Device for cell lysis and method of using the same

Abstract

Methods for isolating intracellular products (e.g., viral particles or nucleic acid molecules) from a cell suspension are provided herein. Methods for mechanically lysing cells are also provided herein. In some embodiments, methods for mechanically lysing cells to isolate intracellular products (e.g., viral particles or nuclear molecules) from a cell suspension are provided herein. Apparatus and cartridges for use in mechanically lysing cells are also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related patent applications This application claims priority to U.S. Provisional Application No. 63 / 456,658 filed on 3 April 2023 and U.S. Provisional Application No. 63 / 532,292 filed on 11 August 2023, each of which is incorporated herein by reference in its entirety.

[0002] This invention relates to a method and system for cell lysis. The invention also relates to a method and system for isolating intracellular products, such as viral particles or nucleic acid molecules, from cells. [Background technology]

[0003] Cell lysis is a method used to recover intact intracellular products. Several cell lysis techniques are available, including mechanical or chemical cell lysis, which can be used across a wide range of cell types. However, not all cell lysis techniques are optimal in terms of lysis efficiency or product recovery. Considerations such as cell volume and quantity, cost, and product preservation are important parameters to consider when selecting a cell lysis technique.

[0004] On an industrial scale, large quantities of cells are grown in bioreactors to obtain bulk products. The lysates obtained from cell lysis are then subjected to further downstream processing for product isolation and purification. While cell lysis for small quantities of cells can be easily optimized, the optimal lysis parameters are not scalable.

[0005] For example, chemical dissolution by surfactant-based methods (e.g., Triton X-100 or Tween-20) is frequently used for large-scale product recovery. Surfactants are a class of molecules whose inherent properties allow for the manipulation (disruption or formation) of hydrophobic-hydrophilic interactions between molecules in biological samples. While surfactants are highly efficient as solvents, dissolution techniques require the use of surfactants by specific weight per unit volume (w / v) for optimal dissolution. However, surfactant-based (e.g., detergent-based) cell lysis is inconsistent and variable across different cell densities, so dissolution efficiency does not scale with cell number and surfactant concentration. Furthermore, while the use of surfactants (e.g., detergent-based) may have been necessary for initial cell lysis or membrane protein extraction, surfactants are difficult to remove from the extracted product, complicating its usefulness in subsequent applications. Other challenges associated with surfactant-based (e.g., detergent-based) dissolution include increased turbidity, increased filter fouling, extreme reduction in clarification and decreased separation fidelity, and process / product instability.

[0006] To circumvent the challenges associated with surfactant-based and / or chemical-based dissolution, physical or mechanical destructive techniques can be used for cell lysis. The most common mechanical method used in this field is microfluidization. This method yields more efficient and consistent dissolution than chemical methods and is commonly used for nucleic acid production and protein product recovery using E. coli and other ectopic expression systems. However, microfluidization involves very high operating pressures (e.g., over 2000 psi). For example, a microfluidizer must operate at 4000 psi for certain eukaryotic cell types to achieve cell lysis, but this process also damages the recovered product. Microfluidizers also require an extremely clean-in-place protocol after each use to avoid cross-contamination of products. Furthermore, microfluidizers are difficult to scale and require high capital expenditure and large investments for various process scales, space, and logistical challenges, posing significant logistical challenges when dealing with cells cultured in large formats such as bioreactors. Dissolution by microfluidization can also require harsh chemicals that are not environmentally friendly. These embodiments reduce the yield of useful products, which is undesirable, especially in the context of biopharmaceuticals. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there is a need to develop novel mechanical lysis methods that do not require high pressure in order to achieve cell lysis in a scalable, controllable, and sterile manner. [Means for solving the problem]

[0008] The inventors have surprisingly discovered that cell lysis and isolation of intracellular products (e.g., polypeptides, viral particles, or nucleic acids) from lysed cells can be achieved using purely mechanical means under mild conditions that do not impair the desired isolated products.

[0009] In a particular embodiment, a method for isolating virus particles or nucleic acid molecules from a cell suspension, (i) Flowing a cell suspension containing viral particles or nucleic acid molecules through a cartridge comprising multiple fibers or tubes, wherein the fibers or tubes have an inner diameter of 0.7 mm or less, and the cells experience a pressure difference of less than 250 psi, thereby lysing some of the cells in the suspension to produce a mixed suspension containing lysed and unlysed cells; (ii) Recirculating the mixed suspension formed in step (i) through a cartridge for one or more further cycles, wherein a further proportion of cells is lysed in each subsequent cycle to produce a target suspension containing lysed cells and cell debris; and (iii) A method for isolating viral particles or nucleic acid molecules from a target suspension containing lysed cells and cell debris is provided herein.

[0010] In some embodiments, the cell suspension includes producer cells expressing viral particles. In some embodiments, the viral particles are rAAV particles. In some embodiments, the viral particles are produced by a triple transfection method. In some embodiments, the cell suspension includes producer cells, and the viral particles are produced by triple transfection of the producer cells. In some embodiments, the viral particles are rAAV particles produced by a triple transfection method, which comprises transfecting host cells with one or more plasmids containing (i) one or more nucleic acids encoding AAV packaging genes (such as nucleic acids encoding AAV Rep protein and nucleic acids encoding AAV Cap protein); (ii) nucleic acid encoding the gene of interest; and (iii) nucleic acid encoding at least one ad helper virus gene. In some embodiments, the viral particles are rAAV particles produced by a transient transfection method. In some embodiments, the viral particles include an AAV serotype, which includes at least one of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and their variants, chimeras, or hybrids thereof.

[0011] In some embodiments, the viral particle includes: (i) AAV packaging gene The virus particles are rAAV particles produced by transfection of a host cell with one or more plasmids containing (i) one of several nucleic acids encoding an AAV Rep protein (such as a nucleic acid encoding an AAV Rep protein and a nucleic acid encoding an AAV Caps protein) and (ii) a nucleic acid encoding the gene of interest. In some embodiments, the virus particles are rAAV particles produced by transfection of a host cell with one or more plasmids containing: (i) one of several nucleic acids encoding an AAV packaging gene (such as a nucleic acid encoding an AAV Rep protein and a nucleic acid encoding an AAV Caps protein); (ii) a nucleic acid encoding the gene of interest; (iii) one or more nucleic acids encoding an AAV ITR (reverse terminal repeat sequence) (e.g., two AAV ITRs); and optionally (iv) a nucleic acid encoding at least one ad helper virus gene. In some embodiments, the ad helper virus gene is a helper gene. In some embodiments, the virus particles are rAAV particles expressed from a producer cell line (PCL). In some embodiments, the PCL comprises a host cell stably incorporating (i) one or more nucleic acids encoding AAV packaging genes (such as nucleic acids encoding AAV Rep protein and nucleic acids encoding AAV Caps protein); (ii) nucleic acid encoding the gene of interest; and (iii) one or more nucleic acids encoding AAV ITRs (e.g., two AAV ITRs). In some embodiments, the viral particles are rAAV particles expressed from a PCL further infected with an ad-helper virus (e.g., adenovirus) expressing one or more helper genes. In some embodiments, the viral particles are rAAV particles produced by a transient transfection method. In some embodiments, the viral particles are lentiviral particles. In some embodiments, the viral particles comprise a recombinant baculovirus expression vector. In some embodiments, the viral particles are rAAV particles expressed from a producer cell line (PCL).In some embodiments, the PCL comprises a host cell stably incorporating (i) one or more nucleic acids encoding an AAV packaging gene; (ii) a nucleic acid (nucleic add) encoding the gene of interest; and (iii) one or more nucleic acids (nucleic add) encoding an AAV TTR (reverse terminal repeat sequence). In some embodiments, the PCL is further infected with an adenovirus. In some embodiments, the adenovirus is an ad helper virus. In some embodiments, the ad helper virus expresses one or more helper genes. In some embodiments, the AAV packaging gene comprises at least an AAV capsid (cap) gene. In some embodiments, the AAV packaging gene comprises at least an AAV replication (rep) gene.

[0012] In some embodiments, the cartridge includes multiple hollow fibers, with the fibers including a tubular side and a shell side. In some embodiments, the multiple hollow fibers constitute a hollow fiber membrane module, the multiple hollow fibers are grouped together to form a hollow fiber bundle, and the hollow fiber bundle is filled into a tube shell.

[0013] In some embodiments, the inner diameter of the fiber containing the hollow fiber membrane is 0.1 mm to about 0.7 mm. In some embodiments, the inner diameter of the fiber containing the hollow fiber membrane is 0.1 mm to about 5 mm. In some embodiments, the inner diameter of the fiber containing the hollow fiber membrane is 0.2 mm to about 0.3 mm.

[0014] In some embodiments, the inner diameter of the fiber containing the hollow fiber membrane is approximately 0.2 mm. In some embodiments, the inner diameter of the fiber containing the hollow fiber membrane is approximately 0.25 mm. In some embodiments, the inner diameter of the fiber containing the hollow fiber membrane is approximately 0.3 mm.

[0015] In some embodiments, cells flowing through the cartridge experience a pressure difference of less than 200 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of less than 150 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of less than 100 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of less than 50 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 5 psi to about 40 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 10 psi to about 40 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 15 psi to about 35 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 20 psi to about 30 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 20 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 22 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 30 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of about 37 psi. In some embodiments, cells flowing through the cartridge experience a pressure difference of approximately 50 psi. In some embodiments, the pressure difference is defined by the difference between the pressure at the cartridge inlet and the pressure at the cartridge outlet.

[0016] In some embodiments, the suspension flows through the cartridge at a certain flow rate. In some embodiments, the flow rate is approximately 10 L / min to approximately 100 L / min. In some embodiments, the flow rate is approximately 50 L / min to approximately 100 L / min. In some embodiments, the flow rate is 200 mL / min. In some embodiments, the flow rate is 360 mL / min. In some embodiments, the flow rate is 600 mL / min. In some embodiments, the suspension flows through the cartridge at a flow rate determined by the cartridge size. In some embodiments, the suspension flows through the cartridge at a flow rate determined by the pressure at the cartridge inlet. In some embodiments, the suspension flows for at least approximately 2000 seconds. -1 The fibers are fed into the cartridge at a shear rate. In some embodiments, the fibers have a thickness of about 0.1 mm to about 0.4 mm.

[0017] In some embodiments, the length of the fibers is approximately 20 cm to 150 cm.

[0018] In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least one cycle. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least five cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least ten cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least twenty cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least thirty cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least forty cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for about one to about fifty cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through a cartridge for about 10 to about 50 cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through a cartridge for about 15 to about 40 cycles. In some embodiments, step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) through a cartridge for about 20 to about 30 cycles.

[0019] In some embodiments, the cell suspension or mixed suspension flows through the luminal side. In some embodiments, the space between the hollow fiber bundle and the tube shell is capped.

[0020] In some embodiments, the total time to complete steps (i) and (ii) is from about 1 hour to about 4 hours.

[0021] In some embodiments, a lysis efficiency of greater than 70% is achieved, and the lysis efficiency is measured by lactate dehydrogenase release, percentage of viable cell count, or percentage of product release per cycle. In some embodiments, a lysis efficiency of greater than 80% is achieved. In some embodiments, a lysis efficiency of greater than 90% is achieved.

[0022] In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are derived from human or a human cell line, and the human cell or human cell line is HEK293 or HeLa. In some embodiments, the mammalian cells are HeLa cells. In some embodiments, the mammalian cells are HEK293 cells. In some embodiments, the mammalian cells are at least one of CHO cells, HEK293 cells, Vero cells, HeLa cells, MDCK cells, BHK cells, or A549 cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are prokaryotic cells. In some embodiments, the prokaryotic cells are Escherichia coli (E. coli) cells.

[0023] In some embodiments, lysis is determined by the percentage of viable cell count. In some embodiments, the percentage of viable cell count is from about 20% to about 99% after 5 cycles. In some embodiments, the percentage of viable cell count is from about 5% to about 90% after 10 cycles. In some embodiments, the percentage of viable cell count is from about 1% to about 70% after 20 cycles.

[0024] In some embodiments, lysis is determined by the percentage of viable cell density. In some embodiments, the percentage of viable cell density is approximately 20% to approximately 99% after 5 cycles. In some embodiments, the percentage of viable cell density is approximately 5% to approximately 90% after 10 cycles. In some embodiments, the percentage of viable cell density is approximately 1% to approximately 70% after 20 cycles.

[0025] In some embodiments, dissolution is determined by the dissolution percentage. In some embodiments, the dissolution percentage is 80% after 18 cycles. In some embodiments, the dissolution percentage is 80% after 11 cycles. In some embodiments, the dissolution percentage is 80% after 8 cycles. In some embodiments, the dissolution percentage is 80% after 5 cycles.

[0026] In some embodiments, a cell suspension is flowed into a container in each continuous cycle. In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is set to a certain temperature. In some embodiments, the temperature is about 25°C to about 37°C. In some embodiments, the bioreactor is 25°C. In some embodiments, the bioreactor is 37°C. In some embodiments, the cartridge and the container are operably connected to form a closed system. In some embodiments, the closed system further includes at least one conduit operably connected to the container and the cartridge, through which the cell suspension flows between the container and the cartridge. In some embodiments, the tubing is made of rubber.

