Cell lysis and plasmid isolation system

Abstract

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

Description

[0001] Related applications

[0002] This application claims priority to U.S. Serial No. 63 / 417,630, filed October 19, 2022, and U.S. Serial No. 63 / 468,322, filed May 23, 2023, each of which is incorporated into this disclosure in its entirety. Technical Field

[0003] The present disclosure relates to a system for isolating plasmids from cells in a closed system comprising disposable materials. Background Art

[0004] The rapid development of cell and gene therapies has led to a demand for systems that provide improved control of the lysis process and that can scale up the lysis process to handle larger quantities of fluids. In addition, if the system could be provided in a disposable form, there would be significant benefits in terms of cost, flexibility, and operation. The present disclosure provides solutions to these problems. For example, in some embodiments, the disposable systems disclosed herein can achieve optimal blending and residence time for multiple input fluids used in cell lysis and plasmid isolation processes. Therefore, the present disclosure provides solutions to these and other recognized and unrecognized problems in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 .First exemplary system design.

[0006] Figure 2 .Second exemplary system design.

[0007] Figure 3 .Exemplary retention piping module (ie, "reactor module").

[0008] <a href="javascript:;" class="see-img-anchor" img-id="HDA0005456164630000041,HDA0005456164630000042,HDA0005456164630000043,HDA0005456164630000044,HDA0005456164630000045,HDA0005456164630000046" img-title="图4A,图4B("A”),图4C("B”),图4D,图4E,图4F"> Figures 4A to 4F .Exemplary preferred "one in, one out" retaining piping module (ie, reactor module).

[0009] <a href="javascript:;" class="see-img-anchor" img-id="HDA0005456164630000051,HDA0005456164630000052,HDA0005456164630000053,HDA0005456164630000054,HDA0005456164630000055,HDA0005456164630000056" img-title="图5A,图5B("A”),图5C("B”),图5D,图5E,图5F"> Figures 5A to 5F .Exemplary preferred "one in, two out" retaining piping module (ie, reactor module).

[0010] Figures 6A to 6F .Exemplary preferred "two in, one out" retaining piping module (ie, reactor module).

[0011] 7A to 7D .Exemplary preferred mixing baffles and their arrangements. Summary of the Invention

[0012] The present disclosure provides a system for performing continuous inline lysis of cells containing plasmid DNA, the system comprising: a disposable flow path, the disposable flow path comprising: a first container, the first container comprising a first fluid, the first fluid comprising cells containing plasmid nucleic acid, the first container being fluidically connected to a first disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) fluid path; a second container, the second container comprising a second fluid, the second fluid being an alkaline lysis buffer fluidically connected to a second disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) fluid path; the first container being fluidically connected to a second disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) fluid path; The first fluid and the second fluid are combined into a third disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) fluid pathway; the third fluid pathway is fluidically connected to at least one third container, the third container comprising disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) tubing, within which the cells are at least partially lysed to release the plasmid nucleic acid, and if there is more than one third container, each third container is fluidically connected to each other in series by one or more additional disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) fluid pathways, The third container further comprises an outlet fluidically connected to a fourth disposable fluid pathway; a fourth container comprising a neutralizing buffer fluidically connected to the disposable (in preferred embodiments, but also in some preferred embodiments, multiple uses and / or reusable) flow path via a third pump through a fifth disposable (in preferred embodiments, but also in some preferred embodiments, multiple uses and / or reusable) fluid pathway; and merging the fourth disposable (in preferred embodiments, but also in some preferred embodiments, multiple uses and / or reusable) fluid pathway and the fifth disposable fluid pathway into a sixth disposable (in preferred embodiments, but also in some preferred embodiments, multiple uses and / or reusable) flow path directly or using a mixer. wherein: the fluids in the first disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) fluid pathway and the second disposable fluid pathway are combined into the third fluid pathway, the third fluid pathway being fluidly connected to the at least one third container; the fluid in the third container is maintained therein for an amount of time sufficient to lyse cells and release plasmids from the cells while minimizing degradation of the plasmids, and the fluid exits the retention tubing segment (i.e., the "reactor module") through the fourth fluid pathway;The fluid of the fourth fluid pathway is combined with the neutralization buffer from the fourth container in the fifth fluid pathway (4a), either directly or using a mixer, and the fluid exits the disposable flow path through a seventh fluid pathway. Other embodiments including methods for using such a system are also provided herein. DETAILED DESCRIPTION

