Cell lysis and plasmid isolation system

Abstract

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

Description

CELL LYSIS AND PLASMID ISOLATION SYSTEMRelated Applications

[0001] This application claims priority to U.S. Ser. No. 63 / 417,630 filed on October 19, 2022 and U.S. Ser. No. 63 / 468,322 filed on May 23, 2023, each of which being incorporated into this disclosure in their entirety.Field of the Disclosure

[0002] This disclosure relates to systems for isolating plasmids from cells in closed systems comprising disposable materials.Background of the Disclosure

[0003] The rapid growth of cell and gene therapy has led to a need for systems providing for improved lysis process control as well as scaling up the lysis process to handle larger volumes of fluids. In addition, there would be significant benefits in cost, flexibility, and operation if the system could be provided in a single use format. This disclosure provides solutions to these problems. For instance, in some embodiments, the single use systems disclosed herein can achieve optimal blending and residence time of multiple input fluids for the cell lysis and plasmid isolation process. This disclosure thereby providing solutions to these and other art-recognized, and unrecognized, problems.Brief Description of the Drawings

[0004] Figure 1. First exemplary system design.

[0005] Figure 2. Second exemplary system design.

[0006] Figure 3. Exemplary retention tubing modules (i.e., “reactor module”).

[0007] Figures 4A-4F. Exemplary, preferred, “one-in one-out” retention tubing module (i.e., reactor module).

[0008] Figure 5A-5F. Exemplary, preferred, “one-in two-out” retention tubing module (i.e., reactor module).

[0009] Figure 6A-6F. Exemplary, preferred, “two-in one-out” retention tubing module(i.e., reactor module).

[0010] Figure 7A-7D. Exemplary, preferred, mixing baffles and arrangements thereof.Summary of the Disclosure

[0011] This disclosure provides systems for continuous in-line lysing of cells containing plasmid DNA, the system comprising a single-use flow path comprising a first container comprising a first fluid comprising cells containing plasmid nucleic acids fluidly to a first single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathway; a second container comprising a second fluid that is an alkaline lysis buffer fluidly connected to the second single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathway; the first and second fluids being combined, directly or using a mixer, into a third single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathway; the third fluidic pathway being fluidly connected to at least one third container comprising single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) tubing within which the cells are at least partially lysed to release the plasmid nucleic acids and, if more than one third container is present, each third container is fluidly connected to one another in series by one or more additional single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathways and comprising an exit fluidly connected to a fourth single-use fluidic pathway; a fourth container comprising neutralization buffer fluidly connected via a third pump to the single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) flow path through a fifth single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathway; and, the fourth and fifth single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathways being combined, directly or using a mixer, into a sixth single-use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathway containing final product comprising released plasmids and cell debris; wherein: the fluids in the first and second single use (in preferred embodiments, but also in some preferred embodiments, multi-use and / or reusable) fluidic pathways arc combined into the third fluidic pathway that is fluidly connected to the at least one third container; the fluid in the third container is maintained therein for a sufficient amount of time to lyse cells and release plasmids from the cells while minimizing degradation of the plasmids, and exits the retention tubing section (i.e., “reactor module”) through the fourth fluidic pathway; and, the fluid of the fourth fluidic pathwayis combined, directly or using a mixer, with neutralization buffer from the fourth container in the fifth fluidic pathway (4a) and exits the single-use flow path through a seventh fluidic pathway. Other embodiments, including methods for using such systems, are also provided herien.Detailed Description