[0027] In some embodiments, separation includes affinity capture. In some embodiments, separation includes ion exchange. In some embodiments, separation includes anion exchange.

[0028] In certain embodiments, a method for mechanically lysing cells is provided herein, comprising flowing a suspension containing a plurality of cells or a population of cells through a cartridge containing a plurality of fibers or tubes, wherein the cells are subjected to a pressure difference of less than 250 psi, thereby lysing a portion of the cells in the suspension.

[0029] In some embodiments, the cells are further recirculated through a cartridge over several cycles to increase the proportion of cells that are lysed.

[0030] In some embodiments, cells are introduced into the cartridge by following a channel.

[0031] In some embodiments, mechanical lysis involves shearing cells. In some embodiments, mechanical cell lysis is achieved by applying one or more external forces to a plurality of cells or cell populations. In some embodiments, one or more external forces are selected from the group consisting of flow, pressure, turbulence, friction, extrusion, impact, and pressure drop.

[0032] In some embodiments, the fibers are hollow fibers. In some embodiments, the hollow fibers constitute a hollow fiber membrane module, the hollow fibers are grouped together to form a hollow fiber bundle, and the hollow fiber bundle is filled into a tube shell. In some embodiments, the hollow fiber membrane bundle is surrounded by a shell that extends longitudinally along the length of the bundle, forming the lumen and shell sides of the hollow fiber membrane module. In some embodiments, the lumen forms a tubular structure. In some embodiments, the space between the hollow fiber bundle and the tube shell is capped. In some embodiments, the hollow fiber membrane module is sterile. In some embodiments, the hollow fiber membrane module is disposable.

[0033] In some embodiments, the number of cycles is determined by the following formula:

number

[0034] In some embodiments, the total dissolution time is approximately 1 to 4 hours.

[0035] In some embodiments, more than 90% of the lysed cells remain.

[0036] In certain embodiments, the present invention provides an apparatus for lysing cells, comprising a container capable of holding a suspension of cells; a cartridge including a plurality of fibers or tubes and an extracapillary space outside the fibers or tubes; and means for connecting the container to the cartridge such that the container and the cartridge form a closed system so that, when operable, the suspension of cells flows through the container and the cartridge multiple times.

[0037] In some embodiments, the means for connecting the container to the cartridge includes a tube. In some embodiments, the fiber or tube has a maximum diameter of about 0.7 mm.

[0038] In some embodiments, the fibers or tubes have a diameter of about 0.1 mm to about 0.5 mm. In some embodiments, the fibers or tubes have a diameter of about 0.2 mm to about 0.3 mm. In some embodiments, the container is a bioreactor.

[0039] In a particular embodiment, a method for isolating virus particles or nucleic acid molecules from cells: (i) Lysing a portion of the cells in a cell suspension containing virus particles or nucleic acid molecules by passing the suspension through a cartridge containing a fibrous porous medium or material, thereby producing a mixed suspension containing lysed and unlysed cells; (ii) Recirculating the mixed suspension of partially lysed cells formed in step (i) through a cartridge for one or more further cycles, wherein further portions of the cells are lysed in each consecutive cycle to produce a target suspension containing lysed cells and cell debris; and (iii) A method for isolating viral particles or nucleic acid molecules from a target suspension containing lysed cells and cell debris is provided herein.

[0040] In some embodiments, the fibrous porous medium or material is a nonwoven material. In some embodiments, the nonwoven material is spunbond, meltblown fiber, felt, or wet-laid material.

[0041] In some embodiments, the fibrous porous medium or material is a woven material. In some embodiments, the fibrous porous medium or material is a membrane.

[0042] In some embodiments, the nonwoven material allows for the retention of at least about 90% of lysed cells and cell debris, including those with a size of at least about 0.1 μm.

[0043] In some embodiments, the pore size of the nonwoven fabric is approximately 20 μm to approximately 60 μm.

[0044] In a particular embodiment, a cartridge for lysing cells, (a) Housing, and (b) A cartridge comprising a plurality of pores, wherein the pores have a maximum or approximately 1 mm in diameter, is provided herein.

[0045] In some embodiments, the pores are embedded within a plurality of hollow fibers having an inner diameter, an outer diameter, and two ends having a length between the ends, with at least one end of each fiber open for a fluid inlet or outlet, and the fibers are arranged parallel to each other.

[0046] In some embodiments, the fibers or tubes do not have pores. In some embodiments, the fibers or tubes include pores. In some embodiments, the pores are less than 300 kDa.

[0047] In some embodiments, the viral particles are produced by a transient transfection method. In some embodiments, the cell suspension includes producer cells that express the viral particles. In some embodiments, the viral particles include an AAV serotype, which includes at least one of AAV1, AAV2, AAV3a, AVV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and their variants, or chimeras, or hybrids thereof.

[0048] In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells include at least one of CHO cells, HEK293 cells, Vero cells, HeLa cells, HEK293 cells, MDCK cells, BHK cells, or A549 cells. [Brief explanation of the drawing]

[0049] [Figure 1A] This is a schematic diagram of a hollow fiber membrane module. [Figure 1B] This diagram shows the flow of buffer and cell suspension through a hollow fiber membrane module. The semicircles indicate cell lysis. [Figure 1C] This is a schematic diagram showing the flow of buffer and cell suspension through a porous membrane. The semicircles represent cell lysis. [Figure 2A] This is a schematic diagram of a container operably connected to a cartridge so that the container and cartridge form a closed system. [Figure 2B] This is a schematic diagram of a container operably connected to a hollow fiber membrane module. [Figure 2C] This is a schematic diagram of a bioreactor operably connected to a hollow fiber membrane module. X indicates a capped outlet. [Figure 2D]This is a schematic diagram of a bioreactor operably connected to a hollow fiber membrane module. A cross-section of the hollow fiber membrane module is shown. X indicates a capped outlet. [Figure 3] This shows the number of viable HEK-293 cells after lysis in various cycles following circulation of HEK-293 cells through a hollow fiber (HF) membrane. [Figure 3-1] Same as above. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4] The percentage of dissolved LDH, the percentage of viable cells, and the percentage of product (rAAV) released are shown as a function of the number of cycles. rAAV release (VG release; square) and LDH release by microfluidization, as well as VG release using surfactant PS-20 (triangle), were used as controls. [Figure 5] This shows the percentage of viable cells as a function of the number of cycles using a 30 psi hollow fiber membrane module with HeLa cells. [Figure 6] This study shows the percentage of viable cells as a function of the number of cycles using HeLa cells and a 40 μm nonwoven fabric at 30 psi. [Figure 7] This shows the lysis percentage as a function of the number of cycles using hollow fiber membrane modules (Cytiva® and Planova® modules) at 20 psi with HEK293 cells. [Figure 8A] The percentage of lysed cells and the percentage of AAV virus genome (vg) released are shown as a function of the number of cycles in a 500L reactor run using a Cytiva® hollow fiber membrane module for HEK293 cells. [Figure 8B] This shows the viable cell density as a function of the mechanical cell lysis cycle. Mechanical cell lysis was performed a total of 6 cycles at 500 L. [Figure 8C]The viability percentages of three different cell lines undergoing mechanical lysis are shown. MCL was performed on 500 L of cells using Cytiva® hollow fibers with a membrane area of ​​150 cm², operated at a pressure of 30 psi and 15 minutes per cycle. [Figure 9] This shows the percentage of viable cells as a function of the number of cycles for HEK293 cells under various conditions (trains 1-6). [Figure 10A] This shows the relationship between differential pressure after lysis based on mechanical cell lysis (MCL) or PS-20 (surfactant) and throughput. MCL was performed for 15 cycles. [Figure 10B] This paper shows the relationship between permeate flux and throughput after mechanical cell lysis (MCL) or PS-20 (surfactant) based lysis. The lysates were subjected to filtration time (TTF) experiments 3 days after lysis at 4°C. NTU is the turbidity unit of turbidimetry. MCL was performed for 15 cycles. [Figure 11] The chromatogram of recombinant AAV purified from cells lysed by mechanical cell lysis is shown. Proteins were quantified using absorbance at 280 nm (UV280); nucleic acids were quantified using absorbance at 254 nm (UV254). [Figure 12] This chromatogram shows the size of recombinant AAV purified from cells lysed by mechanical cell lysis. [Figure 13] This shows dynamic light scattering plots of recombinant AAV purified from cells lysed by mechanical cell lysis or PS-20. Nucleic acids were quantified using absorbance at UV260. [Figure 14A] The results show the increase in lysed percentage and the corresponding relative titer of released rAAV particles using mechanical cell lysis for transient transfection cells (Platform 1A) compared to an un-lysed sample (control). MCL was performed on 5 L of cells using Cytiva® hollow fiber with a membrane area of ​​150 cm² operated at a pressure of 30 psi. [Figure 14B]The results show the increase in lysed percentage and the corresponding relative titer of released rAAV particles using mechanical cell lysis for cell platform 2, compared to an unlysed sample (control). MCL was performed on 5 L of cells using Cytiva® hollow fiber with a membrane area of ​​150 cm² operated at a pressure of 30 psi. [Figure 15] This shows the time course of cell viability when lysed using MCL at various temperatures. The cycle length was set to 15 minutes / cycle. [Figure 16] This shows the amount of full capsid recovered after MCL (microclaving) performed at various temperatures, followed by affinity purification and anion exchange. The complete capsid percentage was determined by mass photometry (MP). [Figure 17A] This shows the lysis percentage (%) of HeLa producer cell line platform (Platform 3) cells over multiple cycles of mechanical cell lysis. Mechanical cell lysis was performed using a 20 cm² Repligen® hollow fiber module (PES, 750 MWCO). Experiments were conducted at 37°C and a pressure of 45–50 psi. [Figure 17B] This shows the percentage viable cell density (VCD%) of HeLa producer cell line platform (Platform 3) cells across multiple cycles of mechanical cell lysis. Mechanical cell lysis was performed using a 20 cm² Repligen® hollow fiber module (PES, 750 MWCO). Experiments were conducted at 37°C and a pressure of 45–50 psi. [Figure 18] This shows the lysis percentage (%) of various HeLa cell lines subjected to different pressure drops over multiple cycles of mechanical cell lysis. Mechanical cell lysis was performed using a 20 cm² Repligen® hollow fiber module (PES, 750 MWCO). [Figure 19] The number of cycles required to achieve 80% lysis of HEK293 cells is shown (Platform 1). Mechanical cell lysis was performed using a 20 cm² Repligen® hollow fiber module (PES, 750 MWCO). [Modes for carrying out the invention]

[0050] Methods for isolating intracellular products (e.g., viral particles or nucleic acid molecules) from cell suspensions are provided herein. Methods for mechanically lysing cells are also provided herein. In some embodiments, methods for mechanically lysing cells to isolate viral particles or nuclear molecules from cell suspensions are provided herein. Apparatus and cartridges for use in mechanically lysing cells are also provided herein.

[0051] Existing methods for isolating intracellular products (e.g., viral particles or nucleic acid molecules) from cell suspensions are inconsistent, not scalable, and can unintentionally damage products during extraction. Therefore, the methods described herein are useful for lysing cells under non-stringent conditions (e.g., lower pressure) to recover viral particles or nucleic acid molecules, compared to existing chemical and mechanical lysis techniques.

[0052] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in their entirety for all purposes, as if each individual publication were incorporated separately by reference. If any definition contained herein contradicts or is inconsistent with any definition contained herein in a patent, application, published application, or other publication incorporated herein by reference, the definition contained herein shall prevail over the definition incorporated herein by reference.

[0053] The section headings used in this specification are for organizational purposes only and should never be interpreted as limiting the subject matter described herein.

[0054] definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter pertains. In some cases, terms that have a generally understood meaning are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from that which is generally understood in the art.

[0055] When used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated: Unless the context requires otherwise, singular terms shall include plurals and plural terms shall include singulars. When used herein and in the appended claims, the singular forms "a," "an," and "it" shall include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "molecule" shall optionally include a combination of two or more such molecules, etc.

[0056] It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments that "comprising," "consisting," and "consisting essentially of."

[0057] As used herein, the term “polynucleotide” refers to a single-stranded or double-stranded nucleic acid polymer with a length of at least 10 nucleotides. In certain embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides, deoxyribonucleotides, or modified forms of any type of nucleotide. The term “polynucleotide” includes, in particular, DNA and / or RNA in single-stranded and double-stranded forms.

[0058] "Isolated" proteins or polypeptides are identified, separated, and / or recovered from components of their natural environment. These contaminants from the natural environment may be materials that interfere with the diagnostic or therapeutic use of the binding protein and may include enzymes, hormones, and other protein or non-protein solutes.

[0059] As used herein, the terms “substantially pure” or “substantially purified” refer to a compound or species that is the dominant species present (i.e., present in abundance on a molar basis compared to any other individual species in the composition). In some embodiments, the species (e.g., a virus or nucleic acid) is purified to an essential homogeneity.

[0060] A "vector" refers to any genetic element that can replicate when associated with appropriate regulatory elements, such as plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions, and can transfer gene sequences between cells. Therefore, this term includes cloning and expression vehicles, as well as viral vectors.

