System and method for recovering target biomolecules from fixed bed bioreactor

Abstract

The present invention relates to a method for obtaining one or more biomolecules of interest from cells cultured in a fixed bed bioreactor, said method comprising: adding to said bioreactor a lysis solution, thereby lysing said cells in said bioreactor, and before, simultaneously with and / or after said cells are lysed, separating said cells from said cells; one or more mechanical effects are applied to the bioreactor after which the one or more target biomolecules are recovered from the bioreactor. The invention further relates to a system for obtaining one or more target biomolecules from cells cultured in a fixed bed bioreactor.

Description

Technical Field

[0001] The following applications are hereby incorporated by reference in their entirety:

[0002] Cell Growth Matrix, PCT / EP2017 / 078775, November 9, 2017, and published as WO2018 / 087235A1 on May 17, 2018.

[0003] Bioreactor and Related Methods, PCT / EP2018 / 086394, December 20, 2018, and published as WO2019 / 122239 on June 27, 2019.

[0004] Bioreactor System with Enhanced Cell Harvesting Capabilities and Related Methods, PCT / EP2022 / 065264, June 3, 2022, and published as WO2022 / 254039 on December 28, 2022.

[0005] Woven cell culture substrates, PCT / US2020 / 016576, February 4, 2020, and published as WO 2020163329A1 on August 13, 2020.

[0006] The systems, compositions, and methods described herein relate to cell culture of adherent or suspension cells in a fixed bed bioreactor, preferably in a structured fixed bed bioreactor such as those described in the patent applications incorporated herein by reference in their entirety above; and the recovery of one or more target biomolecules from the cell culture harvest of the fixed bed bioreactor.

[0007] PCT Publication WO2022 / 254039 is hereby incorporated by reference in its entirety, and it discloses bioreactors and fixed bed configurations, as well as systems and processes for applying mechanical energy in combination with chemical / enzymatic agents to the bioreactors and fixed bed systems described in that document. The bioreactors and fixed bed systems mentioned herein, as well as the systems for applying mechanical energy to the bioreactors and fixed bed systems, include but are not limited to those described in that document.

[0008] The present invention relates to methods for obtaining viral vectors and other target biomolecules from a fixed-bed bioreactor. In a second aspect, the present invention relates to a system for obtaining one or more target biomolecules from cells cultured in a fixed-bed bioreactor. Accordingly, the present invention pertains to the technical field of harvesting target biomolecules from cells cultured in a fixed-bed bioreactor. Background Art

[0009] Fixed-bed bioreactors are effective tools for producing biomolecules such as recombinant proteins, monoclonal antibodies, viral vectors, viruses, and cellular vesicles (such as exosomes). Animal cells can be used to produce such biomolecules, for example, using constitutive expression systems, infection, or transfection. Then, the target biomolecules can be released by the cells (such as secreted recombinant proteins, "secreted" viruses, or lytic viruses), or can be retained within the cells (such as intracellular proteins, viruses, or certain viral vectors). Extracellular target biomolecules can be recovered in the supernatant of the bioreactor and in one or more recycle / perfusion loops by using one or more depletion and wash steps to recover the target biomolecules of interest (referred to herein as target biomolecules). However, intracellular and partially intracellular products remain within the cells and require lysis of the cells using chemical / enzymatic solutions at the end of the process. However, conventional lysis conditions are relatively harsh, and their use may damage the target biomolecules as they are often sensitive to pH, shear stress, enzyme activity, and detergents. In addition, the target biomolecules may be trapped by cell debris or other biomolecules (such as cell membranes, DNA, etc.), which may still clog inside the fixed-bed structure.

[0010] The present invention aims to address at least some of the above problems and disadvantages. The object of the present invention is to provide a method and system that eliminate these disadvantages and improve the recovery rate of intracellular (or partially intracellular) target biomolecules from a fixed-bed bioreactor. Summary of the Invention

[0011] The present invention and its embodiments provide solutions to one or more of the above disadvantages. To this end, the present invention relates to a method for obtaining one or more target biomolecules from cells cultured in a fixed-bed bioreactor according to claim 1. More particularly, the method described herein relates to a method for obtaining one or more target biomolecules from cells cultured in a fixed-bed bioreactor, the method comprising:

[0012] - adding a lysis solution to the bioreactor to lyse the cells in the bioreactor, and applying one or more mechanical actions to the bioreactor before, during, and / or after lysing the cells, and thereafter recovering the one or more target biomolecules from the bioreactor.

[0013] Higher yields of the target biomolecule can be obtained from the bioreactor by combining the lysis solution with one or more mechanical actions.

[0014] Preferred embodiments of the method are shown in any one of claims 2 to 11.

[0015] In a second aspect, the present invention relates to a system according to claim 12. More particularly, the system described herein relates to a system comprising: a stirring device and a fixing system,

[0016] - The stirring device includes a platform adapted to receive the bioreactor; having

[0017] - The fixing system includes at least one fastener for holding the bioreactor to the platform, and a bridge structure adapted to be mounted to the bioreactor and receive the at least one fastener.

[0018] Preferred embodiments of the system are shown in any one of claims 13 to 15.

[0019] In an embodiment, the present invention relates to a system according to claim 12, wherein the fastener includes an adjustable strap, and wherein the fixing system further includes one or more strap guides for positioning and guiding the placement of the strap.

[0020] In an embodiment, the present invention relates to a system according to any one of claims 12 - 13, wherein the fixing system includes an annular part member for engaging a lid or cover of the bioreactor.

[0021] In an embodiment, the present invention relates to a system according to any one of the foregoing claims 12 - 14, wherein the stirring device includes a vibrating table, and the vibrating table includes a placeholder on which the bioreactor is placed.

[0022] In an embodiment, the bridge structure includes a top portion and a depending portion.

[0023] In an embodiment, the fixing system includes a releasable coupling.

[0024] In an embodiment, the stirring device includes a stirrer in the form of a vibrating table, a vortex device, an oscillator, or another device for applying mechanical energy to the bioreactor and / or the fixed bed material.

[0025] In an embodiment, the system forms part of a docking station for the bioreactor.

[0026] In an embodiment, the system further includes a controller for controlling the stirring device.

[0027] In an embodiment, the system further includes a controller for controlling the agitation device and the bioreactor or the cell harvesting process.

[0028] In an embodiment, the bioreactor includes a structured fixed-bed bioreactor.

[0029] In an embodiment, the fixed bed includes a plurality of cell immobilization layers.

[0030] In an embodiment, the plurality of cell immobilization layers are arranged in a stacked or helical configuration.

[0031] In an embodiment, the cell immobilization layers are either arranged in direct contact or have a spacing between adjacent layers.

[0032] In an embodiment, the cell immobilization layers are arranged either in direct contact or have one or more spacer layers between the one or more cell immobilization layers.

[0033] In an embodiment, the fixed bed is a 3D printed fixed bed.

[0034] In an embodiment, the present invention relates to a system for recovering biomolecules from a bioreactor, the system comprising:

[0035] - a bioreactor retained on a platform of an agitation device; and

[0036] - a fixing system for coupling at least a portion of the bioreactor to the agitation device.

[0037] The present invention can be summarized as follows:

[0038] 1. A process for recovering a target biomolecule from cells in a fixed-bed bioreactor, the process comprising the steps of:

[0039] Emptying the cell culture medium from the bioreactor;

[0040] Adding a flushing solution to the bioreactor;

[0041] Emptying the flushing solution from the bioreactor;

[0042] Lysing the cells in the bioreactor by adding a lysis solution to the bioreactor;

[0043] Emptying the lysis solution from the bioreactor;

[0044] Washing the cells in the bioreactor by adding a washing solution to the bioreactor;

[0045] Empty the washing solution from the bioreactor; and

[0046] Recover the target biomolecule from the fixed-bed bioreactor,

[0047] wherein the process further comprises adding a high-conductivity solution to the bioreactor during the lysis step and / or the washing step.

[0048] 2. A process for recovering a target biomolecule from cells in a fixed-bed bioreactor, the process comprising the steps of:

[0049] Empty the cell culture medium from the bioreactor;

[0050] Add a flushing solution to the bioreactor;

[0051] Empty the flushing solution from the bioreactor;

[0052] Lyse the cells in the bioreactor by adding a lysis solution to the bioreactor;

[0053] Empty the lysis solution from the bioreactor;

[0054] Wash the cells in the bioreactor by adding a washing solution to the bioreactor;

[0055] Empty the washing solution from the bioreactor; and

[0056] wherein the process further comprises adding a high-conductivity solution to the bioreactor during the lysis step and / or the washing step.

[0057] 3. The process according to embodiment 2, further comprising recovering the target biomolecule from one or more of the flushing solution, the lysis solution, and the washing solution.

[0058] 4. The process according to embodiment 1 or 2, wherein the lysis solution comprises added DNase, preferably an endonuclease, more preferably benzonase.

[0059] 5. The process according to embodiment 1 or 2, wherein the lysis solution has a low conductivity.

[0060] 6. The process according to embodiment 1 or 2, wherein the conductivity of the lysis solution is increased during the lysis step.

[0061] 7. The process according to embodiment 1 or 2, wherein the conductivity of the lysis solution is increased to up to 1 M by adding NaCl.

[0062] 8. A process for recovering a target biomolecule from cells in a fixed-bed bioreactor, the process comprising one or more of the following steps:

[0063] - Performing a number of cycles with a number of rinses.

[0064] - Having sufficient contact time.

[0065] - Using DNase to degrade free DNA

[0066] - Using a surfactant to limit the adsorption of AAV to the walls of plastic containers

[0067] - Employing a pH below the isoelectric point (pH 3 to 5) or above the isoelectric point (pH 8 to 9).

[0068] - Applying vibration to the bioreactor.

[0069] - Applying vibration to the bioreactor during one or more lysis steps and / or subsequent rinse steps.

[0070] - Applying vibration to the bioreactor during one or more of the processes listed above.

[0071] - Using electrophoresis to extract one or more charged biomolecules from a fixed-bed bioreactor.

[0072] - Operating one or more of the above steps in a recycle mode, preferably using a recycle loop.

[0073] 9. A process for recovering cells or a target biomolecule from cells in a cell culture harvest, the process comprising moving a fixed bed in the cell culture harvest; and recovering the target biomolecule from the cell culture harvest.

[0074] 10. A process for recovering a target biomolecule from a fixed-bed bioreactor comprising cells, the process comprising moving the fixed bed; and recovering the target biomolecule.

[0075] 11. A process for recovering a target biomolecule from a fixed-bed bioreactor comprising cells in a solution, the process comprising moving the solution relative to the fixed bed; and recovering the target biomolecule.

[0076] 12. The process according to any one of the preceding embodiments 2-11, wherein the target biomolecule is selected from the group consisting of: therapeutic proteins, antibodies, viruses, viral vaccines, viral vectors, viral genomes, proteins, virus-like particles (VLPs), microvesicles, exosomes and polysaccharides.

[0077] 13. The process according to embodiment 12, wherein the cells are selected from the group consisting of: adherent cells, suspension cells, and a combination of adherent cells and suspension cells.

[0078] 14. The process according to embodiment 13, wherein the cells are attached or captured in a fixed bed.

[0079] 15. The process according to embodiment 14, wherein the target biomolecule is intracellular.

[0080] 16. The process according to embodiment 15, wherein the target biomolecule is extracellular.