[0012] This disclosure relates to systems for isolating plasmids from cells in closed systems comprising disposable materials (in preferred embodiments single use, but also in some preferred embodiments multi-use and / or reusable). The system provides conduits for moving cells containing plasmids into an area for lysing the cells and releasing plasmid therefrom for a specific amount of time (the retention tubing module or reactor module) to provide a lysis mixture and then combining the lysis mixture with a neutralization buffer to terminate the lysing reaction. The fluids comprising the cells and other materials are moved through the system by pressure (or gravity) from starting points at each respective source (e.g., cellular source, lysis buffer source, and / or neutralization buffer source) through conduits and / or the retention tubing module, to product collection and / or waste vessel(s). The systems include at least one retention tubing module in which the cells are at least partially lysed in the presence of a lysis buffer for a time suitable for releasing plasmid from the cells without damaging the plasmid. In some embodiments, the systems can include a source of cells (e.g., cell vessel), source of lysis buffer (e.g., lysis vessel), source of neutralization buffer (e.g., neutralization buffer vessel), and a retention tubing module for lysing the cells and releasing plasmid therefrom. In preferred embodiments, the cell vessel is fluidly connected to a first conduit (also referred to as fluidic conduit) and at least a first pump, the lysis buffer vessel 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 tubing module (e.g., the the retention tubing module entry port). The retention tubing module is fluidly connected to a fifth conduit through which the solution exits the retention tubing module (e.g., through the retention tubing module exit port). The fifth conduit can be fluidly connected to the third conduit comprising neutralization buffer (e.g., forming a sixth conduit). The alkaline plasmid mixture exiting the retention tubing module is mixed in the sixth conduit with the neutralization buffer to reduce the alkalinity of the plasmid mixture. One or more static mixers can also be included in the fourthand sixth conduits. In preferred embodiments, a static mixer can be included in the fourth conduit between the point at which the first and second conduits merge and the retention tubing module entry port. In preferred embodiments, a static mixer can be included in the sixth conduit after the point at which the fifth and third conduits merge and the retention tubing module entry port. The system can also include final product and waste vessels. The components of the system are preferably constructed of disposable materials (e.g., plastic tubing and the like). These single use (in preferred embodiments, but also in some preferred embodiments multi-use and / or reusable) systems can achieve optimal blending and residence time of multiple input fluids for the lysis process.

[0013] In preferred embodiments (see, e.g., Figs. 1-2), the systems include 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., the retention tubing module (reactor module)) that provides for the lysis of cells comprising plasmids such that the same are released from the cells in intact and undamaged form. Preferably, cell lysis is achieved without completing lysing the cells. In some embodiments, the system can include at least one subsystem (e.g., skid, preferably an automated skid) to perform constant, in-line, lysing of cells wherein plasmids are released from the cells in intact form. In some embodiments, the system would be designed to process batches on the order of 1000L or greater. In some embodiments, flow rates of solutions through the system would be about two to three liters per minute. In some embodiments, the various flow paths (e.g., tubing) for the product (e.g., composition comprising cells containing plasmids) and chemical streams (e.g., buffers) would be single use (at least with respect to that individual system) and changed out between batches. In some embodiments, the system comprises a cell feed pump that transits cells from an upstream pool vessel. In some embodiments, cell feed flow is controlled from an input parameter into the control system and feedback from a flowmeter just downstream of the cell feed pump. In some embodiments, the cell flow path can be combined with a lysing buffer from a second pump fluidly connected to a lysis source (e.g., vessel). This flow would then pass through an in-line mixing device to ensure a robust mixture of the cells and the lysis buffer. In preferred embodiments, lysis chemical exposure time for the cells / plasmid is for only a few minutes and adjustable depending on number of cartridges used on the system. An analytical device such as a pH probe or a conductivity probe could be inserted downstream of the in line mixing device. In some embodiments, a secondflowmeter can be included downstream of the lysis buffer pump. In some embodiments, flow of the lysis buffer can be controlled by proportional flow control from program input and feedback from the second flowmeter from feedback from a downstream analytical device. In preferred embodiments, the combined flow (e.g., cells and lysis buffer) would then enter a retention tubing section. In some embodiments, the retention tubing section is specifically sized based off the flow of the combined streams and the dimensions of the tubing to achieve a specific contact time between the lysing buffer and the cells. In preferred embodiments, the retention tubing module is made up of individually packed “cartridges”, each cartridge comprising a specific length of tubing that may be the same or differ between cartridges. In some embodiments, the retention tubing sections (e.g., cartridge(s), module(s)) can be removable to facilitate installation and provide for the swapping out of tubing after a specific run or between products. In some embodiments, the frame of the skid can hold each cartridge and allow for multiple cartridges to be used. Following the retention tubing section of the system, a neutralization buffer can be introduced from a third pump at the junction of the fluidic conduit leading away (in terms of flow direction) from the retention tubing module and the fluidic conduit comprising the neutralization buffer to produce a neutralization mixture. The neutralization mixture can enter a static mixer to ensure proper neutralization and terminate the lysing reaction. Separate analytical devices such as a pH probe or a conductivity probe can be inserted downstream of the second line mixing device. In some embodiments, a third flowmeter can be included just downstream of the neutralization buffer pump. In some embodiments, flow of the neutralization buffer can be by proportional flow control from program input and feedback from the third flowmeter from feedback from a downstream analytical device. In preferred embodiments, an adequately neutralized cell stream exits the skid for downstream processing.