[0061] "AAV vector" means a vector derived from an adeno-associated virus serotype (including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, and AAV-8). An AAV vector may have one or more AAV wild-type genes deleted, preferably all or part of the rep and / or cap genes, but may retain a functional flanking ITR sequence. The functional ITR sequence is necessary for the rescue, replication, and packaging of the AAV virion. Therefore, an AAV vector is defined herein as containing at least the sequence required in cis for viral replication and packaging (e.g., a functional ITR). The ITR does not have to be a wild-type nucleotide sequence and may be modified, for example, by nucleotide insertion, deletion, or substitution, as long as the sequence provides functional rescue, replication, and packaging.

[0062] The terms “recombinant AAV virion,” “rAAV virion,” “AAV vector particle,” “complete capsid,” “flore,” and “complete particle” are defined herein as infectious replication-deficient viruses containing an AAV protein shell in which the AAV ITR capsidizes the desired heterogeneous nucleotide sequence on both sides. In some examples, rAAV virions are produced in a suitable host cell into which sequences specifying the AAV vector, AAV helper function, and accessory function have been introduced. In this way, the host cell is able to encode the AAV polypeptide necessary to package the AAV vector (containing the desired recombinant nucleotide sequence) into infectious recombinant virion particles for subsequent gene delivery.

[0063] "Operationally linked" refers to an arrangement of elements that are functionally linked in such a way that the components described as such can also perform their normal functions.

[0064] The terms “fiber shape” or “hollow fiber shape” refer to the physical properties of a fiber, tube, hollow fiber filter fiber, or hollow fiber tube. These physical properties include, but are not limited to, the fiber or tube material, length, pore size, pore shape, diameter, surface area, and surface roughness.

[0065] The term "complete capsid titer" as used in relation to viral titer refers to the number of virions containing recombinant AAV DNA genome, regardless of whether they are infectious or functional. In some embodiments, the complete capsid titer of a viral vector may be measured in terms of vg / mL. Suitable methods for measuring this titer are known in the art (e.g., quantitative PCR).

[0066] The term "total capsid titer" as used in relation to viral titer refers to the number of virions or AAV protein shells, regardless of infectivity or functionality. In some embodiments, the capsid titer of a viral vector may be measured in terms of cp / mL. Suitable methods for measuring total capsid titer are known in the art (e.g., ELISA).

[0067] Mechanical lysis method of cells The methods described herein provide a method for mechanically lysing cells. In some embodiments, the method involves flowing a suspension containing a plurality of cells or cell populations through a cartridge containing a plurality of fibers or tubes. In other embodiments, the method involves flowing a suspension containing a plurality of cells or cell populations through a cartridge containing a fibrous porous material.

[0068] The flow path of the system is determined by the opening of the closed system disclosed herein. In some embodiments, cells are introduced into the cartridge by following the flow path.

[0069] Mechanical cell lysis (e.g., mechanical lysis of cells or mechanical dissolution of cells) is the disruption of a cell membrane by using one or more mechanical forces. A mechanical force is a physical activity that disrupts a cell by disrupting its cell membrane. Mechanical forces that may be used in accordance with mechanical lysis may include high shear fluid forces. In some embodiments, mechanical cell lysis is the disruption of a cell membrane by using a shear force (e.g., shear stress). In some embodiments, mechanical lysis includes shearing cells. In some embodiments, mechanical cell lysis is achieved by applying one or more external forces to a plurality of cells or a population of cells.

[0070] In some embodiments, cells may be placed under flow within a closed system. The flow applies shear stress to the cell. Shear stress is a mechanical force induced by the friction of the fluid against the apical cell membrane. In some embodiments, the external force is the flow. In some embodiments, cells may be subjected to pressure, which is the ratio of forces applied over a certain area (e.g., the area of ​​the cell). In some embodiments, the external force is pressure. Turbulence is fluid motion characterized by chaotic changes in pressure and velocity. In some embodiments, the external force is turbulence. Cell extrusion is the act of compressing cells, for example, out of pores or membranes. In some embodiments, the external force is extrusion. Collision can be represented, for example, by one or more cells in contact with each other at high speed. In some embodiments, the external force is collision. Pressure drop is the difference in total pressure between two points in a fluid transport network. In some embodiments, the external force is pressure drop. In some embodiments, one or more external forces are selected from the group consisting of flow, pressure, turbulence, friction, extrusion, collision, and pressure drop. Forces can be applied between a buffer and a cell, between two cells, between a fiber and a cell, between a membrane and a cell, or any combination thereof. In some embodiments, one or more external forces are determined by the geometric shape of the hollow fiber.

[0071] The methods described herein also provide methods for mechanically lysing cells under mild conditions that do not impair the integrity of desired intracellular products. In some embodiments, the methods for mechanically lysing cells are equally efficient in extracting intracellular products from cells compared to surfactant-based cell lysis methods. In some embodiments, the methods for mechanically lysing cells are more efficient in extracting intracellular products compared to surfactant-based cell lysis methods. Efficiency can be measured by standard biochemical and biophysical assays, such as those disclosed herein, by parameters such as the recovery of intact products or the purity of the products. In some embodiments, the surfactant-based cell lysis method uses PS-20. In some embodiments, the method involves flowing a suspension containing multiple cells or cell populations through a cartridge containing multiple fibers or tubes, where the cells are subjected to a pressure difference of less than 1,000 psi, less than 500 psi, less than 250 psi, less than 100 psi, less than 50 psi, or less than 25 psi, thereby lysing a portion of the cells. In some embodiments, the pressure difference is determined by the geometric shape of the hollow fibers. In some embodiments, the cells are further recirculated through a cartridge for several cycles to increase cell lysis.

[0072] In some embodiments, the pressure difference is defined by the difference between the pressure at the cartridge inlet and the pressure at the cartridge outlet. In some embodiments, the cell suspension flowing through the cartridge experiences a pressure difference of less than 250 psi. In some embodiments, the pressure difference is less than 200 psi. In some embodiments, the pressure difference is less than 150 psi. In some embodiments, the pressure difference is less than 100 psi. In some embodiments, the pressure difference is less than 50 psi. In some embodiments, the pressure difference is about 5 psi to about 40 psi. In some embodiments, the pressure difference is about 10 psi to about 40 psi. In some embodiments, the pressure difference is about 15 psi to about 35 psi. In some embodiments, the pressure difference is about 20 psi to about 30 psi. In some embodiments, the pressure difference is about 20 psi. In some embodiments, the pressure difference is about 22 psi. In some embodiments, the pressure difference is about 30 psi. In some embodiments, the pressure difference is about 37 psi. In some embodiments, the pressure difference is about 50 psi. In some embodiments, the pressure difference is less than 250 psi, thereby lysing some of the cells in the suspension to produce a partially lysed cell suspension. In some embodiments, the pressure difference is determined by the geometric shape of the hollow fiber.

[0073] In some embodiments, cells are further recirculated through a cartridge for several cycles to increase cell lysis. In some embodiments, the number of cycles is determined empirically. In some embodiments, the number of cycles is not determined by the total number of cell initiations. In some embodiments, the number of cycles is determined by the following formula:

number

[0074] Methods for isolating products from cells This disclosure provides a method for isolating intracellular products (e.g., polypeptides, viral particles, or nucleic acid molecules) from a cell suspension. In some embodiments, the nucleic acid molecule is a closed-end nucleic acid molecule. In some embodiments, the closed-end nucleic acid molecule is a closed-end double-stranded DNA molecule. In some embodiments, the intracellular product is a polypeptide. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the polypeptide is an antigen-binding protein or a fragment thereof.

[0075] In one embodiment, the present disclosure relates to a method for isolating an intracellular product of interest from a cell suspension, (i) Flowing a cell suspension containing viral particles or nucleic acid molecules through a cartridge comprising multiple fibers or tubes, wherein the fibers or tubes have an inner diameter of 0.7 mm or less, and the cells experience a pressure difference of less than 250 psi, thereby lysing some of the cells in the suspension to produce a mixed suspension containing lysed and unlysed cells; (ii) Recirculating the partially lysed cell suspension formed in step (i) through a cartridge for five or more further cycles, thereby lysing further portions of the cells in each consecutive cycle to produce a suspension containing lysed cells and cell debris; and (iii) A method is provided for isolating the target intracellular product from a suspension containing lysed cells and cell debris.

[0076] In some embodiments, the cell suspension is initially contained in a container connected to a cartridge. Examples of fibrous and fibrous porous materials that can be used for the cartridge are described below. As described above, the cells in the suspension are subjected to a pressure difference as they flow through the cartridge, which can cause lysis of at least some cells and produce a partially lysed cell suspension. The partially lysed cell suspension can then be recirculated through the cartridge for several cycles to induce lysis of more cells. The final suspension contains a mixture of lysed cells and cell debris.

[0077] The pressure difference (e.g., pressure drop) experienced by cells flowing through a cartridge is the difference in total pressure between two points in the fluid transport network. The pressure drop occurs when frictional forces, caused by resistance to flow, act on the fluid as it flows through the tube. Piping networks with high relative roughness (e.g., membranes containing pores) affect the pressure drop. High flow velocity and / or high fluid viscosity result in a greater pressure drop across the cartridge. At low velocities, the pressure drop is small or nonexistent. In some embodiments, the pressure difference is determined by the fiber shape of the cartridge. In some embodiments, the pressure difference is defined by the difference between the pressure at the cartridge inlet and the pressure at the cartridge outlet. In some embodiments, the cell suspension flowing through the cartridge experiences a pressure difference of less than 1,000 psi. In some embodiments, the cell suspension flowing through the cartridge experiences a pressure difference of less than 500 psi. In some embodiments, the cell suspension flowing through the cartridge experiences a pressure difference of less than 250 psi. In some embodiments, the pressure difference is less than 200 psi. In some embodiments, the pressure difference is less than 150 psi. In some embodiments, the pressure difference is less than 100 psi. In some embodiments, the pressure difference is less than 50 psi. In some embodiments, the pressure difference is about 5 psi to about 40 psi. In some embodiments, the pressure difference is about 10 psi to about 40 psi. In some embodiments, the pressure difference is about 15 psi to about 35 psi. In some embodiments, the pressure difference is about 20 psi to about 30 psi.

[0078] The cell suspension can be recirculated through a cartridge. The recirculation step involves reintroducing the partially lysed cell suspension into the cartridge. In some embodiments, the cycle includes circulating the suspension from the container through the cartridge, as shown in step (ii). In some embodiments, the cycle includes recirculating the partially lysed cell suspension into the cartridge. In some embodiments, the method includes recirculating the partially lysed cell suspension for at least one cycle. In some embodiments, the method includes recirculating the partially lysed cell suspension for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more further cycles. In some embodiments, the partially lysed cell suspension is recirculated through the cartridge for at least 10 cycles. In some embodiments, the partially lysed cell suspension is recirculated through the cartridge for at least 20 cycles. In some embodiments, the partially lysed cell suspension is recirculated through the cartridge for at least 30 cycles. In some embodiments, the partially lysed cell suspension is recirculated through the cartridge for at least 40 cycles. In some embodiments, the partially lysed cell suspension is recirculated through the cartridge for at least 50 cycles. In some embodiments, the partially lysed cell suspension is recirculated through a cartridge for approximately 1 to 50 cycles. In some embodiments, the partially lysed cell suspension is recirculated through a cartridge for approximately 10 to 50 cycles. In some embodiments, the partially lysed cell suspension is recirculated through a cartridge for approximately 15 to 40 cycles. In some embodiments, the partially lysed cell suspension is recirculated through a cartridge for approximately 20 to 30 cycles. In some embodiments, the cell suspension is flowed into a container in each continuous cycle. In some embodiments, the container is a bioreactor.In some embodiments, the bioreactor is a closed system.

[0079] In some embodiments, each additional cycle generates an additional portion of cells, which are lysed in each subsequent cycle to produce a suspension containing lysed cells and cell debris.

[0080] In some embodiments, the products that may be generated in a cell suspension (e.g., host cells, e.g., producer cells) include macromolecules such as polypeptides, polypeptide complexes, or polynucleotides. Non-limiting examples of polypeptides include antibodies, enzymes, and signaling peptides. In some embodiments, the products are nucleic acid molecules. Certain nucleic acids include, but are not limited to, DNA molecules, RNA molecules (e.g., mRNA), and siRNA molecules.

[0081] In some embodiments, the isolated product is a viral particle. In some such embodiments, the viral particle is an adenovirus (AV), adeno-associated virus (AAV), or lentivirus particle. In some embodiments, the viral particle is produced by a triple transfection method. In some embodiments, the isolated product is closed-end DNA for gene therapy.

[0082] The cells may comprise a nucleic acid composition and an expression vector containing the nucleic acid used to express the product. In some embodiments, the cells are induced to express the product. In some embodiments, the cells constitutively express the product. The nucleic acid can be encapsulated in an expression vector containing appropriate transcription and translational regulatory sequences, including but not limited to signaling and secretory sequences, regulatory sequences, promoters, origins of replication, and select genes.