[0081] 17. The process according to embodiment 16, wherein the cells are in a cell culture medium.

[0082] 18. The process according to embodiment 17, wherein the cells are in a solution for detaching the cells from the fixed bed, and the process further includes a step of detaching the cells from the fixed bed.

[0083] 19. The process according to embodiment 18, wherein the detachment solution includes a combination of a detergent, DNase, an appropriate pH (different from the isoelectric point), and an appropriate conductivity.

[0084] 20. The process according to embodiment 19, wherein the cells are in a lysis solution, and the process further includes a step of lysing the cells.

[0085] 21. The process according to embodiment 20, wherein the lysis solution includes a combination of a detergent, DNase at an appropriate pH (different from the isoelectric point), and an appropriate conductivity.

[0086] 22. The process according to embodiment 21, wherein moving the fixed bed includes actions selected from the group consisting of: vibrating, stirring, compressing, expanding, and combinations of the foregoing.

[0087] 23. The process according to embodiment 22, wherein moving the solution includes actions selected from the group consisting of: vibrating, stirring, compressing, expanding, and combinations of the foregoing.

[0088] 24. The process according to embodiment 23, wherein the detachment step is before the lysis step.

[0089] 25. The process according to embodiment 24, further includes adding a lysis solution and lysing the cells.

[0090] 26. The process according to embodiment 25, wherein the bioreactor comprises lysed cells and the target biomolecule in solution.

[0091] 27. The process according to embodiment 26, further comprising the step of adding a high conductivity solution, wherein the high conductivity solution has a conductivity between 150 mmol / L NaCl and 2 mol / L NaCl and includes the end point values.

[0092] 28. The process according to embodiment 27, wherein the high conductivity solution is in contact with the lysed cells and the target biomolecule in solution for a time of 5 to 360 minutes and includes the end point values.

[0093] 29. The process according to embodiment 28, further comprising an electrophoresis step.

[0094] 30. The process according to embodiment 29, wherein at least a part of the lysis step is carried out in a recirculation mode.

[0095] 31. The process according to embodiment 30, wherein at least a part of the high conductivity solution step is carried out in a recirculation mode.

[0096] 32. The process according to embodiment 31, wherein the cells are detached from the fixed bed before the lysis step.

[0097] 33. The process according to embodiment 32, wherein the cells are recovered from the fixed bed before the lysis step.

[0098] 34. The process according to embodiment 33, wherein the cells are removed from the fixed bed before the lysis step.

[0099] 35. A process for recovering a target biomolecule from cells in a fixed bed bioreactor, the process comprising the steps of: lysing the cells in the bioreactor by adding a lysis solution to the bioreactor;

[0100] increasing the conductivity of the lysis solution by adding salt to the lysis solution; and recovering the target biomolecule from the bioreactor.

[0101] 36. The process according to embodiment 35, wherein the conductivity of the lysis solution is increased by adding NaCl to the lysis solution.

[0102] 37. The process according to embodiment 36, wherein the concentration of NaCl in the lysis solution is between 1 M and 2 M.

[0103] 38. A process for recovering a target biomolecule from cells in a fixed-bed bioreactor, the process comprising the following steps:

[0104] Lysing the cells in the bioreactor by adding a lysis solution to the bioreactor;

[0105] Emptying the lysis solution from the bioreactor;

[0106] Adding a high-conductivity solution to the bioreactor; and

[0107] Recovering the target biomolecule from the bioreactor.

[0108] 39. The process according to any one of the foregoing embodiments 2-38, wherein the target biomolecule is selected from the group consisting of: therapeutic proteins, antibodies, viruses, viral vaccines, viral vectors, viral genomes, proteins, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides. Brief Description of the Drawings

[0109] Figure 1 Shows a flowchart of the steps carried out in the method for obtaining AAV2 according to the embodiment of the present invention described in Example 1.

[0110] Figure 2 Shows a flowchart of the steps carried out in the method for obtaining AAV2 according to the embodiment of the present invention described in Example 2.

[0111] Figure 3 Shows a flowchart of the steps carried out for harvesting and recovering a target biomolecule from a fixed-bed bioreactor under stirring at 1 cm / s according to an embodiment of the present invention.

[0112] Figure 4 Shows a flowchart of the steps carried out for harvesting and recovering AAV from a fixed-bed bioreactor according to an embodiment of the present invention.

[0113] Figure 5 Shows a flowchart of the steps carried out for harvesting and recovering AAV from a fixed-bed bioreactor when the bioreactor is operating in a recirculation mode according to an embodiment of the present invention.

[0114] Figure 6 A shows a general schematic diagram of an electrophoresis-derived system.

[0115] Figure 6 B shows the detailed structure of the electrode according to an embodiment of the present invention.

[0116] Figure 7A shows the migration of negatively charged biomolecules in an electrophoresis-derived system according to an embodiment of the present invention. The negatively charged particles migrate from the anode to the cathode.

[0117] Figure 7 B shows that during the electrophoresis migration process, the current is reversed according to an embodiment of the present invention to facilitate the collection of biomolecules and avoid their accumulation on the cathode (+).

[0118] Figures 8 - 12 Shows a system for applying a mechanical action to a bioreactor according to an embodiment of the present invention.

[0119] Figure 13 Shows a bridge structure according to an embodiment of the present invention, including a strap guide for positioning and guiding the placement of a strap during the installation process of the strap in a fixing system, and a pushing pin adapted to limit the displacement and / or breakage of the lid of a container or bioreactor.

[0120] Figure 14 Shows a ratchet for pre-installing a strap according to an embodiment of the present invention, for adjusting the length of the strap in a fixing system.

[0121] Figure 15 and Figure 16 Shows a vibrating table according to an embodiment of the present invention. Detailed Embodiments

[0122] The present invention relates to a method for obtaining viral vectors and other target biomolecules from a fixed-bed bioreactor. In a second aspect, the present invention relates to a system including a stirring device and a fixing system for obtaining one or more target biomolecules from cells cultured in a fixed-bed bioreactor.

[0123] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Through further guidance, term definitions are included to better understand the teachings of the present invention.

[0124] As used herein, the following terms have the following meanings:

[0125] Unless the context clearly indicates otherwise, "a", "an", and "the" include singular and plural referents. For example, "compartment" refers to one compartment or more than one compartment.

[0126] References to measurable values such as parameters, amounts, durations, etc., as used herein, "about" is intended to cover variations of + / −20% or less, preferably + / −10% or less, more preferably + / −5% or less, even more preferably + / −1% or less, and still more preferably + / −0.1% or less from or of the specified value, so long as such variations are appropriate to carry out the invention of the present disclosure. However, it should be understood that the value itself to which the modifier "about" refers is also specifically disclosed.

[0127] As used herein, "comprise", "comprising", "comprises" and "comprised of" are synonymous with "include", "including", "includes" or "contain", "containing", "contains", and are inclusive or open-ended terms that specify the presence of the following, e.g., "the component comprises" does not exclude or preclude the presence of additional, unrecited components, features, elements, components, steps known in the art or disclosed therein.

[0128] Furthermore, the terms "first", "second", "third", etc. in the specification and claims are used only to distinguish similar elements and do not necessarily denote an order or a relationship of precedence in time, unless otherwise specified. It should be understood that these terms may be used interchangeably where appropriate, and the order of operation of the embodiments of the invention described herein is not limited to the order described or illustrated in the specification.

[0129] A numerical range recited by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0130] Unless otherwise defined, the expressions "% by weight", "weight percent", "% wt" or "wt%" herein and throughout the specification refer to the relative weight of each component based on the total weight of the formulation.

[0131] In view of the term "one or more" or "at least one", such as one or more members or at least one member of a group of members, which is clear in itself, by way of further example, the term particularly encompasses any one of the recited members, or any two or more of the recited members, e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7, etc. of the members, and up to and including all of the recited members.

[0132] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. Through further guidance, the definitions of the terms used in this specification are incorporated herein to better understand the teachings of the present invention. The terms or definitions used herein are only for the purpose of facilitating the understanding of the present invention.

[0133] References to "an embodiment" or "embodiments" throughout the specification mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in an embodiment" or "in embodiments" that appear in various paragraphs throughout the specification do not necessarily all refer to the same embodiment, but may. Additionally, as will be apparent to those skilled in the art from this disclosure, the particular features, structures, or characteristics that exist in one or more embodiments may be combined in any suitable manner. Moreover, although some of the embodiments described herein include some of the features included in other embodiments and not others, combinations of the features of different embodiments are intended to be within the scope of the present invention and constitute different embodiments, as understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0134] Fixed-bed bioreactors are effective tools for producing biomolecules such as recombinant proteins, monoclonal antibodies, viral vectors, viruses, and cellular vesicles (such as exosomes) (also referred to as "target biomolecules"). Animal cells are used to produce such products (using constitutive expression systems, infection, or transfection). One or more target products can be released by the cells (such as secreted recombinant proteins, "secreted" viruses, or lytic viruses), or can be partially retained intracellularly (such as intracellular proteins, viruses, or some viral vectors).

[0135] Extracellular target biomolecules can be recovered in the supernatant of the bioreactor and in one or more recycle / perfusion loops by using one or more clarification, washing, and concentration steps to recover the target biomolecules of interest (referred to herein as target biomolecules).

[0136] The products within the cells are retained in the cells and require the use of chemical / enzyme solutions at the end of the process to lyse the cells. After lysing the cells, the solution containing cell debris (host cell proteins, host cell DNA, cell membranes, and organelles) and the target biomolecule is harvested by emptying and flushing the bioreactor (similar to how extracellular products are handled). The cell membrane is fragile, and the solutions for lysing the cells are known (hypotonic - hypertonic shock, acidic - basic pH changes, detergents, or combinations thereof). However, most target biomolecules are easily destroyed. Therefore, a solution that can lyse the cells without destroying the target biomolecules is needed.

[0137] Some prior art processes detach the cells (using trypsin or cell dissociation enzyme) and then lyse the cells outside the bioreactor because lysis inside the bioreactor is not efficient. Additionally, it is well known that it is difficult to recover intracellular target biomolecules from cells in a fixed - bed bioreactor using traditional methods. Therefore, effective processes are needed to recover intracellular biomolecules from a fixed - bed bioreactor.

[0138] Among these intracellular products, some target biomolecules are difficult to harvest because they are sensitive to pH, shear force, and detergents. Additionally, many target biomolecules are known to adhere or attach to plastic materials such as T - flasks, cell factories, and storage containers. Moreover, some target biomolecules, including adeno - associated virus serotype 2 (collectively referred to as AAV herein), can attach to plastic bioreactor materials and plastic fixed - bed materials, especially hydrophilic plastic fixed - beds and hydrophilic plastic structured fixed - bed materials. Therefore, a solution is needed that facilitates the recovery of target biomolecules from a fixed - bed bioreactor by reducing the interaction of the target biomolecules with one or more plastic components (including but not limited to bioreactors and fixed - beds). Furthermore, it is believed that some target biomolecules, especially intracellular target biomolecules (including but not limited to AAV), can still be mechanically trapped in or attached to the fixed - bed and cell debris (including organelles such as the nucleus) after the lysis step, making the recovery of the target biomolecules difficult. Therefore, a solution is needed that facilitates the recovery of target biomolecules from a fixed - bed bioreactor by reducing the interaction of the target biomolecules with the fixed - bed and cell debris. It is known in the art to add surfactants (such as Pluronic F68, typically 0.1 - 0.01%) to AAV suspension cell culture harvests to prevent the "loss" of virus remaining on the walls of the bioreactor, pipes, or storage containers.

