outlet port is fluidly connected to a fifth fluid path that is fluidly connected to another portion of the container or device. In some preferred embodiments, the present disclosure provides a method for isolating plasmid DNA from a cell culture, the method comprising processing cells having plasmid DNA through a system disclosed herein.

[0019] The terms "a," "an," and / or "the" typically mean at least one, or more than one. Terms such as "about," "approximately," and the like, when used as modifiers, refer to variations in numbers that normally occur when standard procedures are practiced. In some preferred embodiments, "about," "approximately," and the like refer to a numerical value within 10 percent (i.e., + / - 10%) of the stated numerical value. When preceded by a list of numerical values ​​or ranges, the terms "about," "approximately," and the like refer independently to each individual value in the list or range, as if the term were immediately preceding each individual value in the list or range. These terms mean that the same value is the same as, approximately the same as, or similar to, the value referred to. Optionally or optionally means that the subsequently described event or circumstance may or may not occur, and that the statement includes both cases in which the event or circumstance occurs and cases in which it does not. A range (e.g., 90-100%) is meant to include not only the range itself, but also each individual value within the range, as if each value were listed individually. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about" or "approximately," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include not only the range itself, but also each individual value within the range, as if each value were individually listed.

[0020] All references cited within this disclosure are incorporated herein by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided by way of example only and are not intended to limit the scope of the claims in any way.

[0021] Other embodiments are disclosed and / or contemplated, and / or may be contemplated by one of ordinary skill in the art from the present disclosure. Various embodiments of the present disclosure are described in detail with reference to the drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the claims appended hereto. Moreover, any examples set forth herein are not intended to be limiting and merely illustrate some of the many possible embodiments of the claimed invention.

[0022] While certain embodiments have been described in terms of preferred embodiments, those skilled in the art will recognize that variations and modifications are possible. It is therefore intended that the appended claims cover all such equivalent variations that come within the scope of the following claims.

Claims

1. 1. A system for continuous in-line lysis of cells carrying plasmid DNA, comprising: a single-use flow path; a first container (1) containing a first fluid containing cells having plasmid nucleic acid, said first container (1) being fluidly connected to a first single-use fluid path (1a) via a first pump (P1); a second container (2) containing a second fluid, the second fluid being an alkaline lysis buffer, fluidly connected to a second single-use fluid path (2a) via a second pump (P2); a fourth container (4) containing a neutralization buffer fluidly connected to the single-use flow path through a fifth single-use fluid path (4a) via a third pump (P3); Equipped with the first and second fluids are combined into a third single-use fluid path (3a) either directly or using a mixer; said third fluid pathway (3a) is fluidly connected to at least one third container (3) comprising a single-use tube for at least partially lysing cells to release plasmid nucleic acid, and if there is more than one third container (3b), each third container is fluidly connected in series with one or more further single-use fluid pathways (3b) and comprises an outlet fluidly connected to a fourth single-use fluid pathway (3c); the fourth (3c) and fifth (4a) single-use fluid paths are combined, either directly or using a mixer, into a sixth single-use fluid path (5) containing the final product containing the released plasmids and cell debris; the fluid in the first single-use fluid path (1 a) and the fluid in the second single-use fluid path (2 a) are combined into the third fluid path (3 a) that is fluidly connected to at least one third container; the fluid in said third container (3 / 3b) is maintained in said third container (3 / 3b) for a time sufficient to lyse the cells and release the plasmids from the cells with minimal degradation of the plasmids, and is discharged from the retention tube section through said fourth fluid path (3c); The fluid in the fourth fluid path is combined with neutralization buffer from the fourth container (4) in the fifth fluid path (4a) either directly or using a mixer and is discharged from the single-use flow path through a seventh fluid path (6). system.

2. 2. The system of claim 1, wherein the single-use tube of the third container (3) comprises a single-use tube for at least partially lysing cells to release plasmid nucleic acid, the single-use tube comprising at least one mixing baffle therein.

3. 3. The system of claim 2, wherein the tube comprises a mixing baffle provided by at least one first recess located at approximately a midpoint of the fluid conduit, and optionally further comprises at least one second recess located between the first mixing baffle and a fluid outlet on an opposite side of the retention module from the fluid inlet.

4. 4. The system of claim 3, wherein the third container (3) comprises one fluid inlet and one fluid outlet.

5. 4. The system of claim 3, wherein the third container (3) comprises one fluid inlet and at least two fluid outlets.

6. 10. The system of any preceding claim, wherein the flow of cells, lysis buffer, and / or neutralization buffer is controlled by a process controller, such as a programmable logic controller (PLC) or a distributed control system (DCS), and a human machine interface (HMI) is used to input process recipe parameters, monitor the process, and collect process data.

7. The system of claim 6 , wherein the at least one in-line analytical device controls the pH and / or conductivity of at least one fluid, buffer, and / or stream.

8. 10. The system of any preceding claim, wherein the flow of cells, lysis buffer, and / or neutralization buffer is controlled via feedback from in-line analytical equipment such as, but not limited to, pH and conductivity.

9. 10. The system of any preceding claim, wherein the alkaline lysis buffer has a pH of less than 13 (pH<13), and optionally the alkaline lysis buffer is about 0.1-0.2 N NaOH.

10. 10. The system of any preceding claim, wherein the sufficient time is from about 2 to about 10 minutes.

11. 10. The system of claim 9, wherein the lysis reactor is fluidly connected to a neutralization reactor comprising a fluid path downstream of the lysis reactor, where a neutralization buffer is added to and mixed with the flowing lysis stream.

12. 10. The system of claim 1, wherein the retention tube section comprises a disposable tube pre-packaged in a rigid container.

13. 10. The system of claim 9, wherein the retention tube section includes a plurality of sub-sections fluidly connected to one another, each sub-section containing disposable tubes pre-packaged in a rigid container, the length and / or type of disposable tubes in each sub-section being the same as or different from the length and / or type of disposable tubes in at least one of the other sub-sections.

14. 10. The system of claim 1, wherein the fourth fluid path comprises a static mixer.

15. 10. The system of any preceding claim, wherein the outlet port is fluidly connected to a fifth fluid path that is fluidly connected to another piece of container or equipment.

16. A reactor module including a fluid conduit with at least one mixing baffle therein.

17. 10. A method for isolating plasmid DNA from a cell culture, the method comprising processing cells carrying the plasmid DNA through a system and / or reactor module according to any of the preceding claims.

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