[0083] In some embodiments, the isolated viral particles may be recombinant viral particles containing a gene expressing the protein of interest. In one embodiment, the viral particles are rAAV containing a transgene expressing the product of interest. rAAV is a non-enveloped single-stranded DNA virus modified to be a replication-deficient entity that can only infect cells and deliver DNA to their nuclei. rAAV particles may contain viral proteins and viral nucleic acids of the same or missing serotype. Currently, more than 40 AAV serotypes are known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J.Virol., 2003, 77(12):6799-810. Various AAV serotypes are used to optimize the transduction of specific target cells or to target specific cell types within specific target tissues. In some embodiments, the viral particles include AAV serotypes. In some embodiments, the AAV serotypes include at least one of the following: AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and their variants, or chimeras, or hybrids thereof. In some embodiments, the viral particles are rAAV particles derived from AAV serotypes (including, but not limited to, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and their variants, or chimeras, or hybrids thereof). In some embodiments, the rAAV viral particles are produced by a triple transfection method.In some embodiments, the viral particles are rAAV particles produced by a triple transfection method, which comprises transfecting a host cell with one or more plasmids containing (i) one or more nucleic acids encoding AAV packaging genes (such as nucleic acids encoding AAV Rep protein and nucleic acids encoding AAV Cap protein); (ii) nucleic acid encoding the gene of interest; and (iii) nucleic acid encoding at least one ad helper virus gene. In some embodiments, the viral particles are rAAV particles produced by a transient transfection method. In some embodiments, the viral particles are rAAV particles produced by transfecting a host cell with one or more plasmids containing: (i) one or more nucleic acids encoding AAV packaging genes (such as nucleic acids encoding AAV Rep protein and nucleic acids encoding AAV Caps protein) and (ii) nucleic acid encoding the gene of interest. In some embodiments, the viral particles are rAAV particles produced by transfection of a host cell with one or more plasmids comprising: (i) one of several nucleic acids encoding an AAV packaging gene (such as a nucleic acid encoding an AAV Rep protein and a nucleic acid encoding an AAV Caps protein); (ii) a nucleic acid encoding the gene of interest; (iii) one or more nucleic acids encoding AAV ITRs (reverse terminal repeats) (e.g., two AAV ITRs); and optionally (iv) a nucleic acid encoding at least one ad helper virus gene. In some embodiments, the viral particles are rAAV particles expressed from a producer cell line (PCL). In some embodiments, the PCL comprises a host cell stably incorporating (i) one or more nucleic acids encoding an AAV packaging gene (such as a nucleic acid encoding an AAV Rep protein and a nucleic acid encoding an AAV Caps protein); (ii) a nucleic acid encoding the gene of interest; and (iii) one or more nucleic acids encoding AAV ITRs (e.g., two AAV ITRs).In some embodiments, the viral particles are rAAV particles expressed from PCLs further infected with an ad-helper virus (e.g., adenovirus) expressing one or more helper genes. In some embodiments, the rAAV particles contain a complete capsid. In some embodiments, the rAAV particles are a complete capsid. In some embodiments, the mechanical cell lysis provided in this disclosure improves the percentage of full capsid titer in the total capsid titer compared to chemical lysis (e.g., using a detergent). In some embodiments, the full capsid titer in rAAV preparations produced using mechanical lysis (e.g., using a mechanical lysis apparatus or method described herein) is about 2 to about 20 times higher than that in rAAV preparations produced using chemical lysis (e.g., using a detergent such as PS-20). In some embodiments, after purification, the percentage of full capsid in the total capsid titer is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, and at least about 99%. In some embodiments, after purification, the percentage of complete capsid is approximately 50% to 95%, approximately 50% to 80%, approximately 60% to 90%, and approximately 50% to 70%. In some embodiments, the percentage of complete capsid is approximately 1% to approximately 60%, for example, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, and approximately 59%. In some embodiments, after purification, the percentage of complete capsid in the total capsid titer is approximately 1% to approximately 60%, for example, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, and approximately 59%. In some embodiments, after purification, the percentage of complete capsids in the total capsid titer is about 1% to about 10%, for example, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, and about 4%.

[0084] Intracellular products (e.g., viral particles or nucleic acid molecules or polypeptides) can be isolated from a suspension containing lysed cells and cell debris by several methods, including column chromatography and cesium chloride (CsCl) gradients. Multiple column purification steps can be used, such as purification by anion exchange column, affinity column and / or cation exchange column. See, for example, International Publication No. 02 / 12455. In some embodiments, viral particles or nucleic acid molecules are separated from a suspension containing lysed cells and cell debris, thereby isolating the viral particles or nucleic acid molecules.

[0085] In some embodiments, the total time required to complete steps (i) and (ii) is approximately 1 to 6 hours. In some embodiments, the total time required to complete steps (i) and (ii) is approximately 1 to 4 hours. In some embodiments, the total time required to complete steps (i) and (ii) is approximately 2 to 4 hours.

[0086] A cell suspension can be grown in any container that allows for the culture of a population of cells. In some embodiments, the container is operably connected to a cartridge to form a closed system. In some embodiments, the cartridge is made of a PVC shell or polycarbonate. In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is operably connected to a cartridge. In some embodiments, a container (e.g., a bioreactor) operably connected to a cartridge forms a closed system. In some embodiments, a cell suspension can be grown in a first container and then transferred to a second container operably connected to a cartridge. In such embodiments, the second container is operably connected to a cartridge. In some embodiments, the closed system further includes at least one conduit operably connected to the container and the cartridge, through which the cell suspension flows between the container and the cartridge. In some embodiments, the tubing is rubber. In some embodiments, the tubing is silicone.

[0087] Figure 2A is a schematic diagram of a container operably connected to a cartridge so that the container and cartridge form a closed system. In one embodiment, the cartridge includes a hollow fiber membrane module (Figure 2B). Figures 2C and 2D are schematic diagrams of a bioreactor operably connected to the hollow fiber membrane module. In Figures 2C and 2D, X indicates a capped outlet.

[0088] In some embodiments, the cartridge includes multiple hollow fibers, with the fibers including a tubular side and a shell side. In some embodiments, the multiple hollow fibers constitute a hollow fiber membrane module, the multiple hollow fibers are grouped together to form a hollow fiber bundle, and the hollow fiber bundle is filled into a tube shell.

[0089] In some embodiments, the cells to be lysed are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are at least one of CHO cells, HEK293 cells, Vero cells, HeLa cells, MDCK cells, BHK cells, or A549 cells. In some embodiments, the cells are derived from humans or human cell lines. In some embodiments, the human cells or human cell lines are HEK293 or HeLa. In some embodiments, the cells are yeast cells. In some embodiments, the cells are insect cells.

[0090] In some embodiments, the cells are prokaryotic cells. In some embodiments, the prokaryotic cells are E. coli cells.

[0091] In some embodiments, the cells to be lysed have a diameter of at least or about 15.5 μm. In some embodiments, the cells to be lysed have a diameter of at least or about 16.9 μm. In some embodiments, the cells to be lysed have a diameter of at least or about 20 μm. In some embodiments, the cells to be lysed have a diameter of about 10 μm to about 20 μm.

[0092] In some embodiments, the lysed cells have a high cell density. In some embodiments, the lysed cells have a high live cell density. In some embodiments, the lysed cells are concentrated at a concentration of approximately 100,000 cells / mL to approximately 200 million cells / mL. In some embodiments, the lysed cells are concentrated at a concentration of approximately 500,000 cells / mL to approximately 100 million cells / mL. In some embodiments, the lysed cells are concentrated at a concentration of approximately 500,000 cells / mL to approximately 50 million cells / mL. In some embodiments, the lysed cells are concentrated at a concentration of approximately 500,000 cells / mL to approximately 50 million cells / mL. In some embodiments, the lysed cells are concentrated at a concentration of approximately 1 million cells / mL to approximately 50 million cells / mL. In some embodiments, the lysed cells are concentrated at a concentration of approximately 10 million cells / mL to approximately 50 million cells / mL. In some embodiments, the lysed cells are concentrated at a concentration of approximately 10 million cells / mL to approximately 25 million cells / mL. In some embodiments, the lysed cells are at a concentration of approximately 1 million cells / mL to approximately 25 million cells / mL. In some embodiments, the lysed cells are at a concentration of approximately 5 million cells / mL to approximately 15 million cells / mL. In some embodiments, the lysed cells are at a concentration of approximately 50 million cells / mL to approximately 100 million cells / mL. In some embodiments, the lysed cells are at a concentration of approximately 100 million cells / mL to approximately 200 million cells / mL.In some embodiments, the number of cells lysed is at least about 100,000 cells / mL, at least about 500,000 cells / mL, at least about 1,000,000 cells / mL, at least 2,000,000 cells / mL, at least 3,000,000 cells / mL, at least about 4,000,000 cells / mL, at least about 5,000,000 cells / mL, at least 6,000,000 cells / mL, at least 7,000,000 cells / mL, at least 8,000,000 cells / mL, at least 9,000,000 cells / mL, at least about 10,000,000 cells / mL, at least about 12,000,000 cells / mL, at least about 15,000,000 cells / mL, at least about 20,000,000 cells / mL, at least 25,000,000 cells / mL, and at least The concentrations are 30 million cells / mL, at least 40 million cells / mL, at least 45 million cells / mL, at least 46 million cells / mL, at least 47 million cells / mL, at least 48 million cells / mL, at least 49 million cells / mL, at least about 50 million cells / mL, at least 51 million cells / mL, at least 52 million cells / mL, at least 53 million cells / mL, at least 54 million cells / mL, at least 55 million cells / mL, at least about 75 million cells / mL, at least about 100 million cells / mL, at least 150 million cells / mL, and at least about 200 million cells / mL. In some embodiments, the lysed cells are at a concentration of 2 million cells / mL. In some embodiments, the lysed cells are at a concentration of 4 million cells / mL.

[0093] Flow rate is a measure of the volume of liquid that moves within a given time. In some embodiments, a cell suspension flows through a cartridge at a certain flow rate. In some embodiments, the flow rate is based on various cartridge factors (e.g., fiber shape or hollow fiber shape), such as fiber diameter, fiber length, and number of fibers. For example, the flow rate can be determined based on the shear rate at the fiber walls. The pressure drop and flow rate through the cartridge may be related to the shear rate. In some embodiments, the suspension flows for at least about 2000 seconds. -1 It flows into the cartridge at a shear rate of . In some embodiments, the shear rate is about 50s -1 ~About 100,000 seconds -1 That's all.

[0094] In some embodiments, the cell suspension is flowed into the cartridge at a flow rate of about 10 L / min to about 100 L / min. In some embodiments, the cell suspension is flowed into the cartridge at a flow rate of at least about 0.1 mL / min, at least about 1 mL / min, at least about 10 mL / min, at least about 100 mL / min, at least about 1 L / min, at least about 10 L / min, and at least about 100 L / min. In some embodiments, the partially lysed cell suspension is flowed into the cartridge at a flow rate of about 10 L / min to about 100 L / min. In some embodiments, the partially lysed cell suspension is flowed into the cartridge at a flow rate of about 20 L / min to about 50 L / min. In some embodiments, the partially lysed cell suspension is flowed into the cartridge at a flow rate of at least about 0.1 mL / min, at least about 1 mL / min, at least about 10 mL / min, at least about 100 mL / min, at least about 1 L / min, at least about 10 L / min, and at least about 100 L / min. In some embodiments, the cell suspension flows only through the inside of a fiber or tube.

[0095] Cells can be held in a container operably connected to a cartridge, such that the container and cartridge form a closed system. In some embodiments, the container is temperature-controlled. Temperature control of the container makes it possible to incubate or hold cells at a set temperature. In some embodiments, the temperature is the temperature at which the cells grow. In some embodiments, the temperature is about 25°C to about 37°C. In some embodiments, the temperature is about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, or about 37°C. In some embodiments, the container is a bioreactor. In some embodiments, the bioreactor is set to a certain temperature. In some embodiments, the temperature of the bioreactor is about 25°C to about 37°C. In some embodiments, the temperature of the bioreactor is about 25°C. In some embodiments, the temperature of the bioreactor is 25°C. In some embodiments, the temperature of the bioreactor is about 37°C. In some embodiments, the bioreactor temperature is 37°C. In some embodiments, the bioreactor is not maintained at a certain temperature.

[0096] The separation of viral products (e.g., rAAV) or nucleic acid molecules from a target suspension containing lysed cells and cell debris can be achieved by targeting various properties of the product or molecule to be separated, such as size or charge. One example of a separation technique is affinity capture. In some embodiments, the target viral product or nucleic acid molecule includes a portion for affinity capture. The portion for affinity capture is recognized by a matrix or resin containing a binding portion (e.g., a protein, polypeptide, or ligand) that allows the viral product or nucleic acid to be retained by the immobilized matrix or resin. In some embodiments, the separation includes affinity capture. Another example of a separation technique is ion exchange. Ion exchange uses a charged matrix (e.g., resin or beads) (e.g., negatively charged or positively charged). Under certain buffer conditions, the target viral product or nucleic acid molecule preferentially interacts with and is retained by the charged matrix, while the rest of the suspension is not retained. For example, anion exchange uses a positively charged resin to bind negatively charged molecules. In some embodiments, the separation includes ion exchange. In some embodiments, the separation includes anion exchange.