[0139] The inventors found that when they attempted to recover AAV from cells in a bioreactor with a structured fixed bed after a lysis step using solutions and processes known in the art for suspension cell technology, a large proportion of the AAV could not be recovered because it was trapped in or adhered to the fixed bed, or adhered to cell debris (such as cell host cell proteins / DNA) that was trapped in or adhered to the fixed bed.

[0140] The inventors also found that when the structured fixed bed material included hydrophilized PET, most of the unrecovered target biomolecules (such as AAV) interacted / aggregated with cell debris, adsorbed proteins, adsorbed DNA, and the extracellular cell matrix, and / or were trapped in these substances, which remained on the fixed bed material after chemical (detergent) lysis of the cells.

[0141] Disposable fixed bed bioreactors (examples include Pall's iCELLis, Univercells Technologies' scale-X, and Corning's Ascent) are made of plastic materials, and biomolecules (such as AAV) may adhere to such materials. Due to their large hydrophilized surfaces for cells, the "adhesiveness" of AAV or other target biomolecules is an important issue, making the recovery of target biomolecules more complex than from suspension cell bioreactors.

[0142] The inventors tested the scale-X bioreactor with classical solutions containing 0.1% and 1% Triton and DNase (benzonase), and observed that some virus remained after harvest (by fluorescence microscopy and after in situ harvest followed by freeze / thaw cycles and then ex situ harvest).

[0143] Information obtained from tests on AAV2 harvested from the scale-X bioreactor shows that:

[0144] · Detergent-based "classical solutions" (0.1% and 1% Triton X-100) are sometimes not sufficient to achieve good recovery rates (virus remained after in situ lysis and rinsing, as confirmed by fluorescence microscopy and ex situ harvest after ex situ lysis).

[0145] · The "classical solutions" are sufficient to lyse the cells (evidence: turbidity of the solution), but not sufficient to recover the virus.

[0146] · Adding benzonase (DNase) to degrade DNA to limit virus adhesion is sometimes also insufficient.

[0147] Regarding lysis solutions, it is known that Triton X-100 and Tween (Tween 20 and Tween 80) detergents are effective (at concentrations of 0.1% to 0.5%) in lysing cells and recovering AAV from suspension cells. DNase (such as benzonase) can be added to cleave free DNA and prevent AAV from adhering to the DNA. However, as described above, there are still problems with the recovery of the target biomolecule.

[0148] The inventors have unexpectedly found that combining a chemical lysis step (using a lysis solution) with mechanical action facilitates the recovery of the target biomolecule from the bioreactor. In one embodiment, the present invention includes a novel solution combination to increase the yield of the target biomolecule, preferably to increase the yield of intracellular biomolecules from cells in a cell culture or a cell harvest solution.

[0149] Accordingly, in a first aspect, the present invention relates to a method for obtaining one or more target biomolecules from cells cultured in a fixed-bed bioreactor, the method comprising:

[0150] - adding a lysis solution to the bioreactor to lyse the cells in the bioreactor, and applying one or more mechanical actions to the bioreactor before, during, and / or after lysing the cells, and thereafter recovering the one or more target biomolecules from the bioreactor.

[0151] Although the present disclosure is presented mainly in the context of recovering AAV from cells, the invention described herein should not be limited to the recovery of AAV, but the invention can be used to recover other target biomolecules from adherent cells or suspension cells, where such target biomolecules can include one or more of the following: viruses, non-lytic viruses, viruses with limited cytopathic effects, non-secreted or partially secreted recombinant proteins, non-secreted or partially secreted antibodies, intracellular or partially intracellular viral vectors. The cells can be selected from the group consisting of: adherent cells, suspension cells, and combinations of adherent cells and suspension cells. All types of cells can be used, examples including mammalian cells such as stem cells (e.g., hematopoietic stem cells, skeletal muscle stem cells, mesenchymal stem cells), immune cells (e.g., lymphocytes, dendritic cells), and islet cells. Prokaryotic cells (such as bacterial cells) or viral cells can also be used.

[0152] In an embodiment, the target biomolecule is AAV. Accordingly, in an embodiment, the present invention relates to a method for obtaining AAV from cells cultured in a fixed-bed bioreactor, the method comprising:

[0153] - Add a lysis solution to the bioreactor to lyse the cells therein, and apply one or more mechanical actions to the bioreactor before, during, and / or after lysing the cells, and then recover AAV molecules from the bioreactor.

[0154] In one embodiment, the present invention includes one or more of the following steps to recover a target biomolecule, preferably an AAV virus, from a bioreactor, preferably from a fixed-bed bioreactor.

[0155] As described above, biomolecules can adsorb to the walls of plastic containers. In an embodiment, the method of the present invention thus includes using a surfactant to limit the adsorption of biomolecules such as AAV to the walls of plastic containers. In an embodiment, 0.1% surfactant (more than the usual process) is used. In an embodiment, 0.1% to 0.01% of Pluronic F68 is used. In an embodiment, the method of the present invention further includes using a surfactant to limit the adsorption of biomolecules such as AAV to the walls of plastic containers (as in a suspension process, e.g., 0.1% to 0.01% of Pluronic F68). Pluronic TM F-68 is a non-ionic surfactant commonly used to control shear forces, prevent foaming in agitated cultures, and reduce cell attachment to hydrophilic surfaces. It is also known that adding it can prevent biomolecules such as AAV from adhering to plastics in pipettes and plastic containers during storage. For AAV, the generally recommended usage concentration is between 0.01% and 0.2%.

[0156] In an embodiment, the method of the present invention further includes performing several cycles and several rinses to recover a target biomolecule, preferably an AAV virus, from a bioreactor, preferably from a fixed-bed bioreactor.

[0157] In an embodiment, the bioreactor is emptied before adding the lysis solution to the bioreactor. In other embodiments, 0.1% of Pluronic TM F-68 (v / v) is added to the culture supernatant between 30 minutes and 1 hour before emptying the bioreactor. 0.1% (v / v) of Pluronic TM F-68 is already present in some media. Even if Pluronic TM F-68 is already in the medium, it may be useful to add Pluronic TM F-68 until it reaches 0.2% (v / v). Thus, in an embodiment, Pluronic TM F-68 is added until a concentration of 0.2% (v / v) is reached in the culture supernatant.

[0158] In an embodiment, before adding the lysis solution to the bioreactor, the bioreactor is first emptied and rinsed. This neutral rinse solution is used to eliminate residual culture medium (components that may interfere with the lysis step) and to continue collecting the extracellular portion of biomolecules such as rAAV2. In an embodiment, once the bioreactor is emptied, it is completely filled with the rinse solution and then emptied again, thereby performing a first rinse step. The inventors have observed that filling / emptying the bioreactor has a positive effect on the harvest of biomolecules such as rAAV. Thus, in an embodiment, more than one rinse step is performed.

[0159] In an embodiment, a rinse buffer or a rinse solution formulation is used during the rinse step.

[0160] In other embodiments, the rinse buffer includes a surfactant such as Pluronic. In other embodiments, the rinse buffer includes 0.1% (v / v) Pluronic. In an embodiment, the rinse buffer includes PBS-MK buffer (PBS-magnesium potassium buffer) and Pluronic TM F-68. In an embodiment, the rinse buffer includes PBS, 2.5 mM KCl, 1 mM MgCl2, 0.1% (v / v) Pluronic, with a pH of 7.

[0161] Cell lysis forms the core step of the method of the present invention because this is necessary for releasing the intracellular target biomolecules (e.g., most rAAV2 are intracellular). The contact time between the cells and the lysis solution, as well as the temperature at which lysis is performed, will affect the efficiency of lysis.

[0162] In an embodiment, the lysis step includes adding the lysis solution to the bioreactor and allowing the cells to contact the lysis solution for a long enough time to lyse at least a portion of the cells. In an embodiment, the lysis solution contacts the cells for 30 minutes to 6 hours. In an embodiment, the lysis solution contacts the cells for 1 to 2 hours. In an embodiment, the method of the present invention includes allowing the cells to have sufficient contact time with the lysis solution to recover the target biomolecule, preferably an AAV virus, from the bioreactor, preferably from a fixed-bed bioreactor. During this contact time, one or more mechanical actions should be applied to the bioreactor. For example, the bioreactor can be agitated.

[0163] In a preferred embodiment, the lysis step is carried out at 37 °C for 2 hours (which may depend on the process), while maintaining stirring at 0.5 - 1 cm / s (such that the liquid level moves relative to the fixed bed structure at a speed of 0.5 to 1 cm / s). The agitation can be periodically switched on / off to disrupt the flow. A back-and-forth circulation (empty / fill step) can also be carried out, as the elution effect on the fixed bed helps to recover the target biomolecules, such as viruses. Alternatively, the lysis step can be carried out twice, each time for one (or two) hours. According to the scientific literature, the lysis step may take from 30 min to several hours (up to 6 hours) (e.g., 4 hours), and the raw lysate is sampled regularly (e.g., after 1, 2, 3, 4 hours). These samples can then be analyzed to quantify the virus titer. In these lysis solutions, the virus should remain stable for several hours.

[0164] In an embodiment, the lysis solution comprises a detergent reagent. Detergent reagents for cell lysis are known in the art, and a specific detergent can be selected based on the cell line used, the target biomolecule, and the type of bioreactor. In an embodiment, the lysis solution comprises at least one detergent, such as Triton X-100, Tween 20, or Tween 80.

[0165] Tween 20 or Triton X-100 is used because the detergent disrupts the cell membrane. The use of the detergent is related to the concentration and time. It is reported that the concentration of Triton X-100 is between 0.1% and 0.5%, and the concentration of Tween 20 is between 0.1% and 1% (v / v). Zwittergent 3-14 (Calbiochem) also seems promising. Due to the European REACH regulations tending to prohibit the use of Triton and its derivatives, it is advantageous to use Tween 20 or zwitterionic detergents. Triton is known to be more effective than Tween. A higher concentration of Tween is recommended (the concentration of Triton or zwitterionic detergent required for sufficient virus release is lower than that of Tween 20. This is related to the inherent property of the detergent - the critical micelle concentration).

[0166] In an embodiment, the lysis solution can comprise a detergent reagent or surfactant selected from the group consisting of Triton X-100, Tween-20, Tween-80, zwitterionic detergents, and combinations thereof. In an embodiment, the concentration of the detergent reagent in the lysis solution is 0.05% to 1.5% v / v. In a preferred embodiment, the concentration of the detergent in the lysis solution is between 0.1% and 1.0% v / v.

[0167] In one embodiment, the cell line comprises HEK293T cells. In an embodiment, the bioreactor is a structured fixed-bed bioreactor, and in other embodiments, the target biomolecule is intracellular. In embodiments, the target biomolecule can include intracellular proteins, viruses, or viral vectors. In a preferred embodiment, the target biomolecule is AAV.

[0168] In an embodiment, the lysis solution further comprises DNase and / or surfactant. In an embodiment, the method of the present invention includes using DNase to degrade free DNA and prevent biomolecules such as AAV from adhering to DNA adsorbed on the fixed-bed fibers. In other embodiments, DNase can be used in suspension culture processes, where, for example, it can be treated with 10 - 50 units of Benzonase / ml in classical phosphate buffer at 37 °C in 1 - 2 mM MgCl2 for 1 hour.