[0013] The present disclosure relates to a system for separating plasmid from cells in a closed system comprising disposable (in a preferred embodiment, disposable, but in some preferred embodiments, repeatedly with and / or reusable) material. The system provides conduit, for moving the cell comprising plasmid to the zone (retaining conduit module or reactor module) for lysing cells and therefrom discharging plasmid for a continuous specific time amount, to provide lysis mixture and then mix lysis mixture with neutralization buffer to stop lytic reaction. The fluid comprising cells and other materials moves through the system by conduit and / or retaining conduit module from the starting point of each corresponding source (for example, cell source, lysis buffer source and / or neutralization buffer source) under pressure (or gravity), arrives product collection and / or waste container. The system comprises at least one retaining conduit module, in which cell is at least partially cracked for a period of time in the presence of lysis buffer, and this time is applicable to discharging plasmid from cells and does not destroy plasmid. In some embodiments, system may comprise cell source (for example, cell container), lysis buffer source (for example, lysis container), neutralization buffer source (for example, neutralization buffer container) and for lysing cells and therefrom discharging the retention conduit module of plasmid. In a preferred embodiment, the cell container fluid is connected to the first conduit (also referred to as fluid conduit) and at least one first pump, and this lysis buffer container fluid is connected to the second conduit and at least one second pump, and neutralization buffer fluid is connected to the 3rd conduit and at least one 3rd pump. In a preferred embodiment, the first conduit and the second conduit merge into the 4th conduit, and this 4th conduit fluid is connected to and retains the pipeline module (for example, retains the pipeline module inlet port). Retain the pipeline module fluid and be connected to the 5th conduit, and solution leaves the retention pipeline module by this 5th conduit (for example, by retaining the pipeline module outlet port). The 5th conduit can be fluidically connected to the 3rd conduit (for example, forming the 6th conduit) comprising neutralization buffer. The alkaline plasmid mixture leaving the retention pipeline module is mixed with neutralization buffer in the 6th conduit, to reduce the alkalinity of the plasmid mixture. One or more static mixers also may be included in the 4th conduit and the 6th conduit. In a preferred embodiment, static mixer may be included in the 4th conduit, between the first conduit and the second conduit junction and retain the pipeline module inlet port. In a preferred embodiment, static mixer may be included in the 6th conduit, after the 5th conduit and the 3rd conduit junction and retain the pipeline module inlet port. The system may also include final product and waste container. The components of the system are preferably constructed of disposable materials (e.g., plastic tubing, etc.) These disposable (in preferred embodiments, but also in some preferred embodiments, multiple use and / or reusable) systems allow for optimal mixing and residence time of the various input fluids used in the lysis process.