[0097] Fiber or tube In one embodiment, the present disclosure relates to a method for isolating an intracellular product of interest from a cell suspension, (i) Producing a partially lysed cell suspension by flowing a cell suspension containing the target intracellular product (e.g., viral particles or nucleic acids) through a cartridge comprising multiple fibers or tubes; (ii) Recirculating the partially lysed cell suspension formed in step (i) through a cartridge for five or more further cycles, thereby lysing further portions of the cells in each consecutive cycle to produce a suspension containing lysed cells and cell debris; and (iii) A method is provided for isolating the target intracellular product from a suspension containing lysed cells and cell debris.

[0098] Fibers or tubes generally have an inner diameter and an outer diameter. The space measured by the inner diameter may also be called the lumen. Therefore, the inner diameter may also be called the lumen diameter. The flow path of the cell suspension passes through the inner portion of the fiber or tube. The inner diameter can have a significant effect on the flow velocity of the cell suspension. In some embodiments, the fiber or tube has an inner diameter of 0.7 mm or less. In some embodiments, the fiber or tube has an inner diameter of 0.6 mm or less. In some embodiments, the fiber or tube has an inner diameter of 0.5 mm or less. In some embodiments, the fiber or tube has an inner diameter of 0.4 mm or less. In some embodiments, the fiber or tube has an inner diameter of 0.3 mm or less. In some embodiments, the fiber or tube has an inner diameter of 0.2 mm or less. In some embodiments, the fiber or tube has an inner diameter of about 0.1 mm to about 0.7 mm. In some embodiments, the fiber or tube has an inner diameter of about 0.2 mm to about 0.5 mm. In some embodiments, the fiber or tube has an inner diameter of about 0.2 mm to about 0.3 mm. In some embodiments, the fiber or tube has an inner diameter of about 0.2 mm. In some embodiments, the fiber or tube has an inner diameter of about 0.25 mm. In some embodiments, the fiber or tube has an inner diameter of about 0.3 mm.

[0099] In some embodiments, the cell suspension flowing through the cartridge experiences a pressure difference of less than 1,000 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 750 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 500 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 300 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 250 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 200 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 150 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 100 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 75 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 60 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 50 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 40 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 30 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of less than 20 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of about 5 psi to about 100 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of about 5 psi to about 40 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of about 10 psi to about 40 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of about 15 psi to about 35 psi. In other embodiments, the cell suspension flowing through the fiber or tube experiences a pressure difference of about 20 psi to about 30 psi.In some embodiments, the cell suspension flowing through the fiber or tube is subjected to a pressure difference of about 50 psi, about 45 psi, or about 35 psi.

[0100] In some embodiments, the cartridge contains multiple hollow fibers. In some such embodiments, the multiple hollow fibers are grouped together to form a hollow fiber bundle, which is then filled into a tube shell. In some embodiments, cells flowing through the hollow fiber membrane are subjected to the pressure difference defined above. The hollow fiber membrane module is a liquid-tight housing in which multiple fibers are arranged in parallel (Figure 1A).

[0101] The hollow fiber module contains hollow fibers having various hollow fiber shapes. The hollow fiber shape, while not limited to these, represents fiber properties that affect mechanical cell lysis, such as surface roughness, pore shape, pore diameter, and fiber diameter.

[0102] A hollow fiber module comprises long porous fibers (e.g., filaments) having varying diameters. Pores are embedded within multiple fibers. Each fiber has an inner diameter, an outer diameter, two ends having a length between the ends, at least one end open for a fluid inlet or outlet, and fibers arranged parallel to each other. A hollow fiber module has two compartments within a cartridge. The in-capillary (IC) space represents the space within the hollow fiber, and the out-of-capillary (EC) space represents the space surrounding the hollow fiber. Hollow fibers generally have a membrane coated on the outside of a porous fiber support. Individual fibers are bundled together by joining the fibers to each other. In some embodiments, the hollow fiber membrane is composed of modified polyethersulfone, polysulfone, polyethersulfone, mixed cellulose esters, etc. In some embodiments, the hollow fiber membrane is composed of a compound of materials such as polysulfone, polyethersulfone, polyvinylidene fluoride (PVDF), hydrophilic PVDF membranes, etc. In some embodiments, the hollow fiber membrane is composed of polypropylene, urethane, polyethylene terephthalate, or polyetheretherketone. In some embodiments, the hollow fiber membrane is composed of ceramic, metal film, or mixed matrix device.

[0103] In some embodiments, the hollow fiber has an inner diameter of 0.7 mm or less. In some embodiments, the hollow fiber has an inner diameter of 0.6 mm or less. In some embodiments, the hollow fiber has an inner diameter of 0.5 mm or less. In some embodiments, the hollow fiber has an inner diameter of 0.4 mm or less. In some embodiments, the hollow fiber has an inner diameter of 0.3 mm or less. In some embodiments, the hollow fiber has an inner diameter of 0.2 mm or less. In some embodiments, the hollow fiber has an inner diameter of about 0.2 mm to about 0.5 mm. In some embodiments, the hollow fiber has an inner diameter of about 0.2 mm to about 0.3 mm. In some embodiments, the hollow fiber has an inner diameter of about 0.2 mm. In some embodiments, the hollow fiber has an inner diameter of about 0.25 mm. In some embodiments, the hollow fiber has an inner diameter of about 0.3 mm.

[0104] In some embodiments, the pores of the hollow fibers are embedded in a membrane. In some embodiments, the pores have a diameter of about 0.1 mm to about 1 mm. In some embodiments, the pores have a diameter of about 0.3 mm to about 1 mm. In some embodiments, the pores have a molecular weight cutoff of about 0.3 mm to 0.5 mm. In some embodiments, the pores have a diameter of about 100 kDa to about 700 kDa. In some embodiments, the pores have a molecular weight cutoff of about 100 kDa to about 500 kDa. In some embodiments, the pores have a molecular weight cutoff of about 100 kDa to about 300 kDa. In some embodiments, any of the aforementioned pore diameters is the average pore diameter across the fiber bundle. In some embodiments, the hollow fibers do not have pores.

[0105] In some embodiments, the shell side of the hollow fiber module is completely capped, thus directing the flow through the lumen (e.g., the intercapillary space) without passing through the pores of these fibers. In some embodiments, the extracapillary space is capped. In some embodiments, the flow directs the cell suspension through the length of the fiber rather than through the pores. In some embodiments, the flow through the tubular structure causes intentional damage to the cells in the suspension. An example of an implementation is shown in Figure 1B. In some embodiments, the intentional damage is cell lysis.

[0106] Sterilization is the process of creating a material that is free from contaminants such as other cells. In some embodiments, the hollow fiber membrane module is sterile. In some embodiments, the hollow fiber membrane module is disposable. In some embodiments, the hollow fiber membrane module is not disposable. In some embodiments, the hollow fiber membrane module is reusable. In some embodiments, the hollow fiber membrane module is cleaned before reuse.

[0107] In some embodiments, the hollow fibers are cellulose or synthetic polymers. In some embodiments, the cellulosic hollow fibers are made from cellulose acetate or copper ammonia rayon. In some embodiments, the synthetic polymers are made from polysulfone, polyamide, or polyacrylonitrile. In some embodiments, the hollow fibers are made from 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0108] In some embodiments, the hollow fibers have a thickness of about 0.1 mm to about 0.4 mm. In some embodiments, the hollow fibers have a thickness of about 0.3 mm to about 0.4 mm.

[0109] In some embodiments, the length of the hollow fiber is approximately 20 cm to approximately 150 cm. In some embodiments, the length of the hollow fiber is approximately 50 cm to approximately 150 cm. In some embodiments, the length of the hollow fiber is approximately 100 cm to approximately 150 cm.

[0110] Table 1 contains a table of exemplary hollow fiber membrane modules that may be used in the methods described herein.

[0111] [Table 1]

[0112] Fibrous porous medium or material In another aspect, the present disclosure relates to a method for isolating an intracellular product of interest from a cell suspension, (i) Lysing a portion of the cells by flowing a cell suspension containing the target intracellular product (e.g., viral particles or nucleic acid molecules) through a cartridge containing a fibrous porous medium or material; (ii) Recirculating the partially lysed cell suspension formed in step (i) through a cartridge for five or more further cycles, wherein further portions of the cells are lysed in each subsequent cycle; (iii) A method is provided which includes separating the target intracellular product from the cells and cellular debris, thereby isolating the target intracellular product.

[0113] In some embodiments, the cartridge may include (a) a housing and (b) a fibrous porous material. Non-limiting examples of porous materials include nonwoven fabrics (spunbond, meltblown fibers, felt, wet fiber media), woven fabrics (cloths), membranes, or any porous media. In some embodiments, the nonwoven material includes gradient layers of nonwoven fibers, cellulose, and diatomaceous earth. The nonwoven fibers may optionally include polypropylene, polyethylene, polyester, nylon, or mixtures thereof. In some embodiments, the nonwoven material includes PET, glass fibers, polylactic acid, PVDF, PTFE, or PAN. In some embodiments, the pore size of the fibrous porous material is about 10 μm to about 70 μm. In some embodiments, the pore size of the fibrous porous material is about 20 μm to about 60 μm. In some embodiments, the pore size of the fibrous porous material is about 30 μm to about 50 μm. In some embodiments, the pore size of the fibrous porous material is about 15 μm to about 70 μm. In some embodiments, the pore size of the fibrous porous material is about 20% to about 90% of the cell diameter. In some embodiments, the pore size of the fibrous porous material is about 30% to about 90% of the cell diameter. In some embodiments, the pore size of the fibrous porous material is about 50% to about 80% of the cell diameter. In some embodiments, the pore size of the fibrous porous material is about 70%, about 80%, or about 90% of the cell diameter.

[0114] In some embodiments, the fibrous porous material is a nonwoven fabric material. In some embodiments, the pore size of the nonwoven fabric material is about 10 μm to about 70 μm. In some embodiments, the pore size of the nonwoven fabric material is about 20 μm to about 60 μm. In some embodiments, the pore size of the nonwoven fabric material is about 30 μm to about 50 μm. In some embodiments, the pore size of the nonwoven fabric material is about 15 μm to about 70 μm. In some embodiments, the pore size of the nonwoven fabric material is about 20% to about 90% of the cell diameter. In some embodiments, the pore size of the nonwoven fabric material is about 30% to about 90% of the cell diameter. In some embodiments, the pore size of the nonwoven fabric material is about 50% to about 80% of the cell diameter. In some embodiments, the pore size of the nonwoven fabric material is about 70%, about 80%, and about 90% of the cell diameter. In some embodiments, the nonwoven material allows for the retention of at least about 60% (e.g., at least about 70%, about 80%, about 90%) of lysed cells and cell debris, including sizes of at least about 0.1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, or about 500 μm. In some embodiments, the flow guides the cell suspension through the pores of the porous material. In some embodiments, the flow through the pores causes intentional damage to the cells in the suspension. In some embodiments, the intentional damage is cell lysis. Figure 1C shows the flow of a cell suspension through a fibrous porous material.

[0115] Analytical dissolution efficiency Techniques for analyzing lysis efficiency useful for the apparatus, apparatus and methods provided herein are provided herein. When determining lysis efficiency, an initial sample is isolated and then compared with a sample collected after processing (e.g., after lysis). Lysis efficiency can be determined by measuring cell lysis and analyzing any number of parameters. For example, product release, viable cell number, viable cell density, percentage, lysis percentage (e.g., rate of lysis), relative titer, or intracellular protein release are parameters that can be measured by nucleic acid / protein-based or microscope-based assays to determine lysis efficiency. In some embodiments, lysis efficiency is measured by evaluating the initial viable cell density, the final viable cell density, and determining the rate of decrease between the initial and final viable cell densities (e.g., a 90% lysis efficiency in this method corresponds to a 90% decrease between the initial and final viable cell densities).

[0116] The lysis efficiency percentage can also be expressed as the lysed percentage (e.g., lysis percentage or lysed cell percentage). For example, the lysed percentage can be determined by assaying the number and / or density of viable cells before and after treatment. A decrease in the number or density of viable cells indicates the degree of cell lysis. In some embodiments, the lysis rate achieved by mechanical cell lysis is about 15% and about 20% after 5 cycles or less. In some embodiments, the lysed percentage is about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, and about 70% after 10 cycles. In some embodiments, the lysed percentage is about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, and about 75% after 15 cycles. In some embodiments, the dissolved percentages after 20 cycles are approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, and 80%.

[0117] Intracellular products, such as polypeptides that cannot be secreted and are located at specific cellular locations (e.g., recombinant protein particles), viral particles, or nucleic acids, require cell lysis for product release. In some embodiments, the cells include producer cells. In some embodiments, the lysis efficiency is determined by the release of the product. In some embodiments, the product is viral particles. In some embodiments, the viral particles are produced by triple transfection. In some embodiments, the viral particles are rAAV particles. In some embodiments, the viral particles are produced by the triple transfection method. In some embodiments, the viral particles are lentiviral particles. In some embodiments, the viral particles are produced by triple transfection of producer cells.