[0169] In an embodiment, nuclease treatment is carried out during the lysis step. The addition of DNase is for:

[0170] 1. Digest the nuclear material of host cells during the extraction of biomolecules such as rAAV.

[0171] 2. Avoid the formation of complexes between nuclear material and biomolecules such as AAV.

[0172] 3. Reduce the viscosity of the lysate to facilitate subsequent filtration and chromatography steps.

[0173] To limit the complexes and viscosity induced by genomic DNA, it is preferred to add the enzyme simultaneously with cell rupture. However, this step may be incompatible with the optimal conditions for enzyme activity (pH and salinity, such as NaCl). In an embodiment, Benzonase (Millipore Sigma) is used as DNase. pH 8 does not change the enzyme activity of benzonase as it is active between pH 7 - 9. However, the salt concentration is too high (1M NaCl; it needs to be reduced to 100 - 150 mM to maintain effective activity). In an embodiment, a salt-activated nuclease (SAN-HQ; ArcticZymes) can be used. Its activity at 500 mM NaCl has been reported.

[0174] In an embodiment, 20 - 50 U / mL of Benzonase is added during the lysis step, and the minimum incubation time is 30 - 60 min. Additionally, it may be necessary to add 1 - 2 mM MgCl2 to maintain the enzyme activity. Note that higher concentrations of Benzonase can also be used.

[0175] As described above, in an embodiment, the lysis step may further include adding an enzyme reagent to the lysis solution. In an embodiment, the enzyme reagent is selected from the group consisting of salt-activated endonuclease and DNase. In another embodiment, the enzyme is a DNase enzyme, more likely DNase, perhaps more specifically Benzonase. Thus, the lysis solution further includes the enzyme reagent. It is believed that adding the enzyme reagent can hydrolyze and cleave free DNA and reduce aggregation or viscosity caused by the DNA content during cell lysis. DNase can also help prevent target biomolecules from adhering to DNA. The enzyme or DNase can be added to the lysis solution simultaneously with the detergent reagent or after adding the detergent reagent. In an embodiment, DNase is added to the lysis solution simultaneously with the detergent reagent. In an embodiment, it is added after adding the detergent reagent (lysing agent). In an embodiment, the pH and conductivity are adjusted to optimize the activity of the enzyme. In an embodiment, the pH of the lysis solution is between 7 and 9, and the conductivity of the solution is between 50 and 150 mM. In an embodiment, the lysis solution including DNase (perhaps Benzonase) is incubated for 20 to 90 minutes. The lysis solution including DNase can be incubated for 30 - 60 minutes.

[0176] In an embodiment, the method of the present invention further includes using DNase to degrade free DNA and prevent AAV from adhering to DNA adsorbed on the fixed bed fiber (as in the suspension culture process, for example, in a classical phosphate buffer, 10 - 50 U Benzo / ml, 37 °C, 1 hour, 1 - 2 mM MgCl2) to recover the target biomolecule, preferably the AAV virus, from the bioreactor, preferably from the fixed bed bioreactor.

[0177] In an embodiment, the lysis step may include adding a DNase reagent to the bioreactor and contacting the cells with the DNase reagent for a sufficient length of time to hydrolyze at least a portion of the cells.

[0178] In an embodiment, the lysis solution including DNase such as Benzonase further includes a salt to adjust the conductivity of the lysis solution. Preferably, the conductivity of the lysis solution is optimal for the enzyme. In an embodiment, the salt is selected from the group consisting of NaCl, MgCl2, and combinations thereof. In an embodiment, the lysis solution including benzonase further includes Mg cations with a concentration less than 150 mM, preferably between 100 and 150 mM. In an embodiment, the lysis solution including a detergent, an enzyme, and a salt between 100 and 150 mM, preferably Mg cations or NaCl and having a pH between 7 and 9, is incubated for 30 minutes to 2 hours.

[0179] In a preferred embodiment, the lysis solution comprises DNase, a detergent or surfactant, or any combination of any of the foregoing, and is at an appropriate pH and conductivity.

[0180] In an embodiment, the cell culture harvest of the target biomolecule can include up to three lysis steps, as Figure 3 shown. In an embodiment, the cell culture harvest of the target biomolecule can include a step of separately adding an enzyme (Benzo), as Figure 3 further shown.

[0181] In an embodiment, the method of the present invention further includes using a high-conductivity solution (0.5 to 1 M NaCl) to limit the interaction between biomolecules such as AAV and the adsorbed biomaterial retained on the fiber. When the target biomolecule is AAV, using a high-conductivity solution is particularly useful.

[0182] In an embodiment, the method further includes using a solution comprising 0.5 to 1 M NaCl during the lysis step and / or the washing step. In other embodiments, the solution is obtained by increasing the ionic strength of the lysis solution during the lysis step.

[0183] NaCl is added because sufficient ionic strength must be maintained to avoid aggregation of rAAV2 and binding to other cell components released during lysis. In an embodiment, a high salt concentration is used during the lysis step. We believe this can also reduce the surface interaction between biomolecules such as rAAV2 and cells or cell debris that may still be adsorbed on the surface of the bioreactor.

[0184] In an embodiment, the method of the present invention further includes using a high-conductivity solution (0.5 to 1.0 M NaCl) to limit the interaction between target biomolecules such as AAV and the adsorbed biomaterial retained on the fiber.

[0185] In an embodiment, the present invention relates to a lysis solution comprising 2.0 M NaCl. In an embodiment, the present invention relates to a lysis solution comprising 0.5 to 1.0 M NaCl. In an embodiment, the present invention relates to a lysis solution comprising 0.5 M NaCl. In an embodiment, the present invention relates to a lysis solution comprising 0.6 M NaCl. In an embodiment, the present invention relates to a lysis solution comprising 0.7 M NaCl. In an embodiment, the present invention relates to a lysis solution comprising 0.8 M NaCl. In an embodiment, the present invention relates to a lysis solution comprising 0.9 M NaCl. In an embodiment, the present invention relates to a lysis solution comprising 1.0 M NaCl.

[0186] In an embodiment, the present invention relates to a bioreactor comprising a fixed bed and a lysis solution comprising between 0.5 and 1.0 M NaCl.

[0187] The present disclosure provides processes and chemistries for increasing the yield of a target biomolecule recovered from a cell culture harvest. More specifically, the present disclosure provides a process for recovering a target biomolecule from a cell culture harvest in a bioreactor comprising a fixed bed and a cell culture broth, the process comprising lysing the cells in the bioreactor in a first step ( Figure 1 step 2 in Figure 3 and step 3 in

[0188] ), and increasing the conductivity of the cell culture broth in a second step. Increasing the conductivity of the cell culture broth may help to neutralize the charge of the target biomolecule. In an embodiment, after incubation with the lysis solution, the conductivity of the lysis solution is increased in the second step by adding additional salt to the lysis solution. Increasing the conductivity of the lysis solution helps to increase the solubility of the target biomolecule that aggregates with cell debris during the lysis step, and can further reduce the viscosity of the lysis solution. Increasing the solubility of the target biomolecule increases the concentration of free target biomolecule available for recovery in the lysis solution. In an embodiment, in the second step of the lysis step, the conductivity of the lysis solution is increased to greater than 150 mM. In an embodiment, after incubation with the lysis solution, NaCl is added to the lysis solution. In an embodiment, after incubation with the lysis solution, the NaCl concentration is adjusted up to 1.0 M. In an embodiment, after incubation with the lysis solution, the NaCl concentration is adjusted up to 2.0 M. In embodiments where there are multiple lysis steps, the conductivity of the lysis solution is increased after incubation with the final lysis solution. In an embodiment, the target biomolecule is recovered from the lysis solution having a high conductivity. In an embodiment, the target biomolecule is recovered from the lysis solution having a high conductivity in an evacuation step (see Figure 2Step #3) in. In an embodiment, the target biomolecule is recovered from the lysate solution with high conductivity in the recirculation loop. In an embodiment, where there are more than one lysis step, the target biomolecule is recovered from the lysate solution in one or more evacuation steps. Another possibility is to perform a separate additional step using an enzyme / endonuclease / DNase / Benzonase immediately after lysis to limit the interference between cell lysis and endonuclease activity. Since the lysis step will be carried out at high ionic strength, this additional step using an endonuclease should be completed using a salt-activated nuclease (SAN). Salt-activated nuclease is a general, non-specific endonuclease that can cleave double-stranded and single-stranded DNA as well as RNA. SAN is active at pH above neutral. However, unlike other nucleases, it has optimal activity at high pH and high salt concentration. These properties make SAN very suitable for removing DNA from cell extracts and protein samples.

[0189] In an embodiment, the lysis step can be carried out in different steps. In an embodiment, the addition of the detergent and the enzyme can be carried out in separate steps. In an embodiment, the bioreactor is evacuated between each step, and the target biomolecule is recovered during each evacuation step. In an embodiment, the detergent, the enzyme, and the high-conductivity solution are carried out in separate steps. In an embodiment, the bioreactor is evacuated between each step, and the target biomolecule is recovered during each evacuation step. In an embodiment, the detergent and the enzyme are added in one step, and the high-conductivity solution is added in a separate step. In an embodiment, the bioreactor is evacuated between each step, and the target biomolecule is recovered during each evacuation step.

[0190] In an embodiment, the bioreactor is agitated during the lysis step. The systems and processes for agitating the bioreactor are described below. In an embodiment, any one or more of the lysis steps can be carried out in a recirculation mode. In an embodiment, the recovery of the target biomolecule can be carried out during any step, and / or from any one or more of the solutions described above or below herein, including the case where a step is operating in a recirculation mode. In one or more embodiments, the recovery of the target biomolecule includes the collection of the target biomolecule. In an embodiment, the target biomolecule is recovered and collected from the cell culture harvest supernatant before the lysis step ( Figure 2 Step #1) in. In an embodiment, the bioreactor is evacuated, and the target biomolecule is recovered and collected from the cell culture harvest supernatant before the lysis step ( Figure 2Step #1) in. In an embodiment, a surfactant, preferably poloxamer 188 (also known as Pluronic), is added to the cell culture harvest before or after emptying the cell culture harvest supernatant and before the lysis step ( Figure 3 Step 1) in. A surfactant, preferably poloxamer 188 (also known as Pluronic F68), is used to reduce or control foaming and / or agglomeration, and / or control shear forces, and / or reduce cell attachment to hydrophilic surfaces in the cell culture harvest, particularly in the supernatant. In an embodiment, a surfactant, preferably poloxamer 188, is added to the cell culture harvest supernatant before the lysis step for between 15 and 45 minutes, and the concentration of poloxamer 188 in the cell culture harvest supernatant before lysis is between 0.05% w / v and 5.0% w / v. In an embodiment, the cell culture harvest supernatant comprising poloxamer is emptied from the bioreactor, and the target biomolecule is recovered and collected from the cell culture harvest supernatant after adding poloxamer and before the lysis step ( Figure 2 Steps #1 and Figure 3 Step 1) in. In an embodiment, after emptying the cell culture harvest supernatant from the bioreactor, a rinsing step is performed, which includes adding a rinsing solution to the bioreactor (see Figure 2 Steps 2 and Figure 3 Step 2) in. The rinsing solution can eliminate residual medium that may interfere with the lysis step. In an embodiment, the rinsing solution includes a buffer. In an embodiment, the buffer includes PBS. In an embodiment, the pH of the washing solution is between 6 and 8. Preferably, the pH of the rinsing solution is 7. In an embodiment, the washing solution further includes a surfactant. In an embodiment, the surfactant is poloxamer. In an embodiment, the rinsing solution is emptied from the bioreactor. In an embodiment, the rinsing step is repeated 2 to 4 times. Preferably, the rinsing step is repeated twice.