[0014] In a preferred embodiment (see e.g. Figures 1 to 2), the system comprises at least one subsystem (or section) constructed of disposable (in preferred embodiments, but also in some preferred embodiments multiple use and / or reusable) materials (i.e., retaining tubing modules (reactor modules)), the at least one subsystem providing for lysis of cells comprising a plasmid such that the plasmid is released from the cells in an intact and undamaged form. Preferably, cell lysis is achieved without completely lysing the cells. In some embodiments, the system may comprise at least one subsystem (e.g., a slide, preferably an automated slide) for continuous, inline cell lysis, wherein the plasmid is released from the cells in an intact form. In some embodiments, the system will be designed to process batches of 1000 L or more. In some embodiments, the flow rate of the solution through the system will be about two to three liters per minute. In some embodiments, the various flow paths (e.g., tubing) for the product (e.g., a composition comprising cells containing a plasmid) and chemical streams (e.g., buffer) will be disposable (at least for this separate system) and replaced between batches. In some embodiments, the system includes a cell feed pump that transmits cells from an upstream tank container. In some embodiments, the cell feed flow is controlled according to input parameters entering the control system and feedback from a flow meter just located downstream of the cell feed pump. In some embodiments, the cell flow path can be merged with a lysis buffer from a second pump connected to a lysis source (e.g., a container) by fluid communication. Then, the flow is passed through an inline mixing device to ensure sufficient mixing of the cells and lysis buffer. In a preferred embodiment, the lysis chemical exposure time of the cell / plasmid is only a few minutes and can be adjusted according to the number of boxes used in the system. Analytical devices such as pH probes or conductivity probes can be inserted downstream of the inline mixing device. In some embodiments, a second flow meter can be included downstream of the lysis buffer pump. In some embodiments, the flow of the lysis buffer can be controlled by proportional flow control from a program input and feedback from a second flow meter from a downstream analytical device. In a preferred embodiment, the merged stream (e.g., cells and lysis buffer) will then enter a retention pipe section. In some embodiments, the size of the retention pipe section is specifically determined based on the flow rate of the merged stream and the size of the pipe to achieve a specific contact time between the lysis buffer and the cells. In a preferred embodiment, the retention tubing modules consist of individually packaged "cassettes," each containing a specific length of tubing that can be the same or different between cassettes. In some embodiments, the retention tubing segments (e.g., one or more cassettes, one or more modules) can be removable to facilitate installation and provide for interchange of tubing after a particular run or between products. In some embodiments, the frame of the slide can accommodate each cassette and allow for the use of multiple cassettes.After the retention pipe section of the system, a neutralization buffer can be introduced from a third pump at the junction of a fluid conduit leading from the retention pipe module (in terms of flow direction) and a fluid conduit, the fluid conduit including a neutralization buffer for producing a neutralization mixture. The neutralization mixture can enter a static mixer to ensure proper neutralization and terminate the lysis reaction. A separate analytical device such as a pH probe or a conductivity probe can be inserted downstream of the second inline mixing device. In some embodiments, a third flow meter can be included just downstream of the neutralization buffer pump. In some embodiments, the flow of the neutralization buffer can be achieved 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 fully neutralized cell stream leaves the slide for downstream processing.

[0015] The present disclosure then provides a lysis and plasmid isolation system comprising at least one disposable, disposable fluidically connected to at least one retention tubing module (see e.g. Figures 1 to 2 (Each In various embodiments, such a system may further comprise one or more fluid conduits (e.g., tubing, piping systems), 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 containers (e.g., a reservoir container, a cell source, a buffer source), and / or one or more pressure gauges fluidly connected to transport fluid comprising cells, lysis buffer, neutralization buffer, and / or mixtures thereof through the system. In some embodiments, the system may comprise at least one feed container and at least one disposable retentate container, wherein the at least one feed container and the at least one disposable retentate tubing module are fluidly connected to other components of the system. In some embodiments, the feed container comprising cells comprising plasmids may be a jacketed container comprising at least one heat transfer element.

[0016] In some embodiments, the feed container (preferably comprising a disposable container (DC)) containing cells containing a plasmid can take the form of a standard bioreactor or fermentation vessel, as understood by those of ordinary skill in the art. In some embodiments, the feed container can be insulated, jacketed, and / or include one or more heat transfer systems. In some embodiments, the container can have a form disclosed in, for example, but not limited to, U.S. Patent No. 8,658,419 (ABEC, Inc.), U.S. Patent No. 9,228,165B2 (ABEC, Inc.), and / or WO 2019 / 070648 A2 (ABEC, Inc.). In some embodiments, the disposable feed container can be a cone-bottom or tulip-bottom container, and / or preferably has a capacity of at least 20 liters (L), 100L, 250L, 500L, 750L, 1000L, 1500L, 2000L, 2500L, or 3000L. As will be appreciated by those of ordinary skill in the art, other types of suitable feed containers that may be used as disclosed herein are also known in the art.

[0017] The disposable components of the system can be made of disposable (e.g., single-use), reusable (e.g., sterilizable) and / or replaceable / interchangeable materials that are commercially available to one of ordinary skill in the art. In preferred embodiments, the components of the system are made of disposable materials, preferably disposable disposable materials. In even more preferred embodiments, the components of the system and at least the retaining tubing module and optional components thereof can be made of disposable materials, preferably disposable disposable materials. Exemplary non-limiting components that can be included in the system may include part FlexReady Solution components (EMD Millipore Corporation) and / or Allegro TMComponents (Pall Corporation, Port Washington, NY). In some embodiments, the disposable fluid conduits can be made of disposable tubing. Preferably, the retention tubing module includes at least one disposable flow path, which disposable flow path includes one or more disposable fluid conduits. In some embodiments, the disposable components can be 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 valves or diaphragm valves) and / or can be used therewith. Typically, such components are compatible with the other components of the system, are non-toxic, have high strength, are sanitary and / or are 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 disinfected (e.g., sterilized) prior to use, which can be performed using standard techniques including, but not limited to, gamma irradiation, ethylene oxide (ETO), bleach (e.g., Clorox), chlorine (e.g., NaOCl), peroxides, acids (e.g., peracetic acid), bases (e.g., NaOH), formaldehyde (e.g., formalin solution), or heat, or suitable combinations thereof. The disposable components and disposable reactor containers of the system are made of disposable materials (e.g., plastic, thermoplastic elastomer, rubber, metal). Preferably, the disposable components and containers are made of plastic, thermoplastic elastomer, or a combination of low-density and high-density polyethylene (LDPE and HDPE) or nylon.