[0118] Relative titer is a measure of the change in viral titer of a sample. Relative titer can also be used as an indicator for measuring the amount of virus released from cells. Relative titer may be expressed as a percentage or a change factor and may be determined by methods known in the art. Therefore, relative titer is useful, for example, to determine the amount of viral titer in the supernatant or to determine the amount of virus released from cells before and after sample processing (e.g., lysis, e.g., mechanical cell lysis or surfactant-based lysis). For example, relative titer can be calculated by sampling the supernatant for viral titer after processing and comparing it to the viral titer in the supernatant before lysis. As disclosed herein, determining viral titer for the purpose of evaluating relative titer may be determined by using methods known in the art. In some embodiments, viral titer is determined by qPCR. In some embodiments, viral titer is determined by ELISA. In some embodiments, viral titer is determined by Western blotting. In some embodiments, lysis efficiency is measured by evaluating relative titer. In some embodiments, the relative titer achieved by mechanical cell lysis disclosed herein is about 2 to 10 times that of a non-lysis control. In some embodiments, the relative titer achieved by mechanical cell lysis disclosed herein is about 3 to 8 times that of a non-lysis control. In some embodiments, the relative titer achieved by mechanical cell lysis disclosed herein is about 4 to 8 times that of a non-lysis control. In some embodiments, the relative titer achieved by mechanical cell lysis disclosed herein is about 2, 3, 4, 5, 6, 7, 8, 9 to 10 times that of a non-lysis control. In some embodiments, the relative titer achieved by mechanical cell lysis disclosed herein is about 2, 3, 4, 5, 6, 7, 8, 9 to 10 times that of a non-lysis control. In some embodiments, the relative titer achieved is about 2 or more that of a non-lysis control after 10 cycles of lysis. In some embodiments, the relative titer achieved is about 2.5, 3, 3.5, 4 or more that of a non-lysis control after 15 cycles of lysis.In some embodiments, the relative titer is approximately 2.5 times, 3 times, 3.5 times, 4 times, 5 times, 6 times, or more compared to the non-dissolved control after 20 cycles of dissolution.

[0119] Viral vectors (e.g., rAAV particles) or nucleic acids can be measured using a variety of assays known in the art, including, but not limited to, measuring particle production or nucleic acid production quantity and / or rate, quantifying protein, capsid, or nucleic acid production (e.g., after purification using any of the methods described herein), measuring transduction efficiency, and producing nucleic acids or proteins (e.g., rAAV capsid protein) (e.g., when assayed by Western blotting or PCR-based methods (e.g., qPCR)). The quality of rAAV can be further determined by techniques such as dynamic light scattering (DLS), size exclusion chromatography (SEC), and analytical ultracentrifugation (AUC). In some embodiments, solubility is determined by rAAV quality, which is assessed by dynamic light scattering, size exclusion chromatography, or analytical ultracentrifugation.

[0120] The viable cell count is a method used in cell culture to determine the number of viable cells in a culture and to distinguish between viable and dead cells, and can be expressed as a function of viable cells relative to total cells (e.g., total cells include both viable and dead cells) or as a function of initial viable cell density compared to final viable cell density. Non-limiting examples of methods for determining the viable cell count or percentage of viable cells include the use of trypan blue, a hemocytometer, or an automated cell counter. In some embodiments, lysis efficiency is determined by the viable cell percentage. In some embodiments, lysis efficiency is measured by calculating the viable cell percentage. In some embodiments, the viable cell percentage is determined by evaluating the initial viable cell density, the final viable cell density, and determining the rate of decrease between the initial and final viable cell densities (e.g., a 90% lysis efficiency determined by this method corresponds to a 90% decrease between the initial and final viable cell counts; for example, a 90% lysis efficiency determined by this method corresponds to a 10% viable cell percentage or 90% lysed cells). In some embodiments, the percentage of viable cells decreases after one or more cycles. In some embodiments, the percentage of viable cells decreases after one cycle. In some embodiments, the percentage of viable cells decreases by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, and at least 30% after one cycle. In some embodiments, the percentage of viable cells decreases further after one or more additional cycles. In some embodiments, the percentage of viable cells decreases after one cycle and then decreases further after additional cycles.In some embodiments, the percentage of viable cells decreases by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, and at least 30% in each further cycle. In some embodiments, the percentage of viable cells does not decrease after it has been determined that the percentage of viable cells is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, and about 10%.

[0121] In some embodiments, the percentage of viable cells is approximately 20% to 99% after 5 cycles. In some embodiments, the percentage of viable cells is approximately 5% to 90% after 10 cycles. In some embodiments, the percentage of viable cells is approximately 1% to 70% after 20 cycles.

[0122] In some embodiments, the percentage of viable cells after one cycle is approximately 50%, 55%, 56%, 57%, 58%, and 59%. In some embodiments, the percentage of viable cells after two cycles is approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 56%, 57%, 58%, and 59%. In some embodiments, the percentage of viable cells after four cycles is approximately 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. In some embodiments, the percentage of viable cells after five cycles is approximately 10%, 11%, 12%, 13%, 14%, 15%, and less than 20%.

[0123] In some embodiments, solubility is determined by intracellular protein release. In some embodiments, the protein is lactate dehydrogenase (LDH). Lactate dehydrogenase release can be measured by any number of methods. For example, the identity of the protein is confirmed via antibody, and the concentration released into the suspension relative to the concentration contained in the cell is determined by Western blotting.

[0124] In some embodiments, the methods described herein achieve a solubility of at least 70%, the solubility of which is measured by lactate dehydrogenase release per cycle, percentage of viable cells, or percentage of product release. In some embodiments, the methods described herein achieve a solubility of at least 70%, the solubility of which is measured by lactate dehydrogenase release per cycle, percentage of viable cells, or percentage of product release. In some embodiments, a solubility of at least 70% is achieved within 1 to 4 hours.

[0125] In some embodiments, the method described herein achieves a solubility of at least 80%, the solubility of which is measured by lactate dehydrogenase release, percentage of viable cells, or percentage of product release per cycle. In some embodiments, the method described herein achieves a solubility of at least 80%, the solubility of which is measured by lactate dehydrogenase release, percentage of viable cells, or percentage of product release per cycle. In some embodiments, a solubility of at least 80% is achieved within 1 to 4 hours. In some embodiments, a solubility of at least 80% is achieved within 18 cycles. In some embodiments, a solubility of at least 80% is achieved within 11 cycles. In some embodiments, a solubility of at least 80% is achieved within 8 cycles. In some embodiments, a solubility of at least 80% is achieved within 5 cycles.

[0126] In some embodiments, the methods described herein achieve a solubility of at least 90%, the solubility of which is measured by lactate dehydrogenase release per cycle, percentage of viable cells, or percentage of product release. In some embodiments, the methods described herein achieve a solubility of at least 90%, the solubility of which is measured by lactate dehydrogenase release per cycle, percentage of viable cells, or percentage of product release. In some embodiments, a solubility of at least 90% is achieved within 1 to 4 hours. [Examples]

[0127] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0128] Example 1: Cell lysis by flowing a cell suspension through one or more cartridges. In this example, mammalian cells containing intracellular recombinant adeno-associated virus (rAAV) were lysed using the method disclosed herein. The objective of the experiment was to achieve the maximum recovery rate of rAAV after cell lysis.

[0129] Figure 1A is a schematic diagram showing a commercially available hollow fiber (HF) module, which is a bundle of hollow fibers (e.g., hollowed-out fibers) arranged within a shell. Hollowed fibers contain pores in the fiber walls. The inner surface of the hollow fiber is referred to as the lumen or "lumen side," and the outer surface of the fiber faces the module shell, and is therefore referred to as the "shell side." These hollow fiber modules are available in various configurations (e.g., hollow fiber shapes). For example, variables include lumen diameter (e.g., fiber inner diameter or lumen ID), hollow fiber length, hollow fiber pore diameter, and total fiber area. These modules are fully sterile and can be used in disposable settings. These commercially available hollow fiber modules are essentially plug-and-play devices with very stable and robust operation. Unlike typical methods of operating these modules, the shell side of the module is fully capped, thus directing flow only through the lumen and not through the pores of these fibers. Figure 1B illustrates how cells are lysed through hollow fibers by pressure fluctuations, turbulence, and other external forces as described above. These fibers or tubes have various cross diameters depending on the application and operating time. Figure 2B shows a container (e.g., a bioreactor) operably connected to a hollow fiber module. Cells held within the container (e.g., a bioreactor) are recirculated through the hollow fiber module to achieve cell lysis.

[0130] Various commercially available hollow fiber modules were used in the study (Table 1). These modules were selected based on their different hollow fiber morphologies (e.g., lumen ID, length, pore diameter, and other factors). The shell side of the hollow fiber modules was capped, preventing cells from passing through the pores.

[0131] The assembly consisted of using 24 or larger sizes of tubing for flow rates exceeding 100 mL / min to reduce the effect of the tubing on shear, and the experiments were performed using a peristaltic pump. In particular, the pump alone in the absence of hollow fibers did not make a significant or measurable contribution to cell shear. Pressure was monitored using a Pendotech pressure sensor with a PMAT system. When reusing hollow fiber modules, they were washed with 0.1N NaOH, but in most experiments, the modules were used only once.

[0132] Dissolution efficiency was quantified using various methods and techniques. Total cell count, along with viable cell count, was quantified using ViCell. Lactate dehydrogenase (LDH) levels, a cytosolic protein released during necrosis, were quantified using CEDEX Bio Analyzer. rAAV release was also quantified using qPCR for most of the experiments performed. rAAV quality data were obtained by treating the bioreactor with typical downstream processes. Qualitative techniques included dynamic light scattering (DLS), size exclusion chromatography (SEC), and analytical ultracentrifugation (AUC).

[0133] result Unlysed mammalian cells (HEK293) were observed to have an average diameter in the range of 20 μm. The progression of cell lysis was monitored by the disappearance of a peak around 20 μm. The cell count distribution of HEK293 at various cycle counts using the Repligen® hollow fiber module (HF) was observed to decrease as the number of cycles increased, down to a count in the 10 μm range after 16 cycles (Figure 3). This lysis process was observed to be gradual, requiring several cycles and ensuring that the rAAV was neither damaged nor sheared. The cells were also observed to be efficiently lysed without generating large cell aggregates.

[0134] For HEK293, the percentage of lysation by LDH, the percentage of viable cells, and the percentage of product release as a function of the number of cycles were determined for each parameter (Figure 4). LDH release (using microfluidization) and the percentage of viable cells followed the same trend and were observed to be inversely proportional. The percentage of lysed cells followed a traditional exponential decay model.

number

[0135] The percentage of viable cells was also determined for different mammalian cell lines (HeLa). In HeLa cells, an 80% reduction in viable cell count was reached after 30 cycles (Figure 5). Table 2 summarizes the results observed for HEK293 cells and HeLa cells. To further investigate the effect of initial conditions on lysis efficiency, initial viability and total cell density (TCD) are also reported. For several very different TCDs and initial cell viability, lysis efficiency was within the range of 10% after 17–21 cycles. This further demonstrates the independence of mechanical lysis performance from initial conditions.

[0136] [Table 2]

[0137] The same study was performed with HeLa cells using a nonwoven fabric material with a pore size of 40 μm (Figure 1C). When the nonwoven fabric material was used for cell lysis, the decrease in the number of viable cells decreased from 100% to approximately 40% by cycle 10 and to approximately 20% by cycle 20 (Figure 6). Similar to the hollow fiber membrane approach, it is possible to target the desired lysis efficiency by utilizing optimization based on the number of cycles.

[0138] Next, various hollow fiber modules were also tested using HEK293. In addition to Repligen® HF, Planova® 35N HF and Cytiva® HF were also used. Planova® gave slightly better performance, which may be due to its smaller fiber inner diameter (380 μm compared to 500 μm) (Figure 7). No significant effect of pore size was observed on cell lysis, except for altering the coefficient of friction of the lumen (Table 1, Figure 7).

[0139] Example 2: Mechanical cell lysis was performed on an industrial scale. Using HEK293 cells expressing rAAV, a closed system using hollow fiber membrane modules was assembled (Platform 1) to demonstrate a mechanical cell lysis process at industrial scale (Figures 2A, 2C). For Platform 1, HEK293 cells were transiently transfected with the AAV Rep and Cap genes, as well as the target therapeutic gene (GOI). A 500L bioreactor was operably connected to two Cytiva® size 5HF membranes (Cytiva® UFP-100-C-5S) with the shell sides capped to both permeate outlets. The run was performed at a flow rate of 40 liters per minute (LPM) with a differential pressure of 20 psi. The process took a total time of 2 hours and 10 minutes to achieve a 90% lysis efficiency. Viable cell count (assessed by percent cell lysis and percent viable cell density) and vector genome release showed reproducible trends similar to those of small-scale data (Figures 8A, 8B). Further large-scale (500L) cell cultures were treated with fewer mechanical cells to demonstrate that similar lysis efficiencies were observed across multiple platforms (Figure 8C).

[0140] The parameters listed in Table 3 were optimized, and the dissolution efficiency was analyzed.

[0141] [Table 3]

[0142] Result By cycle 10, the rAAV titer released was about 80% of the titer released by PS-20; by cycle 15, the rAAV titer released exceeded the rAAV titer released by PS-20 (Figure 8A).

[0143] Train 5 functioned best, followed by Train 4 and then Train 1 (Figure 9). This performance behavior suggested that pressure drop (i.e., steady state pressure, psid, or ΔP) was the major mechanism in cell lysis. The shear rates proportional to the flow rates of Train 5 and Train 1 were observed to be the highest. Train 4 showed a shear level between Train 2 and Train 3 but had higher performance than both trains. In particular, it was also observed that when the material, fiber ID, and total length were fixed, the pressure drop (ΔP) could be adjusted or changed using the flow rate (Figure 9, Table 3).