[0014] This disclosure, then, provides lysis and plasmid isolation systems that include at least one single-use, disposable fluidly connected to at least one retention tubing module (see, e.g., Figs. 1-2 (eachrepresenting a section / conduit comprised of single use tubing)). In various embodiments, such systems can also include one or more fluidic conduits (e.g., tubing, piping), a housing or enclosure, valves, gaskets, one or more pumps (e.g., pump module comprising a pump housing, diaphragm and check valve) one or more vessels (e.g., reservoirs containers, cell source, buffer source), and / or one or more pressure gauges, fluidly connected to transport fluids comprising cells, lysis buffer, neutralization buffer, and / or mixtures of the samethrough the system. In some embodiments, the systems can comprise at least one feed vessel, and at least one disposable retentate vessel, where at least one feed vessel and at least one disposable retention tubing module fluidly connected to other components of the system. In some embodiments, the feed vessel comprising the cells comprising plasmids can be a jacketed vessel comprising at least one heat transfer element.

[0015] In some embodiments, the feed vessel containing the cells comprising the plasmids, preferably comprising a disposable container (DC) can take the form of a standard bioreactor or fermentation vessel as these are understood by those of ordinary skill 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 instance and without limitation, 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 cone-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 could be used as disclosed herein are also known in the art as would be understood by those of ordinary skill in the art.

[0016] The disposable components of the system, can be made of disposable (e.g., single use), reusable (e.g., sterilizable), and / or replaceable I swappable materials, the same being commercially available to those of ordinary skill 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 tubing module, and optionally components thereof, can be made of disposable materials, preferably single use disposable materials. Exemplary, non-limiting components that can be included in the systems can include Flexware® components (Mobius®), FlexReady Solution components (EMD Millipore Corporation), and / or Allegro™ components (Pall Corporation, Port Washington, N.Y.) In some embodiments, disposable fluidic conduits can be made from disposable tubing. Preferably, the retention tubing module includes at least one disposable flow path comprising one or more disposable fluidic conduits. In some embodiments, disposable components can be and / or can be used with other disposable components such as a T-line or valve (e.g., feed and / or retentate, or permeate vessel and / or conduit valve, such as a pinch or diaphragm valve). Typically, such components are compatible with other components of the system, are non-toxic,high-strength, sanitary, and / or re-usable. In some embodiments, the system can also comprise one or more pumps, pressure sensor(s) (e.g., comprising a diaphragm), and / or similar components that may or may not be completely or partially disposable and / or re-usable. 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, which can be carried out 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., formaline solution) or heat, or an appropriate combination thereof. The disposable components of the system and disposable reactor vessel are made from disposable materials (e.g., plastic, thermoplastic elastomer, rubber, metal). Preferably, the disposable components and container are made from plastic, thermoplastic elastomer, or a combination of low-density and high-density polyethylene (LDPE and HDPE) or nylon.