[0018] In a preferred embodiment (see e.g. Figures 1 to 2 ), the system provides conduits (i.e., sections comprising disposable tubing) for moving a reaction fluid comprising cells and plasmids from a source container through a reactor module (e.g., in the preferred embodiment shown as component "3" in Figures 4-6) to an area of ​​the system for lysing the cells and releasing the plasmids therefrom by exposing the cells to an alkaline lysis buffer (e.g., preferably having a pH > 13) for a specific amount of time (i.e., a reactor module (or "retention tubing module"; see e.g., Figure 3, Fig. 4 (" one in one out " arrangement) and / or Fig. 5 (" one in two out " arrangement); and Fig. 6 (" two in one out " arrangement) retaining piping module or reactor module) to provide lysis mixture and then lysis mixture is mixed with neutralization buffer to stop lysis reaction and final product (for example, plasmid and cell debris) is moved out of the system. The system comprises at least one (and preferably one in some embodiments) tortuous path or conduit (single use or multiple use) and parts and / or their combination. Fluid comprising cells and other fluids moves through the system from the starting point of each corresponding source (for example, cell source, lysis buffer source and / or neutralization buffer source) under pressure (or gravity) by conduit and / or retaining piping module, arrives product collection and / or waste container. In some embodiments, the system may include, in a preferred embodiment, at least one cell container fluidically connected to a first fluid conduit and optionally a first pump; a lysis buffer container fluidically connected to a second conduit and optionally a first pump or a second pump; wherein the first conduit and the second conduit merge into a third conduit (optionally connected to a static mixer), the third conduit being fluidically connected to a retention conduit module via a retention tubing module inlet port, the retention tubing module comprising at least one segment of pre-packaged disposable tubing within a rigid container (wherein, in embodiments comprising multiple segments, each segment may comprise disposable tubing of the same or different lengths and / or types); the retention tubing module comprising a retention module outlet port fluidically connected to a fourth conduit (optionally connected to a pump), the fourth conduit being connected to a neutralization buffer container, the neutralization buffer container being fluidically connected to a fifth conduit and optionally a third pump; and a sixth conduit, the sixth conduit optionally comprising a static mixer; and finally, a waste container. The fluid exiting the retention tubing module comprises an alkaline plasmid mixture, and the neutralization buffer is mixed to reduce the alkalinity of the plasmid mixture. The system may also include a final product and a waste container. The components of the system are most preferably constructed of disposable materials (e.g., plastic tubing, etc.). In the most preferred embodiment, the components are disposable, but in some preferred embodiments they can also be multiple-use (eg, or reusable). These disposable systems allow for optimal blending and residence time of the various input fluids used in the lysis process.

[0019] The system consists of a tortuous path within a fluid conduit (e.g., a fabricated or piping (disposable or multiple use) component or a combination of both). The system includes baffles designed to improve the mixing efficiency within the pathway. The system provides a set time for the flow of the reaction fluid from the inlet of the fluid conduit to the outlet of the fluid conduit to process the reaction fluid into a more completely mixed (preferably uniformly mixed) solution. The set time can be determined by the length of the fluid conduit, the flow rate of the reaction fluid through the reactor module, the temperature and / or adjustments to other parameters. The design includes single or multiple inlets and / or outlets (at least one of each) to and from the reactor module, depending on the application and how much fluid needs to be added to the system. The design will also consist of single or multiple outlets that can be connected to another The static mixer may be connected to another downstream process. The inlet and / or outlet may be aligned with each other (e.g., horizontal relative to each other to provide a balanced, geometrically uniform flow path), or misaligned with each other (e.g., to provide a downwardly sloping flow path so that gravity drives the reactant fluid toward the outlet).