[0144] Example 3: The reduction in turbidity of the sample was achieved by mechanical cell lysis compared to surfactant-based lysis. Method: Exemplary rAAV was lysed and purified from 5 L cultures of HEK293 (Platform 1) or HeLa (Platform 2) cells using either mechanical cell lysis or PS-20. For Platform 2, HeLa cells were stably incorporated with the AAV Rep and Cap genes, which are the therapeutic genes of interest, and further infected with Adhelper virus. Mechanical cell lysis (two Cytiva (trademark) 20 cm 2 hollow fiber modules arranged in parallel) was performed for the indicated number of cycles. For detergent-based lysis, 0.2% PS-20 was used. TFF was operated using a Repligen (trademark) flat sheet cassette at an area of 0.02 m 2 with a cross-flow flux of 200 L / hr / m 2 and a transmembrane pressure of 6 psi. Both diafiltration buffers were 380 mM Tris containing 50 mM NaCl at pH 8. Turbidity measurements were performed using an Orion AQ4500 turbidimeter.

[0145] The viable cell density (VCD) was determined, and then used to calculate the lysis percentage. The lysates were sampled, unlysed cells were pelleted, and the relative titer was determined by assaying the supernatant for vg using qPCR.

[0146] result Exemplary rAAV was extracted, purified from mechanically lysed platform 1 cells, and then analyzed by dynamic light scattering. Lysates obtained from mechanical cell lysis were observed to be less turbid and did not significantly increase the differential pressure during deep filtration compared to lysates obtained from PS-20 lysated cells (Figures 10A-10B). PS-20 yielded 60-70% (v / v) floc after 30 minutes of sedimentation, while mechanical lysis yielded 20-30% (v / v) floc after 12 minutes of sedimentation (data not shown). Lysates were filtered using various commercially available depth filters, and their performance was compared (Table 4). Deep filtration was observed to process a larger volume of mechanically lysed (MCL) treated lysates compared to PS-20 lysates (Table 4). MCL treated samples were also observed to have a higher density of floc sedimentation.

[0147] [Table 4]

[0148] The capsids co-migrated with nucleic acids, indicating that a portion of the purified capsids were complete capsids (Figure 11). Similar analysis was performed using PS-20. Mechanical cell lysis extracted more exemplary rAAV capsids (both whole and whole) compared to PS-20 (Table 5). The observed percentage complete capsid titer obtained by mechanical cell lysis (3% of the total isolated capsids) was also comparable to that of surfactant-based methods (2% and 4% of the total isolated capsids) (Table 5). In particular, the amount of full capsid isolated from mechanical cell lysis (1.01E+13vg / mL) was greater than the amount of full capsid isolated from surfactant-based lysis (8.66E+12vg / mL and 5.77E+12vg / mL), even under conditions where the percentage of full capsid obtained (4%) was higher than that obtained by mechanical cell lysis (3%).

[0149] To verify that exemplary rAAVs obtained by mechanical cell lysis were at least similar in quality to the same rAAVs obtained by surfactant-based methods, dynamic light scattering data were obtained for purified capsids. The purified Platform 1 capsids were determined to have a diameter of approximately 30 nm, exhibited low polydispersity, and the rAAV particles in the sample did not have a wide size distribution (Figure 12). Full capsids obtained by mechanical cell lysis showed a similar elution profile compared to PS-20, indicating that the rAAVs were consistent with (e.g., at least of similar quality) the rAAVs obtained by surfactant-based lysis methods (Figure 13).

[0150] [Table 5]

[0151] To demonstrate the reproducibility of mechanical cell lysis, further exemplary rAAVs were released by mechanical cell lysis and purified from human cells (Platform 1 and Platform 2) (Figure 14A, Figure 14B). Downstream data evaluating rAAV quality were observed to be similar across platforms compared to control lysis (data not shown).

[0152] Relative release is one method for evaluating the efficiency of rAAV particle release from cells. Relative release of rAAV particles from cells was measured by qPCR (Figures 13, 14A, and 14B) by sampling the cell suspension again before and after lysis.

[0153] Mechanical cell lysis across multiple platforms was observed to consistently achieve high rAAV particle release, as indicated by the accompanying increase in relative titer and cell lysis percentage. For example, at least 50% of cells were observed to be lysed within 10–15 cycles (Figures 14A and 14B). For platform 1A, nearly 80–90% cell lysis could be achieved by cycle 15 (Figure 14A).

[0154] Example 4: Mechanical cell lysis is effective at various temperatures. Method: The bioreactor temperature was controlled by a water bath or heated blanket. Mechanical cell lysis was performed on 5 L cultures (Platform 1) using a hollow fiber membrane (Cytiva® UFP-100-C-3MA), and the capsid was then purified by affinity chromatography followed by anion exchange. The percentage of complete capsid was quantified by mass photometry.

[0155] Results: The temperature of mechanical cell lysis was investigated, including its efficiency at various temperatures and whether this affects the yield and quality of the product.

[0156] While not bound by any particular theory, it was hypothesized that cell lysis performed at lower temperatures increases product yield in three ways. The first is transmembrane efficiency. The fragility of the cell membrane increases at lower temperatures, resulting in greater cell lysis and product release compared to lysis performed at higher temperatures. The second is by reducing the amount of product impurities. At higher temperatures, cells continue to produce rAAV, which is more likely to be empty. The third is by reducing the amount of product modification. Higher temperatures drive post-translational modifications of the product, such as deamidation. Deamidation converts neutral residues into negatively charged residues, altering anion exchange capacity. Therefore, products lysed at lower temperatures may be recovered more productively because they are less likely to undergo post-translational modification.

[0157] MCL was observed to achieve similar solubility at 37°C–25°C (Figure 15). The percentage of complete product recovered after purification by affinity chromatography and anion exchange chromatography was also comparable (Figure 16). Lowering the temperature for mechanical cell lysis did not adversely affect cell lysis efficiency or product recovery.

[0158] Example 5: Efficiency of mechanical cell lysis at various starting cell densities Method: Platform 3 (Producer Line Platform) is 1.5 L of HeLa cells containing Platform 3 cells at 2 million cells / mL or 4 million cells / mL, and is filtered at 37°C at a pressure of 45-50 psi using a Repligen® hollow fiber PES filter (20 cm). 2 Processed by MCL using ).

[0159] Results: The number of cycles required to achieve comparable levels of cell lysis and viable cell density was similar for 2 million cells / mL and 4 million cells / mL cultures (Figure 17A, Figure 17B). This result demonstrates that MCL is suitable for cultures of various densities.

[0160] Example 6: The efficiency of mechanical cell lysis is related to pressure drop. Method: 5L cell cultures of three different producer cell line platform cells (Platform 2, Platform 3, and Platform 4) were filtered using a Repligen® hollow fiber PES filter (20cm). 2 The samples were processed by MCL using [a specific method]. Different degrees of pressure drop were introduced by adjusting the flow rate. The flow rates were 200 mL / min at Δ20 psi, 360 mL / min at Δ30 psi, and 60 mL / min at Δ50 psi.

[0161] Results: Cells that experienced a higher pressure drop lysed more efficiently compared to cells lysed using a lower pressure drop (Figure 18). In particular, lower pressure drops failed to achieve comparable levels of cell lysis, despite an extended cycle count (Figure 18).

[0162] Methods: Cell cultures of HEK293 cells (Platform 1, a transient transfection platform) on a 5L scale were also treated with various pressure drops and plotted against the number of cycles required to reach 80% lysis. In particular, it was observed that lysis using higher pressure drops required fewer cycles to achieve 80% lysis, while lysis using lower pressure drops required more cycles to achieve 80% lysis (Figure 19). Pressure was adjusted using flow rate as described above.

[0163] These results demonstrate that the pressure drop applied during mechanical cell lysis is a crucial determinant of the degree and efficiency of cell lysis.

[0164] The robustness of mechanical cell lysis has been demonstrated for multiple volumes (1.5 L to 500 L), cell types, and AAV platforms. Mechanical lysis has been demonstrated to offer various advantages, as described above, in both transient transfection (Platform 1, Platform 1A) and producer cell line (Platform 2, Platform 3, and Platform 4) based rAAV production systems. Platforms 1, 2, 3, and 4 each contained different target genes. The target gene for Platform 1A was the same as that for Platform 3; however, for Platform 1A, a transient transfection-based rAAV production system was tested using HEK293 cells, while for Platform 3, a producer cell line-based rAAV production system was tested using HeLa cells. The presented data demonstrate that this cell lysis method is consistent and efficient for surfactant-free cell lysis and the release and recovery of intracellular products.

[0165] The present invention is not intended to be limited to specific disclosed embodiments provided, for example, to illustrate various embodiments of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such modifications can be carried out without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the disclosure.

Claims

1. A method for isolating virus particles or nucleic acid molecules from a cell suspension, (i) Flowing a suspension of cells containing viral particles or nucleic acid molecules through a cartridge comprising a plurality of fibers or tubes, wherein the fibers or tubes have an inner diameter of 0.7 mm or less, and the cells experience a pressure difference of less than 250 psi, thereby lysing a portion of the cells in the suspension to produce a mixed suspension containing lysed and unlysed cells; (ii) Recirculating the mixed suspension formed in step (i) through the cartridge for one or more further cycles, wherein a further proportion of the cells is lysed in each consecutive cycle to produce a target suspension containing lysed cells and cell debris; and (iii) A method comprising separating the virus particles or nucleic acid molecules from the target suspension containing lysed cells and cell debris, thereby isolating the virus particles or nucleic acid molecules.

2. The method according to claim 1, wherein the virus particles are rAAV particles.

3. The method according to claim 2, wherein the virus particles are produced by a triple transfection method.

4. The method according to claim 1 or 2, wherein the cell suspension comprises producer cells, and the virus particles are produced by triple transfection of the producer cells.

5. The method according to claim 1, wherein the virus particle is a lentivirus particle.

6. The method according to claim 1, wherein the virus particles include a recombinant baculovirus expression vector.

7. The method according to any one of claims 1 to 6, wherein the cartridge includes a plurality of hollow fibers, and the fibers include a tubular side and a shell side.

8. The method according to claim 7, wherein the plurality of hollow fibers are configured in a hollow fiber membrane module, the plurality of hollow fibers are grouped together to form a hollow fiber bundle, and the hollow fiber bundle is filled into a tube shell.

9. The method according to claim 8, wherein the inner diameter of the fibers including the hollow fiber membrane is 0.1 mm to about 0.7 mm.

10. The method according to claim 8, wherein the inner diameter of the fibers including the hollow fiber membrane is 0.1 mm to about 5 mm.

11. The method according to claim 8, wherein the inner diameter of the fibers including the hollow fiber membrane is 0.2 mm to about 0.3 mm.

12. The method according to claim 8, wherein the inner diameter of the fibers including the hollow fiber membrane is about 0.2 mm.

13. The method according to claim 8, wherein the inner diameter of the fibers including the hollow fiber membrane is about 0.25 mm.

14. The method according to claim 8, wherein the inner diameter of the fibers including the hollow fiber membrane is about 0.3 mm.

15. The method according to any one of claims 1 to 14, wherein the cells flowing through the cartridge are subjected to a pressure difference of less than 200 psi.

16. The method according to any one of claims 1 to 14, wherein the cells flowing through the cartridge are subjected to a pressure difference of less than 150 psi.

17. The method according to any one of claims 1 to 14, wherein the cells flowing through the cartridge are subjected to a pressure difference of less than 100 psi.

18. The method according to any one of claims 1 to 14, wherein the cells flowing through the cartridge are subjected to a pressure difference of less than 50 psi.

19. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of about 5 psi to about 40 psi.

20. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of about 10 psi to about 40 psi.

21. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of approximately 15 psi to approximately 35 psi.

22. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of about 20 psi to about 30 psi.

23. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of approximately 20 psi.

24. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of approximately 22 psi.

25. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of approximately 30 psi.

26. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of approximately 37 psi.

27. The method according to claim 18, wherein the cells flowing through the cartridge are subjected to a pressure difference of approximately 50 psi.

28. The method according to any one of claims 15 to 27, wherein the pressure difference is defined by the difference between the pressure at the cartridge inlet and the pressure at the cartridge outlet.

29. The method according to any one of claims 1 to 28, wherein the suspension flows through the cartridge at a certain flow rate.

30. The method according to claim 29, wherein the flow rate is approximately 10 L / min to approximately 100 L / min.

31. The method according to claim 29, wherein the flow rate is approximately 50 mL / min to approximately 100 L / min.

32. The method according to claim 31, wherein the flow rate is approximately 200 mL / min.

33. The method according to claim 31, wherein the flow rate is approximately 360 mL / min.

34. The method according to claim 31, wherein the flow rate is approximately 600 mL / min.

35. The method according to any one of claims 1 to 34, wherein the suspension flows through the cartridge at a flow rate determined by the cartridge size.

36. The method according to any one of claims 1 to 35, wherein the suspension flows through the cartridge at a flow rate determined by the pressure at the cartridge inlet.