[0017] In preferred embodiments (see, e.g., Figs. 1-2), the system provides conduits (i.e., sections comprised of single-use tubing) for moving a reaction fluid through a reactor module (e.g., in preferred embodiments shown as component “3” in Figs. 4-6) including cells comprising plasmids from a source vessel into an area of the system (i.e., the reactor module (or “retention tubing module”; see, e.g.. Fig. 3, Fig. 4 (the “one-in one-out” arrangement) and / or Fig. 5 (the one-in two-out” arrangement); and Fig. 6 (“two-in one-out” arrangement)) for lysing the cells and releasing plasmid therefrom by exposing the cells to a alkaline lysis buffer (e.g,. preferably having a pH>13) for a specific amount of time to provide a lysis mixture and then combining the lysis mixture with a neutralization buffer to terminate the lysing reaction and move the final products (e.g., plasmids and cell debris) out of the system. The system comprises at least one, and in some embodiments preferably one, tortuous path or conduit (single or multi-use), and components, and / or combinations thereof. The fluids comprising the cells and other fluids are moved through the system by pressure (or gravity) from starting points at each respective source (e.g., cellular source, lysis buffer source, and / or neutralization buffer source) through the conduits and / or the retention tubing module, to product collection and / or waste vessel(s). In some embodiments, the system can In preferred embodiments, the system includes at least one cell vessel fluidly connected to a first fluidic conduit and optionally a first pump; a lysis buffer vessel fluidly connected to a second conduit and optionally the first or a second pump; where the first and second conduits merge into a third conduit (optionally connected to static mixer) fluidlyconnected to the retention tubing module that comprises at least one section of pre-packaged disposable tubing within a rigid container (where in embodiments in which multiple sections are included, each section may contain the same or different lengths and / or types of single-use tubing therein) through a retention tubing module entry port; the retention tubing module including a retention module exit port fluidly connected to a fourth conduit (optionally connected to a pump) connected to a neutralization buffer vessel fluidly connected to a fifth conduit and optionally a third pump; and a sixth conduit optionally including static mixer; and, finally, a waste vessel. The fluid exiting the retention tubing module comprises an alkaline plasmid mixture and is mixed with the neutralization buffer to reduce the alkalinity of the plasmid mixture. The system can also include final product and waste vessels. The components of the system are most preferably constructed of disposable materials (e.g., plastic tubing and the like). In the most preferred embodiments, the componetns are single use but can also, in some preferred embodiments, be multi-use (e.g., or reusable). These single use systems can achieve optimal blending and residence time of multiple input fluids for the lysis process.

[0018] The system consists of a tortuous path within a fluidic conduit, e.g., manufactured or tubing (single or multi-use) components or a combination of the two. The system includes baffles which are designed to increase the efficiency of mixing within the pathway. The system provides a set time of flow of a reaction fluid from entrance into the fluidic conduit to the exit from the fluidic conduit to process a reaction fluid into a more completely mixed (preferably a homogenously mixed) solution. That set time can be determined by fluidic conduit length, rate of flow of the reaction fluid through the reactor module, temperature, and / or the adjustment of other parameters. The design includes single or multiple inlets and / or outlets (at least one of each) leading to and from a reactor module comprising the depending on the application and how many fluids need to be added to the system. The design will also consist of a singular or multiple exits which can be connected to another CSR® Static Mixer or can be connected to a further downstream process. The inlets and / or outlets can be aligned with one another (e.g., horizontal relative to each other to provide a balanced, geometrically even flow path) or out of alignment with one another (e.g., providing a downward sloping flowpath such that gravity drives the reaction fluid toward the exit).