[0020] In a preferred embodiment, the fluid conduit (e.g., a pipe) includes one or more mixing baffles therein. Within the present disclosure, a mixing baffle is any structure provided by the fluid conduit that provides a change in the geometry of the fluid conduit, preferably a change in the interior of the fluid conduit, which allows or causes the reaction fluid to become turbid (or mixed), thereby allowing for improved mixing. The relatively consistent flow of the reaction fluid within the fluid conduit is interrupted by the one or more mixing baffles to create a limited region of fluid turbulence and / or eddy flow within the fluid conduit, which is used to assist in lysing the cells, thereby releasing the plasmids therefrom, and / or further separating the released plasmids from any cell debris that may be attached thereto. During transmission through the reactor module 3, the flow of the reaction fluid past more than one mixing baffle provides further turbulence of the cells and plasmids contained in the reaction fluid, and further release of the plasmids from the cells and / or cell debris attached to the plasmids. In some embodiments, the one or more mixing baffles may be formed by the geometry (or shape) of the internal channel of the fluid conduit. The geometry can be altered in any suitable manner, but in preferred embodiments, can be provided by protrusions (or extensions) of material projecting from the interior surface toward the center of the fluid conduit and / or by a partial deformation of the normal shape of the fluid conduit (e.g., from a circle to a curve, such as a "kink"). In some embodiments, one or more protrusions are positioned directly across from each other relative to the inner diameter of the fluid conduit (i.e., positioned perpendicularly relative to each other). In some embodiments, one or more protrusions are not positioned directly across from each other relative to the inner diameter of the fluid conduit (i.e., not positioned perpendicularly relative to each other). In some embodiments, one or more combinations of perpendicularly positioned extensions and non-perpendicularly positioned extensions can be included. One or more mixing baffles can be located at any suitable location within the fluid conduit. In preferred embodiments, a first mixing baffle can be located within approximately 25% of the length of the fluid conduit from its inlet and / or outlet (i.e., 25% of the distance along the length of the fluid conduit). In some such embodiments, a second mixing baffle can be located within approximately 25% of the length of the fluid conduit from its relative inlet and / or outlet. In some embodiments, a plurality of mixing baffles may be positioned along the length of the fluid conduit, such as at distances equal to about 25% of the length of the fluid conduit and / or subsections thereof (e.g., at Figure 6B, within the horizontal section shown). Other locations may also be suitable, as will be appreciated by one of ordinary skill in the art. In some preferred embodiments, the mixing baffle comprises a triangular indentation (e.g., a kink type) that extends partially toward an interior center point of the fluid conduit and may extend around the entire circumference of the pipe or conduit, or may only be present over less than the entire circumference (e.g., half of the circumference). In a preferred embodiment, opposite to or in addition to one or more extensions, a first section of the fluid conduit (near the entry point) extends from its interior (and exterior) surface at an angle of approximately 90 degrees to reduce the diameter of the fluid conduit, and a second section extends from the first section that gradually expands the circumference of the fluid conduit (e.g., in a ramp shape; see Figure 6A and Figure 6B This type of mixing baffle provides a vortex-type mixing action in which the fluid moves from an upwardly extending ramp section and over a sharp edge (ie, which extends at an angle of approximately 90 degrees from the surface of the fluid conduit). Figure 6F An exemplary illustration of fluid flow through a ramp section of a vortex type mixing baffle is provided (arrows indicate fluid flow through a ramp section of a vortex type mixing baffle). Figure 6A (See 6-3 thereof) in the design of the mixing baffle. In some embodiments, such as 7A to 7B As illustrated, the fluid conduit may include a plurality of extensions extending from the edge of the fluid conduit toward the center, which extensions may take any suitable shape (e.g., rectangular or triangular protrusions). In some embodiments, a mixture of different types of protrusions may extend from the inner wall of the fluid conduit (e.g., see Figure 7C In some preferred embodiments, the mixing baffles may extend from the interior surface of the fluid conduit in an approximately uniform distribution or in an alternating pattern, with each "first" row of one or more mixing baffles offset from the next adjacent row (see, e.g., Figure 7D mode shown).