37. The suspension lasts for at least about 2000 seconds. －１ The method according to any one of claims 1 to 36, wherein the material is flowed through the cartridge at a shear rate.

38. The method according to any one of claims 7 to 37, wherein the fiber has a thickness of about 0.1 mm to about 0.4 mm.

39. The method according to any one of claims 7 to 37, wherein the length of the fiber is approximately 20 cm to approximately 150 cm.

40. The method according to any one of claims 1 to 39, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least one cycle.

41. The method according to any one of claims 1 to 40, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least five cycles.

42. The method according to any one of claims 1 to 41, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least 10 cycles.

43. The method according to any one of claims 1 to 42, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least 20 cycles.

44. The method according to any one of claims 1 to 43, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least 30 cycles.

45. The method according to any one of claims 1 to 44, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for at least 40 cycles.

46. The method according to any one of claims 1 to 45, wherein step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) into the cartridge for about 1 to about 50 cycles.

47. The method according to any one of claims 1 to 46, wherein step (ii) includes recirculating the mixed suspension of partially lysed cells formed in step (i) into the cartridge for about 10 to about 50 cycles.

48. The method according to any one of claims 1 to 47, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) into the cartridge for about 15 to about 40 cycles.

49. The method according to any one of claims 1 to 48, wherein step (ii) comprises recirculating the mixed suspension of partially lysed cells formed in step (i) into the cartridge for about 20 to about 30 cycles.

50. The method according to any one of claims 7 to 49, wherein the cell suspension or the mixed suspension flows through the luminal side.

51. The method according to any one of claims 8 to 50, wherein the space between the hollow fiber bundle and the tube shell is capped.

52. The method according to any one of claims 1 to 51, wherein the total time required to complete steps (i) and (ii) is approximately 1 hour to approximately 4 hours.

53. The method according to any one of claims 1 to 52, wherein a dissolution efficiency of more than 70% is achieved, and the dissolution efficiency is measured by lactate dehydrogenase release per cycle, percentage of viable cells, or percentage of product release.

54. The method according to claim 53, wherein a dissolution efficiency of more than 80% is achieved.

55. The method according to claim 53, wherein a dissolution efficiency of more than 90% is achieved.

56. The method according to any one of claims 1 to 55, wherein the cells are eukaryotic cells.

57. The method according to any one of claims 1 to 56, wherein the cells are mammalian cells.

58. The method according to claim 57, wherein the cells are derived from a human or human cell line, and the human cells or human cell line is HEK293 or HeLa.

59. The method according to claim 58, wherein the mammalian cell is a HeLa cell.

60. The method according to claim 58, wherein the mammalian cells are HEK293 cells.

61. The method according to claim 57, wherein the mammalian cell is at least one of CHO cells, HEK293 cells, Vero cells, HeLa cells, MDCK cells, BHK cells, or A549 cells.

62. The method according to any one of claims 1 to 61, wherein the cells are insect cells.

63. The method according to any one of claims 1 to 61, wherein the cells are yeast cells.

64. The method according to any one of claims 1 to 55, wherein the cells are prokaryotic cells.

65. The method according to claim 64, wherein the prokaryotic cell is an E. coli cell.

66. The method according to any one of claims 1 to 65, wherein lysis is determined by the percentage of viable cells.

67. The method according to claim 66, wherein the percentage of viable cells after 5 cycles is approximately 20% to approximately 99%.

68. The method according to claim 66, wherein the percentage of viable cells is approximately 5% to approximately 90% after 10 cycles.

69. The method according to claim 66, wherein the percentage of viable cells after 20 cycles is approximately 1% to approximately 70%.

70. The method according to any one of claims 1 to 65, wherein lysis is determined by the percentage of living cell density.

71. The method according to claim 70, wherein the percentage of viable cells after 5 cycles is approximately 20% to approximately 99%.

72. The method according to claim 70, wherein the percentage of viable cells is approximately 5% to approximately 90% after 10 cycles.

73. The method according to claim 70, wherein the percentage of viable cells is approximately 1% to approximately 70% after 20 cycles.

74. The method according to any one of claims 1 to 65, wherein dissolution is determined by the dissolution percentage.

75. The method according to claim 74, wherein the dissolution percentage is 80% after 18 cycles.

76. The method according to claim 74, wherein the dissolution percentage is 80% after 11 cycles.

77. The method according to claim 74, wherein the dissolution percentage is 80% after 8 cycles.

78. The method according to claim 74, wherein the dissolution percentage is 80% after 5 cycles.

79. The method according to any one of claims 1 to 78, wherein the suspension of cells is poured into a container in each continuous cycle.

80. The method according to claim 79, wherein the container is a bioreactor.

81. The method according to claim 80, wherein the bioreactor is set to a certain temperature.

82. The method according to claim 81, wherein the temperature is approximately 25°C to approximately 37°C.

83. The method according to claim 81 or 82, wherein the bioreactor is at 25°C.

84. The method according to claim 81 or 82, wherein the bioreactor is 37°C.

85. The method according to claim 79 or 80, wherein the cartridge and the container are operably connected to form a closed system.

86. The method according to claim 85, wherein the closed system further comprises at least one tubular line operably connected to the container and the cartridge, and the cell suspension flows between the container and the cartridge through the tubular line.

87. The method according to claim 86, wherein the tube is made of rubber.

88. The method according to any one of claims 1 to 87, wherein the separation includes affinity capture.

89. The method according to any one of claims 1 to 88, wherein the separation includes ion exchange.

90. The method according to claim 89, wherein the separation includes anion exchange.

91. A method for mechanically lysing cells, comprising flowing a suspension containing a plurality of cells or a population of cells through a cartridge containing a plurality of fibers or tubes, wherein the cells are subjected to a pressure difference of less than 250 psi, thereby lysing a portion of the cells in the suspension.

92. The method according to claim 91, wherein the cells are further recirculated through the cartridge over several cycles to increase the proportion of cells that are lysed.

93. The method according to claim 91 or 92, wherein the cells are introduced into the cartridge by following a channel.

94. The method according to any one of claims 91 to 93, wherein the mechanical dissolution includes shearing the cells.

95. The method according to any one of claims 91 to 94, wherein mechanical cell lysis is achieved by applying one or more external forces to the plurality of cells or cell populations.

96. The method according to claim 95, wherein the one or more external forces are selected from the group consisting of flow, pressure, turbulence, friction, extrusion, collision, and pressure drop.

97. The method according to any one of claims 91 to 96, wherein the plurality of fibers are hollow fibers.

98. The method according to claim 97, wherein the plurality of hollow fibers are configured in a hollow fiber membrane module, the plurality of hollow fibers are grouped together to form a hollow fiber bundle, and the hollow fiber bundle is filled into a tube shell.

99. The method according to claim 98, wherein the hollow fiber membrane bundle is surrounded by a shell extending longitudinally along the length of the bundle, forming the lumen side and the shell side of the hollow fiber membrane module.

100. The method according to claim 99, wherein the lumen forms a tubular structure.

101. The method according to any one of claims 98 to 100, wherein the space between the hollow fiber bundle and the tube shell is capped.

102. The method according to any one of claims 91 to 101, wherein the hollow fiber membrane module is sterile.

103. The method according to any one of claims 91 to 102, wherein the hollow fiber membrane module is disposable.

104. The aforementioned number of cycles is given by the formula [Math 1] (In the formula, q フィード is the flow rate passing through the hollow fiber module, t is the operating time, and V リアクター The method according to any one of claims 91 to 103, wherein is the volume of an aqueous solution containing the plurality of cells or a population of cells.

105. The method according to any one of claims 91 to 104, wherein the total dissolution time is approximately 1 hour to approximately 4 hours.

106. The method according to any one of claims 91 to 105, wherein the aforementioned portion of the lysed cells is more than 90%.

107. A device for lysing cells, comprising: a container capable of holding a cell suspension; a cartridge including a plurality of fibers or tubes and an extracapillary space outside the fibers or tubes; and means for connecting the container to the cartridge such that the container and the cartridge form a closed system, so that the cell suspension flows through the container and the cartridge multiple times when operable.

108. The apparatus according to claim 107, wherein the means for connecting the container to the cartridge includes a tube.

109. The apparatus according to claim 107 or 108, wherein the fiber or tube has a maximum diameter of approximately 0.7 mm.

110. The apparatus according to claim 107 or 108, wherein the fiber or tube has a diameter of about 0.1 mm to about 0.5 mm.

111. The apparatus according to claim 107 or 108, wherein the fiber or tube has a diameter of about 0.2 mm to about 0.3 mm.

112. The apparatus according to any one of claims 107 to 111, wherein the container is a bioreactor.

113. A method for isolating virus particles or nucleic acid molecules from cells, (i) Dissolving a portion of the cells in a suspension of cells containing virus particles or nucleic acid molecules by flowing the suspension through a cartridge containing a fibrous porous medium or material, thereby producing a mixed suspension containing lysed and unlysed cells; (ii) Recirculating the mixed suspension of partially lysed cells formed in step (i) through the cartridge for one or more further cycles, wherein further portions of the cells are lysed in each consecutive cycle to produce a target suspension containing lysed cells and cell debris; and (iii) A method comprising separating the virus particles or nucleic acid molecules from the target suspension containing lysed cells and cell debris, thereby isolating the virus particles or nucleic acid molecules.

114. The method according to claim 113, wherein the fibrous porous medium or material is a nonwoven fabric material.

115. The method according to claim 114, wherein the nonwoven material is spunbond, meltblown fiber, felt, or wet-laid material.

116. The method according to claim 113 or 114, wherein the nonwoven material is capable of retaining at least about 90% of lysed cells and cell debris including at least about 0.1 μm in size.

117. The method according to any one of claims 114 to 116, wherein the pore size of the nonwoven fabric is approximately 20 μm to approximately 60 μm.

118. The method according to claim 113, wherein the fibrous porous medium or material is a woven fabric material.

119. The method according to claim 113, wherein the fibrous porous medium or material is a film.

120. A cartridge for lysing cells, (a) Housing, and (b) A cartridge comprising a plurality of pores, the pores having a maximum or approximately 1 mm in diameter.

121. The cartridge according to claim 120, wherein the pores are embedded within a plurality of hollow fibers having an inner diameter, an outer diameter, and two ends having a length between the ends, and at least one end of each fiber is open for a fluid inlet or outlet, and the fibers are arranged parallel to each other.

122. The method or apparatus according to claims 1 to 111, wherein the fiber or tube does not have pores.

123. The method or apparatus according to claims 1 to 111, wherein the fiber or tube includes pores.

124. The method or apparatus according to claim 123, wherein the pores are less than 300 kDa.

125. The method according to any one of claims 1 to 90 or 113 to 119, wherein the virus particles are produced by a transient transfection method.

126. The method according to any one of claims 1 to 90 or 113 to 119, wherein the cell suspension comprises producer cells expressing the virus particles.

127. The method according to claim 125 or 126, wherein the virus particle comprises an AAV serotype, and the AAV serotype comprises at least one of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and their variants, chimeras, or hybrids thereof.

128. The method according to any one of claims 113 to 119, wherein the cells are mammalian cells.

129. The method according to claim 128, wherein the mammalian cell is at least one of CHO cells, HEK293 cells, Vero cells, HeLa cells, HEK293 cells, MDCK cells, BHK cells, or A549 cells.

130. The method according to any one of claims 91 to 106, wherein the cells are mammalian cells.

131. The method according to claim 128, wherein the mammalian cell is at least one of CHO cells, HEK293 cells, Vero cells, HeLa cells, HEK293 cells, MDCK cells, BHK cells, or A549 cells.

132. The method according to claim 2, wherein the virus particles are rAAV particles produced by a transient transfection method.

133. The method according to claim 1 or 2, wherein the viral particles are rAAV particles produced by transfection of a host cell with one or more plasmids comprising (i) one or more nucleic acids encoding an AAV packaging gene and (ii) a nucleic acid encoding a gene of interest.

134. The method according to claim 1 or 2, wherein the virus particles are rAAV particles produced by transfection of the host cell with one or more plasmids comprising (i) one or more nucleic acids encoding an AAV packaging gene; (ii) a nucleic acid encoding the gene of interest; (iii) one or more nucleic acids encoding an AAV ITR (reverse terminal repeat sequence); and optionally (iv) a nucleic acid encoding at least one ad helper virus gene.

135. The method according to claim 2, wherein the virus particles are rAAV particles expressed from a producer cell line (PCL).

136. The method according to claim 135, wherein the PCL comprises a host cell stably incorporating (i) one or more nucleic acids encoding an AAV packaging gene; (ii) a nucleic acid encoding a gene of interest; and (iii) one or more nucleic acids encoding an AAV ITR (reverse terminal repeat sequence).

137. The method according to claim 136, wherein the PCL is further infected with adenovirus.

138. The method according to claim 137, wherein the adenovirus is an ad helper virus.

139. The method according to claim 138, wherein the ad helper virus expresses one of a plurality of helper genes.

140. The method according to any one of claims 133 to 139, wherein the AAV packaging gene comprises at least an AAV capsid (cap) gene.

141. The method according to any one of claims 133 to 140, wherein the AAV packaging gene includes at least an AAV replication (rep) gene.

142. The method according to claim 134, wherein the ad helper virus gene is a helper gene.

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