[0019] In preferred embodiments, the fluid conduit (e.g., tubing) includes one more mixing baffles therein. Within this disclosure, a mixing baffle is any structure provided by the fluidconduit that provides a change in the geometry of the fluid conduit, preferably the interior of the fluid conduit, that allows or provides for turbidity (or mixing) of the reaction fluid, which allows for improved mixing. The relatively consistent flow of the reaction fluid within the fluid conduit is interrupted by the one or more mixing baffle(s) to produce a limited area of fluid turbulence and / or vortexing within the fluidic conduit that serves to assist in lysing the cells, thereby releasing plasmid therefrom, and / or to further separate released plasmid from any cell debris that may be attached thereto. The flow of a reaction fluid more than one mixing baffle during transit through the reactor module 3 provides further vortexing of the cells and plasmids contained in the reaction fluid, and the further release of plasmid from cells and / or cell debris attached to the plasmid. 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 geometry can be changed by any suitable means, but in preferred embodiments can be a projection (or an extension) of material projecting from the interior surface toward the center of the fluid conduit and / or provided by a partial distortion of the normal shape of the fluid conduit (e.g., from circular to bent such as in a “kink”). In some embodiments, one or more projections are positioned directly across from one another relative to the inner diameter of the fluid conduit (i.e., positioned perpendicularly relative to each other). In some embodiments, the one or more projections are not positioned directly across from one another relative to the inner diameter of the fluid conduit (i.e., not positioned perpendicularly relative to each other). In some embodiments, a combination(s) of perpendicularly positioned extension and non-perpendicularly positioned extensions can be included. The mixing baffle or mixing baffles can be positioned at any suitable position within the fluid conduit. In preferred embodiments, a first mixing baffle may be positioned within about 25% of the length of the fluid conduit from the entry and / or exit thereof (i.e., 25% of the distance of the fluid conduit length). In some such embodiments, a second mixing baffle may be positioned within about 25% of the length of the fluid conduit from the opposite entry and / or exit thereof. In some embodiments, multiple mixing baffles may be positioned along the length of the fluid conduit, such as at distance equivalent to about 25% of the length of the fluid conduit and / or subsection thereof (e.g., within a horizontal section as shown in Fig. 6B). Other positions may also be suitable, as could be determined by those of ordinary skill in the art. In some preferred embodiments, a mixing baffle includes a triangular- shaped indent (e.g., a type of kink) that extends partially toward the interior center point of the fluidic conduit and can extend around theentire circumference of the tubing or conduit, or only be present within less than the entire circumference (e.g., half of the circumference). In preferred embodiments, as opposed to or in addition to one or more extension, a first section of the fluid conduit (proximal to the entry point) extends at about a 90-degree angle from the interior (and exterior) surface thereof to decrease the diameter of the fluidic conduit, and a second section extends from the first section that gradually enlarges the circumference of the fluidic conduit (e.g., in a ramp shape; see the illustrations in Figs. 6A and 6B). This type of mixing baffle provides an eddy-type mixing action in which fluid moves from the upwardly extending ramp section and over the sharp edge (i.e., which extends at about a 90-degree angle from the fluid conduit surface). Fig. 6F provides an exemplary illustration of the fluid flow over the ramp section of the eddy-type mixing baffle (the arrows indicating fluid flow over a mixing baffle designed as in Fig. 6A (see 6-3 thereof). In some embodiments, as illustrated in Figs. 7A-7B, the fluid conduit can comprise multiple extensions from the edge toward the center of the fluid conduit that can take any suitable shape (e.g., rectangular or triangular- projections). In some embodiments, a mixture of different types of projections can extend from the inner wall of the fluid conduit (see, e.g., Fig. 7C). In some preferred embodiments, the mixing baffles can extend from the interior surface of the fluid conduit in an approximately even distribution or in an alternating pattern, each “first” row of one or more mixing baffles offset with the adjacent following row (see, e.g., the pattern shown in Fig. 7D).