[0021] In the preferred embodiments of the reactor module 3 illustrated in FIG4 (a "one in, one out" arrangement), FIG5 (a "one in, two out" arrangement), the fluid conduit (e.g., pipe) therein includes at least one, preferably two, mixing baffles (e.g., recesses within the conduit or pipe) therein, and FIG6 (preferably including more than two mixing baffles along the length of the fluid conduit or pipe). The exemplary but preferred mixing baffles are Figure 4A Figure 4B Figure 5A Figure 5B Figure 6A and shown in FIG7 .

[0022] Figure 4( Figure 4A) is a "one in, one out" design, wherein a single fluid conduit introduces fluid into the reactor module ("fluid inlet point") and a single fluid conduit provides for the exit of fluid from the reactor module ("fluid outlet point"). FIG. 4B and FIG. 4C illustrate a recess within a fluid conduit, wherein a first mixing baffle ("A") is positioned approximately midway through the fluid conduit, and a second mixing baffle ("B") is positioned between the first ("A") and the fluid outlet located opposite the fluid inlet of the retention module.

[0023] Figure 5 Figure 5A ) is a "one in, two out" design, wherein a single fluid conduit introduces fluid into the reactor module 3 ("fluid inlet point"), and at least two fluid conduits provide outlets for fluid from the reactor module ("fluid outlet points"). Figures 5B and 5C illustrate a recess within a fluid conduit, wherein a first mixing baffle ("A") is positioned at approximately the midpoint of the fluid conduit, and a second mixing baffle ("B") is positioned between the first ("A") and the fluid outlet located opposite the fluid inlet of the retention module. In this preferred embodiment, the presence of two fluid outlet points provides another point in the system where components can be mixed, effectively functionally providing a third mixing baffle.

[0024] Another preferred embodiment of Figure 6 illustrates a "two in, one out" design, where two fluid conduits introduce the reaction fluid into the reactor module 3 ("fluid inlet points"), which exits through one outlet ("fluid outlet point"). Figure 6A The preferred design of the mixing baffle arrangement in this embodiment is illustrated. In this embodiment, Figure 6B The mixing baffles of the embodiment exemplify this embodiment, in which a plurality of alternating sections of the fluid conduit are arranged within the reactor module. Each of the alternating sections may or may not include at least one mixing baffle (1, Figure 6A ). Each mixing baffle ("A") is located approximately at the midpoint of the fluid conduit segment in which it exists. The segment is connected to the following segment that does not include a mixing baffle. This arrangement is repeated throughout the reactor module. In the illustrated example, seven (7) alternating segments are included, wherein the first segment, the third segment, the fifth segment, and the seventh segment include mixing baffles, while the second segment, the fourth segment, and the sixth segment do not. Thus, this embodiment provides a plurality of mixing baffles distributed throughout the fluid conduit. In this preferred embodiment, the presence of two fluid outlet points provides another point in the system where the components can be mixed, effectively providing a third mixing baffle in function.