[0191] In an embodiment, the rinsing solution includes:

[0192] · PBS

[0193] · 2.5 mM KCl

[0194] · 1 mM MgCl2

[0195] · 0.1% (v / v) Pluronic

[0196] · pH 7

[0197] In an embodiment, a washing step is performed after each lysis step (see Figure 2 Steps #4 and Figure 3(in the intermittent flushing step). The washing step recovers the target biomolecules retained in the bioreactor and the fixed bed of the bioreactor after the lysis step. In an embodiment, the washing step recovers the target biomolecules retained in one or more of the fixed beds (fibers, cell debris, extracellular matrix) and interacting with the bioreactor wall or internal components. In an embodiment, a washing solution is added to the bioreactor. In an embodiment, the washing solution contacts the interior of the bioreactor and the fixed bed for a sufficient length of time to recover the target biomolecules from the bioreactor and the fixed bed into the washing solution. In an embodiment, the flushing solution is emptied from the bioreactor, and the target biomolecules are recovered from the washing solution. In an embodiment, the washing and emptying steps are repeated. In an embodiment, the washing and emptying steps are repeated 2 to 4 times. In an embodiment, the washing and emptying steps are repeated twice.

[0198] In an embodiment, the washing solution comprises:

[0199] · PBS

[0200] · 1.0 to 3.0 mM KCl

[0201] · 0.5 to 2.5 mM MgCl2

[0202] · 0.01 to 0.2% (v / v) Pluronic

[0203] · 5 to 15 mM Tris - pH8

[0204] In another embodiment, the washing solution comprises:

[0205] · PBS

[0206] · 2.5 mM KCl

[0207] · 1 mM MgCl2

[0208] · 0.1% (v / v) Pluronic

[0209] · 10 mM Tris - pH8

[0210] In an embodiment, during any one or more of the cell culture harvest steps described above or below herein, the bioreactor can be agitated using any of the techniques or processes described below herein. In an embodiment, agitation increases the amount of target biomolecules recovered from the bioreactor.

[0211] In one embodiment, the inventors observed that when the pH is below or above the isoelectric point of AAV (between 5 and 6 depending on the serotype), the recovery rate is better than at neutral pH (ectopic harvest was performed at pH 3.0 and 8.0). The inventors also showed that high conductivity allows for better recovery.

[0212] As described above, it is believed that AAV adheres to biomaterials adsorbed on fixed bed fibers. The inventors have shown that non-neutral pH (low: 3.0 - 4.0 and high: 8.0 - 9.0) and high conductivity solutions (1M NaCl) contribute to the recovery of the virus, indicating that electrostatic interaction is a key factor in the attachment of AAV to biomaterials adsorbed on fixed bed fibers. The inventors conducted some tests to prove this and observed that when the pH is below or above the isoelectric point of AAV (between 5 and 6 depending on the serotype), the recovery rate is better than at neutral pH (ectopic harvest tests were performed at pH 3.0 and 8.0). The inventors also showed that high conductivity allows for better recovery (ectopic harvest tests were performed at 0.5 and 1M NaCl). These solutions are very similar to the solutions used to elute the virus from IEX (ion exchange chromatography) and CEX (cation exchange chromatography) and are suitable for maintaining the stability of AAV. Thus, in one embodiment, the present invention combines a cell lysis chemical solution (detergent) with a solution commonly used to elute the virus from IEX and CEX chromatography systems to recover the virus from a fixed bed.

[0213] In an embodiment, the method of the present invention includes using a pH below the isoelectric point (pH 3 to 5) or above the isoelectric point (pH 8 to 9).

[0214] In an embodiment, the method of the present invention includes using a pH below or above the isoelectric point of the target biomolecule. By using a pH below or above the isoelectric point of the target biomolecule, the interaction between the target biomolecule and the hydrophilic fixed bed material is prevented.

[0215] Note that since the virus is less stable at pH 3, the solution should be quenched and buffered after lysis.

[0216] Thus, in other embodiments, after the lysis step, the solution is quenched and buffered. In an embodiment, an inactivating solution is added after the lysis step to quench or inhibit the lysis solution, thereby improving the stability of the biomolecule after harvest.

[0217] High ionic strength (NaCl > 150 mM) is important for avoiding viral vector aggregation, but the concentration of these monovalent salts greatly reduces the activity of Benzonase (e.g., at 150 mM NaCl, relative activity loss + / - 70%). To ensure efficient harvest, the following compromise can be used:

[0218] In an embodiment, first, the lysis step is started without adding NaCl (N.B.: PBS buffer provides 137 mM) to maintain the effective activity of Benzonase (manufacturer's manual). Since Mg 2+ is a cofactor of Benzonase, its concentration must be accurate. Thus, in an embodiment, 2 mM MgCl2 is added, which is optimal for enzyme activity. Under these salinity conditions, Benzonase retains effective activity, and aggregation of the vector should be limited. In the next step, after incubation for 1 (or 2) hours, the ionic strength of the lysis buffer can be increased by adjusting the NaCl concentration up to 1 M. Under these conditions, Benzonase is inhibited, but the risk of viral vector aggregation is reduced, and / or aggregates of viral particles are dissolved (if this process is reversible). At such salinity, the interaction of the virus with plastic materials should be reduced.

[0219] In another embodiment, an additional separate step using an endonuclease / Benzonase is performed immediately after lysis to limit the interference between cell lysis and endonuclease activity. Since the lysis step will be carried out at high ionic strength, this additional step using the endonuclease should be done with a salt-activated nuclease.

[0220] In an embodiment, the lysis buffer is formulated as follows:

[0221] - 1% (v / v) Tween 20 or Triton X-100

[0222] - 1 M NaCl

[0223] - 10 mM Tris - pH8

[0224] - 0.1% Pluronic (v / v)

[0225] - 20 - 50 U / mL endonuclease (Dnase) and 1 - 2 mM MgCl2

[0226] A buffer solution of 10 mM Tris - pH8 is used because it has been observed that an alkaline solution plus an appropriate amount of detergents such as Triton X - 100 or Tween 20 is sufficient to lyse cells encapsulating biomolecules (such as cells encapsulating rAAV) and release the biomolecules (such as virus particles) from the cells. According to different processes, the pH reported for chemical cell lysis protocols is usually between 8 - 9. Acidic pH (3 - 4) also seems promising for harvesting biomolecules (such as virus particles), but it may affect the integrity of (viral) proteins.

[0227] The role of 0.1% Pluronic (v / v) is similar to its role in the rinsing solution. Pluronic TM F - 68 is a non - ionic surfactant that is commonly used to control shear forces, prevent foaming in agitated cultures, and reduce cell attachment to hydrophilic surfaces. It is also known that adding it can prevent AAV from adhering to the plastic of pipettes and plastic containers during storage.

[0228] In one embodiment, the present invention includes a method for recovering a target biomolecule (preferably AAV, more preferably intracellular AAV) through a combination of detergents, Pluronic (to prevent AAV adsorption), high conductivity, pH, and benzonase (to cleave host cell DNA).

[0229] As described above, in a preferred embodiment, after the lysis of the cells, one or more washing steps are performed by filling and / or emptying the bioreactor with a washing solution.

[0230] The washing steps are performed to remove biomolecules such as rAAV2 that are blocked in the fixed bed (fibers, cell debris, extracellular matrix) and / or interact with the bioreactor wall. Once the bioreactor is empty, it is filled with the washing solution and then emptied again, thereby performing one washing step. As previously mentioned, filling / emptying the bioreactor has a positive impact on the harvest of biomolecules such as rAAV. Therefore, in a preferred embodiment, the washing steps are performed more than once, for example, twice. In an embodiment, the washing steps are performed 2 hours after cell lysis.

[0231] In an embodiment, the washing solution is formulated as follows:

[0232] - PBS

[0233] - 2.5 mM KCl

[0234] - 1 mM MgCl2

[0235] - 0.1% (v / v) Pluronic

[0236] -10 mM Tris-pH8

[0237] In a preferred embodiment, the pH of the washing solution is more alkaline compared to the rinsing solution.

[0238] When the cells grown on the surface are lysed, a mechanical action or movement should be applied to recover the cell debris and target products that may still adhere to the surface. In some cases, gentle agitation alone is sufficient. Sometimes, just the movement of the liquid is enough to recover the target product. But sometimes stronger mechanical action is required. Recovering biomolecules such as AAV from a fixed-bed bioreactor is more complex than from suspension cells because the biomolecules such as AAV may adhere to the surface of the bioreactor (the surface available for cell growth).

[0239] Accordingly, the invention described above herein includes a mechanical action for recovering and / or increasing the recovery rate of cells (adherent or suspension cells) from a fixed bed and / or a bioreactor. The inventors tested the harvesting of AAV2 from a fixed-bed bioreactor and showed that AAV does not adhere to the "fresh" hydrophilic PET / PP material (the uncolonized fixed-bed fibers), indicating that AAV does not adhere to the fixed-bed material (PET / PP) itself but remains captured by cell debris or other biomolecules (such as cell membranes, DNA, proteins, etc.), which still clog the interior of the fixed-bed structure after chemical (detergent) lysis.

[0240] (Ectopic) AAV harvesting tests conducted under static, gentle agitation, and strong mechanical action (vortex) indicated that mechanical action plays a role in the harvesting yield of AAV.

[0241] The present invention relates to a method for obtaining one or more target biomolecules from cells cultured in a fixed-bed bioreactor, the method comprising:

[0242] - adding a lysis solution to the bioreactor to lyse the cells in the bioreactor, and applying one or more mechanical actions to the bioreactor before, during, and / or after lysing the cells, and recovering the one or more target biomolecules from the bioreactor thereafter.

[0243] The present invention includes a harvesting process for recovering target biomolecules from a cell culture harvest in a bioreactor comprising a fixed bed, wherein the process comprises applying a mechanical action to the bioreactor, the fixed bed, or a combination thereof for a sufficient length of time to increase the recovery rate of the target biomolecules.

[0244] In an embodiment, the mechanical action may be selected from moving a fixed-bed bioreactor or moving the solution within the fixed-bed bioreactor such that the liquid level moves relative to the fixed-bed structure. The liquid level may move at a certain speed, which represents the distance that the liquid level moves relative to the fixed-bed structure within a certain time range (e.g., expressed in cm / s).

[0245] In other embodiments, moving the bioreactor includes actions selected from the group consisting of vibrating, agitating, compressing, expanding, shaking, applying ultrasonic waves, and combinations of the foregoing.

[0246] In an embodiment, moving the fixed-bed bioreactor includes moving the fixed bed within the bioreactor. In other embodiments, moving the fixed bed includes actions selected from the group consisting of vibrating, agitating, compressing, expanding, and combinations of the foregoing.