[0020] In the preferred embodiments of the reactor module 3 illustrated in Fig. 4 (the “one-in one-out” arrangement), Fig. 5 (the one-in two-out” arrangement), the fluidic conduit (e.g., tubing) therein includes at least one (preferably two mixing baffles therein (e.g., indentations within the conduit or tubing)), and Fig. 6 (preferably including more than two mixing baffles along the length of the fluid conduit or tubing). Exemplary, but preferred, mixing baffles are shown in Figs. 4A, 4B. 5A, 5B, 6A and 7.

[0021] A preferred embodiment illustrated in Fig. 4 (Fig. 4A) is a “one-in, one-out” design in which a single fluidic conduit introduces fluid into the reactor module (“fluid entry point”) and a single fluidic conduit provides for exit of the fluid from the reactor module (“fluid exit point”). Figs. 4B and 4C illustrate the indentations positioned within the fluidic conduit, with a first mixing baffle (“A”) being positioned at approximately the midpoint of the fluidic conduit and a second mixing baffle (“B”) being positioned between the first (“A) and the fluid exit locatedopposite the fluid entry of the retention module.

[0022] Another preferred embodiment illustrated in Fig. 5 (Fig. 5A) is a “one-in, two-out” design in which a single fluidic conduit introduces fluid into the reactor module 3 (“fluid entry point”) and at least two fluidic conduits provide for exit of the fluid from the reactor module (“fluid exit points”). Figs. 5B and 5C illustrate the indentations positioned within the fluidic conduit, with a first mixing baffle (“A”) being positioned at approximately the midpoint of the fluidic conduit and a second mixing baffle (“B”) being positioned between the first (“A) and the fluid exits located opposite the fluid entry of the retention module. The presence of two fluid exit points in this preferred embodiment provides another point in the system at which the components can be mixed, thereby in effect functionally providing a third mixing baffle.

[0023] Another preferred embodiment of Fig. 6 illustrates a “two-in, one-out” design in which a two fluidic conduits introduce reaction fluid into reactor module 3 (“fluid entry point”) which exits through one exit (“fluid exit point”). Fig. 6A illustrates a preferred design of the mixing baffle arrangement in this embodiment. In this embodiment, the mixing baffle Fig. 6B illustrates this embodiment in which multiple alternating sections of fluidic conduit are arranged within the reactor module. Each of the alternating sections include or do not include at least one mixing baffle (1, Fig. 6A). Each mixing baffle (“A”) is positioned at approximately the midpoint of the section of fluidic conduit in which it is present. That section is connected to the following section that does not include a mixing baffle. This arrangement is repeated throughout the reactor module. In the illustrated example, seven (7) alternating sections are included, with the first, third, fifth, and seventh sections include a mixing baffle, while the second, fourth and sixth sections do not include a mixing baffle. This embodiment therefore provides multiple mixing baffles distributed throughout the fluidic conduit. The presence of two fluid exit points in this preferred embodiment provides another point in the system at which the components can be mixed, thereby in effect functionally providing a third mixing baffle.