[0025] In a preferred embodiment, the present disclosure provides a system for continuous inline lysis of cells containing plasmid DNA ( Figure 1The system comprises: a disposable flow path, the disposable flow path comprising: a first container, the first container comprising a first fluid, the first fluid comprising cells containing a plasmid nucleic acid (1), the first container being fluidically connected to a first disposable fluid path (1a) via a first pump (P1); a second container (2), the second container comprising a second fluid, the second fluid being an alkaline lysis buffer (lysis buffer input) being fluidically connected to a second disposable fluid path (2a) via a second pump (P2); the first fluid and the second fluid being combined directly or using a mixer into a third disposable fluid path the third fluid pathway (3a) being fluidically connected to at least one third container (3) comprising disposable (e.g., preferably disposable) tubing within which the cells are at least partially lysed to release the plasmid nucleic acid, and if more than one third container (3b) is present, each third container is fluidically connected in series to one another via one or more additional disposable fluid pathways (3b) and comprises an outlet fluidically connected to a fourth disposable fluid pathway (3c) (the third container being a retention tubing cassette); a fourth container (4) comprising a pump (P3) which pumps the cells through the pump (P4) and the outlet (P5) of the fourth container. The invention further comprises a method for merging the fluid in the first disposable fluid pathway (1a) and the fluid in the second disposable fluid pathway (2a) (alkaline lysis buffer) into the third fluid pathway (3a), which is fluidically connected to the at least one disposable flow path; and merging the fourth disposable fluid pathway (3c) and the fifth disposable fluid pathway (4a) directly or using a mixer into a sixth disposable fluid pathway (5), which contains the final product (released plasmids and cell fragments); wherein: the fluid in the first disposable fluid pathway (1a) (cells) and the fluid in the second disposable fluid pathway (2a) (alkaline lysis buffer) are combined into the third fluid pathway (3a), which is fluidically connected to the at least one disposable flow path; a third container comprising disposable retention tubing (retention tubing cassette); maintaining the fluid in the third container (3 / 3b) therein (retention tubing segment) for a sufficient amount of time to lyse cells and release plasmids from the cells while minimizing degradation of the plasmids, and the fluid exiting the retention tubing segment through the fourth fluid pathway (3c); and combining the fluid of the fourth fluid pathway with neutralization buffer from the fourth container (4) in the fifth fluid pathway (4a) either directly or using a mixer, and the fluid exiting the disposable flow path through the seventh fluid pathway (6). In some preferred embodiments, the retention tubing segment (3 / 3b) comprises a plurality of sub-segments fluidically connected to one another, each sub-segment comprising pre-packaged disposable tubing within a rigid container, wherein each sub-segment may contain the same or different lengths and / or types of disposable tubing therein. In a preferred embodiment, the retention tubing segment (3 / 3b) comprises four such sub-segments.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 control (PLC) or a distributed control system (DCS), which has a human-machine interface (HMI) for inputting process recipe parameters, monitoring processes, and collecting process data. In some embodiments, at least one inline analytical instrument 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 such as, but not limited to, pH and conductivity from an inline analytical instrument. In some embodiments, the pH of the alkaline lysis buffer is less than 13 (i.e., pH < 13; for example, preferably any value of about 7 or more, such as 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, or 12.5), optionally wherein the alkaline lysis buffer is about 0.1N to 0.2N NaOH. In some embodiments, the sufficient amount of time is any one of about 2 minutes to about 10 minutes. In some embodiments, the cleavage reactor fluid is connected to a neutralization reactor consisting of a fluid path located downstream of the cleavage reactor, in which a neutralization buffer is added to the cleavage stream and blended with the cleavage stream. In some preferred embodiments, the retaining pipe section comprises pre-packaged disposable tubing within a rigid container. In some preferred embodiments, the fourth fluid pathway comprises a static mixer. In some preferred embodiments, the outlet port fluid is connected to a fifth fluid pathway, which is fluidically connected to a container or another piece of equipment. In some preferred embodiments, the present disclosure provides a method for isolating plasmid DNA from a cell culture, the method comprising: processing cells comprising plasmid DNA by a system disclosed herein.

[0026] The terms "a", "an" and / or "the" generally represent at least one, or one or more. When used as modifiers, the terms "about", "approximately" and the like refer to variations of the numbers that typically occur when performing standard procedures. In some preferred embodiments, "about", "approximately" and the like indicate values ​​within ten percent (i.e., + / - 10%) of the listed values. When preceding a series of numerical values ​​or ranges, the terms "about", "approximately" and the like refer individually to each individual value in the list or range, as if each individual value in the list or range were immediately preceding the term. The terms mean that the values ​​referred to are exact, close or similar. "Optional" or "optionally" means that the event or situation described subsequently may or may not occur, and that the description includes instances in which the event or situation occurs and instances in which the event or situation does not occur. A range (e.g., 90% to 100%) is meant to include the range itself and each individual value within the range, as if each value were listed individually. When expressing such a range, on the other hand, it includes from one specific value and / or to another specific value. Similarly, when values ​​are expressed as approximate values ​​by using the antecedents "about" or "approximately," it should be understood that the particular value forms another aspect. It should also be understood that the endpoints of each range are significant relative to the other endpoint, as well as independently of the other endpoint. A range (e.g., 90% to 100%) is meant to include the range itself and each individual value within the range, as if each value were individually recited.

[0027] All references cited in 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.

[0028] Other embodiments are also disclosed and / or contemplated, and / or may be conceived by one of ordinary skill in the art from this disclosure. Various embodiments of the present disclosure, if any, will be described in detail with reference to the accompanying drawings. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the appended claims. In addition, any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible embodiments of the claimed invention.

[0029] Although certain embodiments have been described in terms of preferred embodiments, it will be understood that changes and modifications will occur to those skilled in the art. It is therefore intended that the appended claims cover all such equivalent variations that fall within the scope of the following claims.