[0247] In other embodiments, moving the solution includes actions selected from the group consisting of vibrating, agitating, compressing, expanding, shaking, applying ultrasonic waves, at least partially emptying the liquid in the bioreactor, adding liquid to the bioreactor, and combinations of the foregoing. The solution may be any type of suitable solution, such as a lysis solution, a wash solution, or an inactivation solution. The solution (e.g., a lysis solution) may be moved internally by using an internal circulation within the bioreactor (such as by a stirrer) or by using an external recirculation (circulation or perfusion). In other embodiments, the solution may be moved internally within the bioreactor by an impeller using internal circulation.

[0248] In a preferred embodiment, the method includes agitating the bioreactor and moving the solution within the fixed-bed bioreactor such that the liquid level moves relative to the fixed-bed structure. In other preferred embodiments, the agitating and moving steps are performed simultaneously. By moving the liquid level relative to the fixed-bed structure while agitating the bioreactor (e.g., by vibration), the energy of agitation can be transferred to the air-liquid interface (ALI) of the fixed-bed structure. The moving step may include at least partially emptying the liquid in the bioreactor, such as by moving the liquid level from near the top of the fixed-bed structure to near the bottom of the fixed bed. The moving step may include adding liquid to the bioreactor, such as, for example, by adding a cell lysis solution to the bioreactor. The moving step may include moving the fixed bed relative to the bioreactor to move the position of the liquid level. Prior to the moving step, the liquid level may be positioned above the fixed-bed structure, which may involve raising and lowering the liquid level multiple times (but may also be only once) (such as, for example, from the top of the fixed-bed structure to the bottom of the fixed-bed structure). The agitating step may include vibrating the bioreactor.

[0249] The method can include adjusting the position of the liquid level in a fixed-bed bioreactor while vibrating the bioreactor. The adjusting step can include filling and flushing the bioreactor with liquid, including repeatedly filling and flushing the bioreactor with liquid.

[0250] Further, the method can include tilting the bioreactor and / or compacting the fixed bed in the bioreactor. The adjusting step can include moving the fixed bed relative to the bioreactor.

[0251] The method can include vibrating, tilting, and emptying the bioreactor. The vibrating, tilting, and emptying steps can be performed simultaneously.

[0252] In a preferred embodiment, the biomolecule is an intracellular biomolecule. In a more preferred embodiment, the target biomolecule is an intracellular protein, virus, or viral vector. This mechanical action can be applied continuously or intermittently and can be applied during one or more target biomolecule harvesting steps; for a sufficient length of time to increase the recovery rate of the target biomolecule. In an embodiment, the mechanical action is vibration. In another embodiment, the mechanical action is rotation. In yet another embodiment, the mechanical action is a combination of different actions.

[0253] In one embodiment, the mechanical action is applied after cell culture. In another embodiment, the mechanical action is applied during the cell harvesting process. In one embodiment, the cell culture harvest includes animal cells. In other embodiments, the cell culture harvest includes HEK293T cells. In another embodiment, the mechanical action is applied during one or more target biomolecule harvesting steps of the target biomolecule harvesting process. In another embodiment, the mechanical action is applied during one or more target biomolecule harvesting steps, the harvesting steps including one or more of the following: before emptying the culture medium from the bioreactor, when emptying the culture medium from the bioreactor, after emptying the culture medium from the bioreactor, during the addition of a rinsing solution, during the rinsing step, during the emptying of the rinsing solution, before the addition of a lysis solution, during the addition of the lysis solution, during the lysis step, during the emptying of the lysis solution, after the emptying of the lysis solution, before the addition of a washing solution, during the addition of the washing solution, during the washing step, during the emptying of the washing solution, and after the emptying of the washing solution.

[0254] In one embodiment, the mechanical action applied during any one or more steps in the process of harvesting target biomolecules (including cell vesicles), which includes the steps listed above and below herein, may include any one or more processes or devices known to those skilled in the art for applying mechanical action to a bioreactor, a fixed bed, or a combination thereof. In one embodiment, the mechanical action applied during any one or more steps in the process of harvesting target biomolecules, which includes the steps listed above and below herein, may include any one or more processes or devices disclosed in PCT Publication WO2022 / 254039, titled "Bioreactor System with Enhanced Cell Harvesting Capabilities and Related Methods", the content of which is incorporated herein by reference in its entirety. Where possible, the description herein uses reference numerals corresponding to equivalent elements in the above PCT application and its accompanying drawings.

[0255] In a preferred embodiment, the mechanical action and its application include vibrating the bioreactor, wherein the bioreactor includes a fixed bed material to which cells are attached or captured therein, and wherein these cells include the target biomolecule. In one embodiment, a modified version of the system shown and described in A of PCT Publication WO2022 / 254039 (titled "Bioreactor System with Enhanced Cell Harvesting Capabilities and Related Methods", the content of which is incorporated herein by reference in its entirety) is used to apply mechanical action to a bioreactor containing the target biomolecule. Additionally, the systems for providing mechanical action as described above and below herein are not limited to the recovery of target biomolecules and can also be readily adapted for cell collection, as well as for generating seed cultures for adherent cell production from small to large scale, and / or any application that requires or benefits from agitation of a container. Figure 2 and Figure 2 A in the PCT publication WO2022 / 254039, titled "Bioreactor System with Enhanced Cell Harvesting Capabilities and Related Methods", the content of which is incorporated herein by reference in its entirety, is used to apply mechanical action to a bioreactor containing the target biomolecule. Additionally, the systems for providing mechanical action as described above and below herein are not limited to the recovery of target biomolecules and can also be readily adapted for cell collection, as well as for generating seed cultures for adherent cell production from small to large scale, and / or any application that requires or benefits from agitation of a container.

[0256] In an embodiment of the present invention, the mechanical action or movement may be selected from stirring, vibrating, vortexing, or moving the solution inside the bioreactor. In an embodiment, moving the solution includes filling and / or emptying the bioreactor with the solution. In an embodiment, the mechanical action or movement is selected from the group consisting of vibrating, stirring, compressing, vortexing, shaking, applying ultrasonic waves, filling and / or emptying the bioreactor with the solution, expanding, and combinations of the foregoing. In a preferred embodiment, harvesting may be carried out under stirring conditions. In other embodiments, the stirring is carried out at a speed of 0.5 to 2 cm / s. Thus, in an embodiment, the movement of the liquid level relative to the fixed bed structure occurs at a speed of 0.5 to 2 cm / s. This refers to the (vertical) movement speed of the liquid inside the bioreactor (e.g., during emptying and / or filling of the bioreactor).

[0257] In an embodiment, the method of the present invention includes applying vibration to the bioreactor. In an embodiment, the vibration is applied during one or more of the lysis step and / or subsequent washing steps. In this way, biomolecules such as AAV that are still trapped in the fixed bed matrix can be recovered.

[0258] In an embodiment, the vibration is applied during one or more of the processes listed above herein. In an embodiment, the vibration is applied during the washing step, or during the contact time between the lysis solution and the cells, or during the application of DNase, or during the application of surfactant, or during the harvesting of cells, or when using a pH below or above the isoelectric point, or when using a solution with high conductivity.

[0259] In a preferred embodiment, a cell harvesting shaker described in PCT / EP2022 / 065264 ("Bioreactor System with Enhanced Cell Harvesting Capabilities and Related Method", June 03, 2022, the content of which is incorporated herein by reference in its entirety) is used to harvest biomolecules such as AAV from a fixed bed bioreactor. Vibrations of 20 to 100 Hz can be combined with filling and / or emptying cycles to transfer the vibrations to the ALI (air-liquid interface) of the fixed bed structure. Since such vibrations are suitable for recovering fragile live cells from a fixed bed bioreactor using vibration and detachment enzymes such as trypsin, they should also be suitable for sensitive products such as AAV. Since detergents are used to lyse the cells, one drawback may be the formation of foam. Therefore, after chemical lysis, vibration may be more appropriate during the washing step using a buffer solution that does not contain a high concentration of detergent.

[0260] Thus, in an embodiment, the mechanical action or movement is vibration applied to the bioreactor, wherein the frequency of the vibration is 20 to 100 Hz. In a preferred embodiment, a liquid (such as a filling and / or emptying cycle) is moved inside the bioreactor while the vibration is being carried out to transfer the vibration to the fixed bed structure. In an embodiment, the vibration is applied by a shaker table.

[0261] In an embodiment, the method further comprises agitating the bioreactor, moving the liquid level relative to the fixed bed structure, and introducing a cell lysis solution into the bioreactor. In one embodiment, the agitation and movement steps are carried out simultaneously. The movement step may include moving the liquid level to a higher or lower position than the previous liquid level. In an embodiment, the movement step includes at least partially emptying the liquid in the bioreactor, such as by moving the liquid level from near the top of the fixed bed structure to near the bottom of the fixed bed. The movement step may include adding liquid to the bioreactor, such as by adding a cell lysis solution to the bioreactor. The movement step may include moving the structure (fixed bed) for cell retention / attachment and growth relative to the bioreactor to move the position of the liquid level. Prior to the movement step, the liquid level may be positioned above the fixed bed structure, which may involve raising and lowering the liquid level multiple times (but may also be only once), such as from the top of the fixed bed structure to the bottom of the fixed bed structure. Moving the liquid level relative to the fixed bed structure may also include using a stirrer. In some embodiments, the stirrer may be a rotating, non-contact magnetic impeller, blade or helical stirring system, or an external circulation system. In some embodiments, the stirrer may include a disk blade turbine, a curved blade turbine, an open blade fluid foil axial impeller, an inclined blade turbine type impeller or a three-blade propeller.

[0262] The agitation step may include vibrating the bioreactor. The introducing step may include an enzyme reagent selected from the group consisting of salt-activated endonuclease and DNase.

[0263] The method may include adjusting the position of the liquid level in a fixed bed bioreactor while applying one or more additional mechanical actions to the bioreactor. In a preferred embodiment, the one or more additional mechanical actions include vibrating the bioreactor. The adjusting step may include filling and flushing the bioreactor with liquid, including repeatedly filling and flushing the bioreactor with liquid. Adjusting the position of the liquid level in the fixed bed bioreactor may include using a stirrer, such as an impeller, inside the bioreactor.

[0264] Furthermore, the method may include tilting the bioreactor and / or compacting the fixed bed in the bioreactor. The adjusting step may include moving the fixed bed relative to the bioreactor.

[0265] The method can include vibrating, tilting, and emptying a bioreactor. The vibrating, tilting, and emptying steps can be performed simultaneously.

[0266] For data on process steps and condition examples for harvesting and recovering biomolecules such as AAV cells from a fixed-bed bioreactor, see Example 2 and the related Figure 2 .

[0267] In an embodiment, the present invention relates to harvesting biomolecules (such as viral vectors, particularly AAV) from a fixed-bed bioreactor in a recirculation mode.

[0268] The invention disclosed herein includes processes, compositions, and devices for increasing the yield and recovery of target biomolecules obtained from cell cultures. Any step in such processes and systems for implementing such systems and processes can be operated in a batch mode, a perfusion mode, a recirculation mode, or a combination thereof. In one embodiment, the system disclosed herein can include, for example, one or more liquid transfer devices, such as a two-way pump or a reversible pump, or other devices, to transfer fluid to and from the bioreactor. The liquid transfer device can recirculate the fluid emptied during the emptying mode back into the bioreactor during the filling mode, or can introduce fresh fluid into the bioreactor during such filling mode. The liquid transfer device can also perform only emptying, clearing, or filling cycles, and can complete with only one such cycle (e.g., one emptying or filling) or multiple cycles.