[0024] In a preferred embodiment, this disclosure provides a system (illustrated in Fig. 1) for continuous in-line lysing of cells containing plasmid DNA, the system comprising: a single-use flow path comprising: first container comprising a first fluid comprising cells containing plasmid nucleic acids (1), the first container being fluidly connected via a first pump (Pl) to a first singleuse fluidic pathway (la); a second container (2) comprising a second fluid that is an alkaline lysisbuffer (Lysis Buffer In) fluidly connected via a second pump (P2) to the second single-use fluidic pathway (2a); the first and second fluids being combined, directly or using a mixer, into a third single-use fluidic pathway (3a); the third fluidic pathway (3a) being fluidly connected to at least one third container (3) comprising disposable (e.g., preferably single-use) tubing within which the cells are at least partially lysed to release the plasmid nucleic acids and, if more than one third container (3b) is present, each third container being fluidly connected to one another in series by one or more additional single-use fluidic pathways (3b) and comprising an exit fluidly connected to a fourth single-use fluidic pathway (3c) (the third container being a retention tubing cartridge); a fourth container (4) comprising neutralization buffer fluidly connected via a third pump (P3) to the single-use flow path through a fifth single-use fluidic pathway (4a); and, the fourth (3c) and fifth (4a) single-use fluidic pathways being combined, directly or using a mixer, into a sixth single-use fluidic pathway (5) containing final product (released plasmids and cell debris); wherein: the fluids in the first (cells) (la) and second (alkaline lysis buffer) single use fluidic pathways (2a) are combined into the third fluidic pathway (3a) that is fluidly connected to the at least one third container comprising disposable retention tubing (retention tubing cartridge); the fluid in the third container (3 / 3b) is maintained therein (retention tubing section) for a sufficient amount of time to lyse cells and release plasmids from the cells while minimizing degradation of the plasmids, and exits the retention tubing section through the fourth fluidic pathway (3c); and, the fluid of the fourth fluidic pathway is combined, directly or using a mixer, with neutralization buffer from the fourth container (4) in the fifth fluidic pathway (4a) and exits the single-use flow path through a seventh fluidic pathway (6) (Product Out). In some preferred embodiments, the retention tubing section (3 / 3b) comprises multiple subsections fluidly connected to one another, each subsection comprising pre-packaged disposable tubing within a rigid container wherein each may contain the same or different lengths and / or types of disposable tubing therein. In preferred embodiments, the retention tubing section (3 / 3b) includes four such subsections. 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), with a Human-Machine Interface (HMI) used for inputting process recipe parameters, monitoring the process, and collecting process data. In some embodiments, the at least one inline analytical instrument controls 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 bufferis controlled via feedback from inline analytical instruments such as but not limited to pH and conductivity. In some embodiments, the pH of the alkaline lysis buffer is less than 13 (i.e., pH<13; e.g., preferably about any of above 7, such as about any of 8, 8.5 9, 9.5 10, 10.5 11, 11.5 12, or 12.5), optionally wherein the alkaline lysis buffer is about 0.1-0.2 N NaOH. In some embodiments, the sufficient amount of time is any of about two to about ten minutes. In some embodiments, the lysis reactor is fluidly connected to a neutralization reactor consisting of a fluid path downstream of the lysis reactor where neutralization buffer is added to and blended with the lysis flow stream. In some preferred embodiments, the retention tubing section comprises prepackaged disposable tubing within a rigid container. In some preferred embodiments, the fourth fluidic pathway comprises a static mixer. In some preferred embodiments, the exit port is fluidly connected to a fifth fluidic pathway that is fluidly connected a container or another piece of equipment. In some preferred embodiments, this disclosure provides methods for isolating plasmid DNA from a cell culture, the method comprising processing cells comprising plasmid DNA through a system disclosed herein.

[0025] The terms “a”, “an”, and / or “the” typically means at least one, or one or more. The terms “about”, “approximately”, and the like, when used as a modifier refers to a variant of a number that typically occurs in carrying out standard procedures. In some preferred embodiments, “about”, “approximately”, and the like, indicates a numerical value of within ten percent (i.e., + / -10%) of the listed numerical value. The terms “about”, “approximately”, and the like, when preceding a list of numerical values or range, refer to each individual value in the list or range independently as if each individual value in the list or range was immediately preceded by that term. The terms mean that the values to which the same refer are exactly, close to, or similar thereto. Optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed. When such a range is expressed, another aspect 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 aspect. 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 otherendpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed.

[0026] All references cited within this disclosure are hereby incorporated by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.

[0027] Other embodiments are also disclosed and / or contemplated and / or can be contemplated from this disclosure by those of ordinary skill in the art. Various embodiments of the present disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible embodiments for the claimed invention.