Claims

1. A system for performing continuous inline lysis of cells containing plasmid DNA, the system comprising: A disposable flow path, the disposable flow path comprising: a first container comprising a first fluid containing cells containing a plasmid nucleic acid (1), the first container being fluidically connected to a first disposable fluid passage (1a) via a first pump (P1); a second container (2) comprising a second fluid, the second fluid being an alkaline lysis buffer fluidically connected to a second disposable fluid passage (2a) via a second pump (P2); combining the first fluid and the second fluid into a third disposable fluid passage (3a) directly or using a mixer; The third fluid pathway (3a) is fluidically connected to at least one third container (3), the third container comprising disposable tubing within which the cells are at least partially lysed to release the plasmid nucleic acid, and if more than one third container (3b) is present, each third container is fluidically connected in series to one another via one or more additional disposable fluid pathways (3b), the third containers further comprising an outlet fluidically connected to a fourth disposable fluid pathway (3c); a fourth container (4) comprising a neutralization buffer fluidly connected to the disposable flow path through a fifth disposable fluid passage (4a) via a third pump (P3); and merging the fourth disposable fluid pathway (3c) and the fifth disposable fluid pathway (4a) into a sixth disposable fluid pathway (5), either directly or using a mixer, the sixth disposable fluid pathway containing a final product comprising released plasmids and cell debris; in: merging the fluids in the first disposable fluid pathway (1a) and the second disposable fluid pathway (2a) into the third fluid pathway (3a), the third fluid pathway being fluidically connected to the at least one third container; maintaining the fluid in the third container (3 / 3b) therein for a sufficient amount of time to lyse cells and release plasmids from the cells while minimizing degradation of the plasmids, and the fluid exits the retention tubing segment through the fourth fluid pathway (3c); and The fluid of the fourth fluid pathway is combined with neutralization buffer from the fourth container (4) in the fifth fluid pathway (4a), either directly or using a mixer, and the fluid exits the disposable flow path through a seventh fluid pathway (6).

2. The system of claim 1 , wherein the disposable tubing of the third container (3) comprises a disposable tubing within which the cells are at least partially lysed to release the plasmid nucleic acid, wherein the disposable tubing comprises at least one mixing baffle therein.

3. The system of claim 2 , wherein the conduit includes a mixing baffle provided by at least one first recess located approximately at a midpoint of the fluid conduit, the conduit optionally including at least one second recess positioned between the first recess and a fluid outlet located opposite the fluid inlet of the retention module.

4. The system according to claim 3, wherein the third container (3) comprises a fluid inlet and a fluid outlet.

5. The system according to claim 3, wherein the third container (3) comprises a fluid inlet and at least two fluid outlets.

6. A system according to 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 control (PLC) or a distributed control system (DCS), wherein the process controller has a human machine interface (HMI) for inputting process recipe parameters, monitoring the process and collecting process data.

7. The system of claim 6, wherein at least one inline analytical instrument controls the pH and / or conductivity of at least one fluid, buffer and / or stream.

8. A system according to any preceding claim, wherein the flow of cells, lysis buffer and / or neutralisation buffer is controlled via feedback from an inline analytical instrument, such as but not limited to pH and conductivity.

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

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

11. The system of any preceding claim, wherein the cleavage reactor is fluidly connected to a neutralization reactor consisting of a fluid path located downstream of the cleavage reactor, in which neutralization buffer is added to and blended with the cleavage stream.

12. The system of any preceding claim, wherein the retained tubing section comprises pre-packaged disposable tubing within a rigid container.

13. A system according to any preceding claim, wherein the retention tubing segment comprises a plurality of sub-segments fluidly connected to each other, each sub-segment comprising pre-packaged disposable tubing within a rigid container, the length and / or type of disposable tubing in each sub-segment being the same as or different from the length and / or type of disposable tubing in at least one of the other sub-segments.

14. The system of any preceding claim, wherein the fourth fluid pathway comprises a static mixer.

15. A system according to any preceding claim, wherein the outlet port is fluidly connected to a fifth fluid pathway, the fifth fluid pathway being fluidly connected to a container or another piece of equipment.

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

17. A method for isolating plasmid DNA from a cell culture, the method comprising: Cells comprising plasmid DNA are processed by a system and / or reactor module according to any preceding claim.

Images