[0269] In an embodiment, target biomolecules are obtained from a fixed-bed bioreactor in a recirculation mode. In one embodiment, the present invention includes recovering target biomolecules from a fixed-bed bioreactor having a V / S ratio of about 0.3 to about 0.1 and a void volume of about 10% to about 55%, including the following steps (see also the schematic overview in Figure 4 ), and repeating one or more of the steps 1 to 10 times, preferably 1 to 5 times.

[0270] - Add 0.1% Pluronic (30 min)

[0271] - Rinse 2 times, PBS-MK (5 min - 37 °C)

[0272] - Cell lysis #1, Triton / benzonase (2 h - 37 °C)

[0273] - Increase conductivity (final concentration 1 M NaCl - 30 min)

[0274] - Cell lysis #2 and #3, Triton / salt (30 min - 37 °C)

[0275] - Washing, PBS-MK (5 min)

[0276] In one embodiment, the present invention includes a method for harvesting an intracellular target biomolecule, preferably AAV, from a fixed-bed bioreactor. When the bioreactor is operating in a recirculation mode, the method includes one or more of the steps described above herein. In one embodiment, the recirculation mode includes a recirculation loop. This "artificially" increases the volume of the bioreactor, allowing each surface (and thus each cell and virus) to be exposed to a similar amount of enzyme and lysis buffer without increasing the impurity concentration. The recirculation mode can be used throughout the harvesting process, intermittently, or only during one or more steps.

[0277] Operating the bioreactor in a recirculation mode during harvesting also allows for the implementation of back-and-forth cycling (performed during harvesting, see Figure 5 ) to generate a mechanical effect, which can increase and / or enhance the yield of the target biomolecule, preferably the AAV virus, recovered from the bioreactor. The recirculation loop can include a sampling section for sampling during operation of the recirculation loop. The recirculation loop can also or alternatively be used to empty or evacuate the bioreactor, including during back-and-forth cycling (combining on / off agitation cycling with fixed-bed emptying / filling). In another embodiment, the recirculation loop includes means for performing a medium exchange operation.

[0278] In one embodiment, the present invention includes one or more of the following steps to recover a target biomolecule, preferably an AAV virus, from a bioreactor, preferably from a fixed-bed bioreactor.

[0279] · Performing a number of cycles with several rinses.

[0280] · Having sufficient contact time.

[0281] · Using DNase to degrade free DNA and prevent AAV from adhering to DNA adsorbed on the fixed-bed fibers.

[0282] · Using a surfactant to limit the adsorption process of AAV on the walls of plastic containers.

[0283] · Performing harvesting under agitation.

[0284] · Applying vibration to the bioreactor.

[0285] · Applying vibration to the bioreactor during one or more lysis steps and / or subsequent rinse steps.

[0286] · Applying vibration to the bioreactor during one or more of the processes listed above herein.

[0287] · Run one or more of the above steps in a recirculation mode, preferably using a recirculation loop.

[0288] In an embodiment, one or more of the following steps are run in a recirculation mode, preferably using a recirculation loop: the rinsing step, the contact time between the lysis solution and the cells, the application of DNase or surfactant, cell harvesting, or when using a pH below or above the isoelectric point, when using a solution with high conductivity, or when extracting one or more charged biomolecules from a fixed-bed bioreactor using electrophoresis.

[0289] For a schematic diagram of a preferred embodiment of the present invention, see Figure 2 . In an embodiment of the method, electrophoresis is used to extract one or more charged biomolecules from a fixed-bed bioreactor. In an embodiment, an alternative solution based on electrophoresis is used to extract biological particles.

[0290] In one embodiment, the present invention includes using electrophoresis to extract one or more charged biomolecules from a fixed-bed bioreactor, preferably from a fixed bed. When electrophoresis is applied to the fixed bed, a constant and uniform electric field is applied to cause the biomolecules in the fixed bed to flow.

[0291] In one embodiment, the present invention includes positioning two electrodes in the bioreactor: (A) an anode, located on top of the fixed bed and in direct contact with it; (B) a cathode, located on the bottom of the bioreactor, below the impeller ( Figure 6 A). Depending on the type of biomolecule and its charge, the electrode charges can be easily reversed.

[0292] In one embodiment, the electrode structure can be similar to a mesh to ensure a uniform electric field is generated ( Figure 6 B).

[0293] The present invention demonstrates a derivative electrophoresis structure implemented in a fixed-bed bioreactor, where the fixed bed is composed of assembled elements such as PET FB layers or 3D structures. After cultivation, cell lysis is performed if necessary, and a constant potential difference is applied between two electrodes, which causes any charged biomolecules (including charged viruses, proteins, and DNA) to migrate ( Figure 7 A).

[0294] In one embodiment, the impeller is started in reverse to reverse the direction of liquid circulation ( Figure 7 B), while adding PBS buffer to supplement the shortage of liquid in the fixed bed. Since the liquid and biological materials will flow towards the impeller and the center of the bioreactor, it is necessary to pump out the excess liquid and collect the biological product into a harvest bottle.

[0295] Advantages:

[0296] The present invention requires only minimal modifications to existing bioreactor hardware and software systems, including: (1) the implementation of electrodes; and (2) adjustment of the software to allow for reversal of the rotational direction of the magnetic impeller.

[0297] In one embodiment, the present invention includes one or more of the following steps to recover a target biomolecule, preferably an AAV virus, from a bioreactor, preferably from a fixed-bed bioreactor.

[0298] · Perform a number of cycles with a number of rinses.

[0299] · Have sufficient contact time.

[0300] · Use electrophoresis to extract one or more charged biomolecules from the fixed-bed bioreactor.

[0301] In one embodiment, the present invention includes a method for recovering a target biomolecule (preferably AAV, more preferably intracellular AAV) by a combination of detergent, Pluronic (to prevent AAV adsorption), high conductivity, pH, and benzonase (to cleave host cell DNA).

[0302] In a second aspect, the present invention relates to a system comprising: a stirring device and a fixing system, the stirring device comprising a platform adapted to receive a bioreactor; and, the fixing system comprising at least one fastener for holding the bioreactor to the platform, and a bridge structure adapted to be mounted to the bioreactor and receive the at least one fastener. In a preferred embodiment, the system further includes a bioreactor.

[0303] In one example, the system may include a stirring device for stirring the bioreactor and a second device for moving or changing the liquid level within the bioreactor. For example, in one instance, by filling the bioreactor with liquid such that the liquid level is at or above the top portion of the fixed bed, and emptying the bioreactor, thereby moving the liquid level from the filling level to the bottom of the fixed bed or below the bottom. Alternatively or additionally, the second device may create a reciprocating or "back-and-forth" movement of a portion of the liquid between the inside and outside of the fixed bed bioreactor. This back-and-forth movement of the liquid may be generated by an actuator such as one or more pumps to create a pulsating action, where the liquid is partially emptied and then partially introduced into the bioreactor, or it may involve a complete emptying and refilling of the bioreactor. For this purpose, the bioreactor may be associated with an inlet, an outlet, or a discharge port, each of which may be associated with a suitable pump and vent. The bioreactor may include a rigid container, or may include a disposable or single-use container or bag. The stirring device may be any device that applies stirring energy to the bioreactor or to the fixed bed, for example. The portion of the bioreactor to which energy is applied may include any part of the bioreactor as long as it can cause the fixed bed to vibrate. The stirring device may include, for example, a stirrer in the form of a vibrating table, a vortex device, an oscillator, or another device for applying mechanical energy to the bioreactor and / or for adherent cell growth / interception such as the fixed bed. The stirring device may be inside or outside the bioreactor. The vibrating action may be oscillatory, reciprocating, or periodic, harmonic or random. The frequency may be between 0.5 and 200 Hz. The frequency may be 20 - 100 Hz, or more specifically, 50 - 80 Hz. The amplitude may be low, such as 0.5 - 5.0 mm, or more specifically, 2 - 3 mm. The second device may include, for example, one or more liquid transfer devices, such as a two-way or reversible pump, or other devices for transferring fluid to and from the bioreactor. The second device may recycle the fluid emptied during the emptying mode back into the bioreactor during the filling mode, or may introduce fresh fluid into the bioreactor during such filling mode. The second device may also perform only emptying, clearing, or filling cycles, and may complete these using only one such cycle (e.g., one emptying or filling) or multiple cycles. The second device may be integrated with the stirring device so that these devices work in concert or in parallel. Alternatively, the devices may be part of a single device.

[0304] In some embodiments, the bioreactors disclosed herein may include a process controller. In some embodiments, the systems disclosed herein may include one or more process controllers. In an embodiment, the one or more process controllers are configured to control both the bioreactor and the system. In some embodiments, the process controller is configured to control the operation of the bioreactor and / or the system, and may include a plurality of sensors, a local computer, a local server, a remote computer, a remote server, or a network. In some embodiments, the bioreactor and / or the system may include one or more sensors, such as a temperature sensor (e.g., a thermocouple), a flow rate sensor, a gas sensor, a liquid level sensor, or any other sensor. In some embodiments, the process controller may operably control aspects of the biomolecule production and harvesting process and may be coupled to sensors disposed in the bioreactor and / or the system, e.g., to control in real time the temperature, volumetric flow rate, or gas flow rate entering the bioreactor and / or the system. In an embodiment, the process controller is divided into two parts, namely a programmable logic controller (PLC) and a supervisory control and data acquisition (SCADA). The PLC is the intelligence of the system and is connected to sensors and actuators. The PLC only contains data and does not contain power. SCADA is important for visualization, data historian, and audit trail. This SCADA system runs on a server that stores the data historian and supports visualization. In an embodiment, the information can also be visualized from a client tablet. In an embodiment, the client network can be directly connected to the server for remote access. In some embodiments, the process controller may include a human machine interface (HMI), such as a display, e.g., a computer monitor, a smartphone application, a tablet application, or an analog display, that a user can access to determine the state of the system (based on the sensors included in the system) and to control the system via various actuators such as pumps, valves, heaters, and stirrers. In some embodiments, the process controller may include an input, e.g., a keyboard, a separate smart tablet, a keypad, a mouse, or a touch screen, to allow a user to input control parameters to control the operation of the bioreactor. In some embodiments, the process controller may control access to the bioreactor.

[0305] In either case, a controller may be provided to manage the method of the present invention, wherein a lysis solution is added to the bioreactor to lyse the cells in the bioreactor, and one or more mechanical actions are applied to the bioreactor before, during, and / or after lysing the cells, and one or more target biomolecules are recovered from the bioreactor thereafter. In an embodiment, the controller manages an algorithm or process to perform a combined operation of agitation and liquid movement back and forth into and out of the bioreactor in an automated manner or according to operator instructions.