[0028] While certain embodiments have been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A system for continuous in-line lysing of cells containing plasmid DNA, the system comprising: a single-use flow path comprising: a first container comprising a first fluid comprising cells containing plasmid nucleic acids (1), the first container being fluidly connected via a first pump (Pl) to a first single-use fluidic pathway (la); a second container (2) comprising a second fluid that is an alkaline lysis buffer fluidly connected via a second pump (P2) to the second single-use fluidic pathway (2a); the first and second fluids being combined, directly or using a mixer, into a third single-use fluidic pathway (3a); the third fluidic pathway (3a) being fluidly connected to at least one third container (3) comprising single-use tubing within which the cells are at least partially lysed to release the plasmid nucleic acids and, if more than one third container (3b) is present, each third container being fluidly connected to one another in series by one or more additional single-use fluidic pathways (3b) and comprising an exit fluidly connected to a fourth single-use fluidic pathway (3c); a fourth container (4) comprising neutralization buffer fluidly connected via a third pump (P3) to the single-use flow path through a fifth single-use fluidic pathway (4a); and, the fourth (3c) and fifth (4a) single-use fluidic pathways being combined, directly or using a mixer, into a sixth single-use fluidic pathway (5) containing final product comprising released plasmids and cell debris; wherein: the fluids in the first (la) and second single use fluidic pathways (2a) are combined into the third fluidic pathway (3a) that is fluidly connected to the at least one third container; the fluid in the third container (3 / 3b) is maintained therein for a sufficient amount of time to lyse cells and release plasmids from the cells while minimizingdegradation of the plasmids, and exits the retention tubing section through the fourth fluidic pathway (3c); and, the fluid of the fourth fluidic pathway is combined, directly or using a mixer, with neutralization buffer from the fourth container (4) in the fifth fluidic pathway (4a) and exits the single-use flow path through a seventh fluidic pathway (6). The system of claim 1 wherein the single-use tubing of the third container (3) comprising single-use tubing within which the cells are at least partially lysed to release the plasmid nucleic acids, wherein the single-use tubing comprises at least one mixing baffle therein. The system of claim 2 wherein the tubing comprises a mixing baffle provided by at least one first indentation positioned at approximately the midpoint of the fluidic conduit, optionally further comprising at least one second indentation being positioned between the first and the fluid exit located opposite the fluid entry of the retention module. The system of claim 3 wherein the third container (3) comprises on fluidic entrance and one fluidic exit. The system of claim 3 wherein the third container (3) comprises on fluidic entrance and at least two fluidic exits. 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 Distributed Control System (DCS), with a Human-Machine Interface (HMI) used for inputting process recipe parameters, monitoring the process, and collecting process data. The system of claim 6 wherein the at least one inline analytical instrument controls pH and / or conductivity of at least one fluid, buffer and / or stream. The system of any preceding claim wherein the flow of cells, lysis buffer, and / or neutralization buffer is controlled via feedback from inline analytical instruments such as but not limited to pH and conductivity.The system of any preceding claim wherein the pH of the alkaline lysis buffer is less than 13 (pH<13), optionally wherein the alkaline lysis buffer is about 0.1-0.2 N NaOH. The system of any preceding claim wherein the sufficient amount of time is about two to about ten minutes. The system of any preceding claim wherein the lysis reactor is fluidly connected to a neutralization reactor consisting of a fluid path downstream of the lysis reactor where neutralization buffer is added to and blended with the lysis flow stream. The system of any preceding claim wherein the retention tubing section comprises prepackaged disposable tubing within a rigid container. The system of any preceding claim wherein the retention tubing section comprises multiple subsections fluidly connected to one another, each subsection comprising pre-packaged disposable tubing within a rigid container, the length and / or type of disposable tubing of each subsection being the same or different from the length and / or type of disposable tubing of at least one of the other subsections. The system of any preceding claim wherein the fourth fluidic pathway comprises a static mixer. The system of any preceding claim wherein the exit port is fluidly connected to a fifth fluidic pathway that is fluidly connected a container or another piece of equipment. A reactor module comprising a fluid conduit comprising therein at least one mixing baffle. A method for isolating plasmid DNA from a cell culture, the method comprising processing cells comprising plasmid DNA through a system and / or reactor module of any preceding claim.

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