[0306] Using the above system, the method of the present invention can be carried out, wherein a lysis solution is added to the bioreactor to lyse the cells in the bioreactor, and one or more mechanical actions are applied to the bioreactor before, during and / or after lysing the cells, and one or more target biomolecules are recovered from the bioreactor thereafter. As previously mentioned, the mechanical action can include a combination of two or more mechanical actions. In a preferred embodiment, the system of the present invention allows for the combination of agitation of the bioreactor with the movement of the liquid inside the bioreactor to increase the yield of biomolecules harvested. For example, the system can vibrate, pulse or oscillate the bioreactor vessel while circulating the lysis solution with a pulsed or back-and-forth liquid movement via external pumping. Alternatively, the lysis solution can be moved internally by using internal circulation within the bioreactor (such as via a stirrer) or by using external recirculation - cycling or perfusion. For example, the vibration can be at a selected frequency (e.g., 20 - 300 Hz, including for example 60 - 80 Hz), and the pulsed application of the liquid is carried out for multiple cycles (e.g., between 1 and 10, and at a flow rate between 0.1 - 5 L / min). Such agitation creates the maximum energy transfer at the liquid level of the gas phase adjacent to the bioreactor. By dynamically adjusting the liquid level inside the bioreactor and along the fixed bed during the vibration / oscillation / stirring process, such as by using a second device (e.g., a pump), the biomolecules are more effectively detached from the fixed bed material. Thus, the yield or harvest of biomolecules from the bioreactor is increased in an easy and relatively inexpensive manner without a significant increase in cost or complexity.

[0307] The system may further include a harvesting container, a waste container and a supply container containing a cell lysis solution, each of which can be fluidically connected to the bioreactor or to each other. An optional container for supplying a flushing solution and a deactivation solution may also be arranged to be fluidically connected to the bioreactor, or to any of the aforementioned containers. A filter or other device may be positioned between any of these containers and / or the bioreactor. Any or all of these containers (and the solution therein) may be optionally stirred and may be part of a recirculation loop to allow recirculation with the bioreactor, possibly also with a reservoir. The system may also be suitable for preheating the lysis solution and / or maintaining the temperature of the cell lysis solution (usually 37° C.). The system is designed for the growth of adherent cells and non-adherent cells. In an embodiment, the bioreactor is a batch bioreactor. In another embodiment, the bioreactor is a perfusion bioreactor. In a perfusion bioreactor, equal volumes of culture medium are simultaneously added to and removed from the bioreactor, while the cells remain in the bioreactor. This provides a stable source of fresh nutrients and can continuously remove cell (waste) products. Perfusion allows for higher cell densities and thus higher volumetric productivity compared to conventional bioreactors. In addition, perfusion bioreactors allow for continuous harvesting of secreted products during the process of removing culture medium.

[0308] In embodiments, the system comprises one or more devices for concentrating / purifying the target biomolecule by filtration such as tangential flow filtration (TFF), coated magnetic beads (affinity), packed or expanded bed chromatography, or specific purification affinity columns or ultracentrifugation steps.

[0309] In an embodiment, the system includes a concentrator. The concentrator of the system can be selected from a variety of devices known to those skilled in the art that are suitable for reducing the volume of the liquid in which the target biomolecule is retained. In some embodiments, the concentrator includes one type of concentration device (e.g., a tangential flow filter). In some embodiments, the concentrator includes more than one type of concentration device (e.g., a tangential flow filter and a dead-end filter). Most of these devices are based on filtration and / or size exclusion chromatography. In one embodiment, the concentrator is a filtration device, more preferably a microfiltration device, or an ultrafiltration device, or a combination of both a microfiltration and an ultrafiltration device. When the system is equipped with an ultrafiltration device for reducing the volume of the liquid in which the target biomolecule is retained, the membrane of the device is adapted to allow water and low molecular weight solutes (commonly referred to as permeate) to pass through, while macromolecules such as biomolecules are retained on the retentate side of the membrane. In other embodiments, the system is provided with a tangential flow filtration device (TFF). In an embodiment, the TFF is equipped with at least one hollow fiber having pores with a porosity sufficient to retain almost all of the target biomolecules while allowing smaller contaminants such as growth media and solutes to pass through the pores of the membrane. Different from dead-end filtration, in which the liquid passes through the membrane or bed and the solids are captured on the filter, in the TFF device, a tangential flow is allowed to pass across the filter surface rather than directly through the filter. Thus, the formation of a filter cake can be avoided in the TFF. In another embodiment, the TFF can be equipped with a cassette / cartridge that allows tangential flow filtration. In yet another embodiment, the TFF is a single-pass tangential flow filtration (SP-TFF). This device is particularly advantageous when purifying proteins such as antibodies. In some embodiments, the TFF device includes a membrane with an area between about 1000 cm 2 and 2000 cm 2 , preferably about 1500 cm 2 . The TFF can be reusable, single-use, and / or disposable. In some embodiments, the TFF is plug-and-play.

[0310] In an embodiment, the system is provided with one or more membrane-based chromatography devices. The membranes can have various shape factors, such as columns, well plates, and cartridges, with volumes as small as 5.5 microliters and up to several liters. The membrane-based chromatography devices include, for example: a protein A-based affinity membrane chromatography device for antibody purification in the capture step; a device utilizing affinity membrane adsorption technology; a device utilizing a multimodal strong anion exchange membrane; a device utilizing a weak anion exchange membrane; a device for selectively purifying biologics such as sialic acid-binding lectins, biomarkers, and many virus surface proteins (e.g., AAV4 and AAV5) and recombinant proteins that have a binding affinity for sialic acid using affinity membrane chromatography.

[0311] As described above, the present invention further relates to a system for recovering a target biomolecule from a fixed-bed bioreactor.

[0312] To maintain the integrity of the bioreactor during agitation, a fixing system can be used to attach the bioreactor to the system, particularly to the agitation device.

[0313] The fixing system can include a structure that couples the agitation device to the bioreactor, which should have sufficient rigidity to transfer mechanical energy to the bioreactor.

[0314] The fixing system includes at least one fastener for holding the bioreactor to the platform of the agitation device, and a bridge structure adapted to be mounted to the bioreactor and receive the at least one fastener. In an embodiment, the fastener includes a strap. In other embodiments, the fastener includes an adjustable strap. In an embodiment, the fixing system further includes one or more strap adapters or guides for positioning and guiding the placement of the strap.

[0315] To prevent mechanical damage, the fixing system should be suitably fitted to the bioreactor and should maintain and protect any fragile components (e.g., pH and DO probes) of the bioreactor from damage.

[0316] In an embodiment, the agitation device includes a shaking table, and the shaking table includes a placeholder on which the bioreactor is placed. In an embodiment, the shaking table is adapted to receive a container, preferably a bioreactor.

[0317] In an embodiment, the bridge includes a top portion and a hanging portion. As used herein, the top portion is referred to as the spider portion. In an embodiment, the spider portion includes two or more straps that intersect at a common connection point at the center and top of the bridge. In an embodiment, each strap includes two legs that hang down from the spider portion. In a preferred embodiment, the straps are perpendicular to each other and intersect, the straps and legs are of the same length, and the bridge is symmetric. In an embodiment, the straps and the hanging portion can have any width, length, and depth, and such a bridge structure can be adapted to any size of bioreactor or container. In an embodiment, the hanging portion can extend to and contact a part of the bioreactor or the platform of the agitation device for connection therewith. In an embodiment, the hanging portion contacts and is supported by a part of the bioreactor or the platform of the agitation device.

[0318] In an embodiment, the bridge further includes an annular portion to which one or more legs of the bridge are connected. The annular portion includes a flat surface adapted to mount the bridge to a part of the bioreactor or to a platform of the agitation device. In an embodiment, the dimensions of the annular portion are suitable for a part of the bioreactor so as to mount (or couple) the bridge to the bioreactor. In a preferred embodiment, the dimensions of the annular portion are suitable to surround and mount to a lid or a part of the lid of the bioreactor. In an embodiment, the annular portion of the bridge rests on the annular portion of the bioreactor lid. The annular portion may extend completely or partially around the lid of the bioreactor or container. This allows the bridge to be placed on the lid of the bioreactor without removing the pipes, probes, manifolds or other facilities attached to the bioreactor or container. In an embodiment, the annular portion is coupled to the lid of the bioreactor. In an embodiment, the annular portion rests on a part of the lid of the bioreactor. In an embodiment, the annular portion is positioned around the base of the bioreactor. The annular portion may be coupled to a part of the bioreactor using a clamp or other mounting means.

[0319] The annular receiving portion and the bridge may be coupled to the bioreactor, to each other, to a part thereof, to the platform or to a combination thereof using any technique known in the art (including but not limited to clips, latches, locking latches and screws). In an embodiment, such coupling is releasable. Thus, in an embodiment, the fixation system includes a releasable coupling.

[0320] Thus, in an embodiment, the fixation system includes an annular portion component for engaging with the lid or cover of the bioreactor.

[0321] In an embodiment, the agitation device includes a stirrer in the form of a shaking table, a vortex device, an oscillator, or another device for applying mechanical energy to the bioreactor and / or the fixed bed material.

[0322] In an embodiment, the system further includes a controller for controlling the agitation device. In an embodiment, the controller controls the bioreactor or the cell harvesting process.

[0323] The agitation device may be manually controlled or controlled in an automated manner. In an embodiment, the agitation device is remotely controlled.

[0324] The stirring device can be controlled independently or by a controller. In an embodiment, the controller is contained within the PDG box. In an embodiment, the controller can be integrally formed with the stirring device (such as, for example, included within a shaking table). In an embodiment, the controller forms part of a docking station of the bioreactor. The docking station can include a controller that has a display for displaying various parameters associated with the ongoing bioprocessing operation and also allows input for controlling various aspects of the bioprocessing operation. For example, the docking station can include various auxiliary containers associated with pumps connected by conduits. The controller can be used to control these pumps in order to control the fluid flow into or out of the bioreactor and to control the mixing of the fluid within the bioreactor, such as by controlling the stirring device.

[0325] In an embodiment, the bioreactor includes a structured fixed-bed bioreactor.

[0326] The fixed bed can include, for example, microcarriers (such as beads made of different materials such as gelatin, dextran, cellulose, plastic, or glass), structured fixed beds, 3D printed matrices, beds including one or more woven or non-woven materials, such as, for example, direct contact with inserted spacers, beads, hollow fibers, or one or more sheets of such materials with the inserted spacers, beads, hollow fibers, or any other suitable cell culture structure for promoting adherent cell growth or cell growth via entrapment. The bed can be designed in any desired shape, orientation, or form, such as, for example, a 3D porous monolithic structure, stacked layers (see, for example, U.S. Patent No. 11,111,470), vertically arranged parallel layers, layers arranged in a spiral or wound configuration, or a packed bed (see, for example, U.S. Patent No. 8,137,959). In an embodiment, the structured fixed bed includes a three-dimensional (3D) monolithic structure, such as a scaffold or lattice form formed by a plurality of interconnected units or objects. Such objects have surfaces for cell attachment. The fixed bed can be disposable in nature to avoid the costs and complexities involved in cleaning according to bioprocessing standards. Such an integral structured fixed bed can prevent the generation of particles (a fixed bed containing PET fibers may release some free fibers), which makes it suitable for processes where the final product can be filtered (such as, for example, stem cell applications for large virus production that cannot be aseptically filtered).

[0327] Various treatments can also be applied to the fixed bed material to impart certain properties, such as, for example, making certain parts of the bed hydrophilic while certain parts are hydrophobic. Similarly, certain parts can be made cell-adhesive, such as, for example, by providing binding ligands. In a preferred embodiment, the fixed bed includes a hydrophilic material.

[0328] In an embodiment, the fixed bed includes a plurality of cell immobilization layers. In an embodiment, the plurality of cell immobilization layers are arranged in a stacked or helical configuration. In an embodiment, the cell immobilization layers are either arranged in direct contact or have a gap between adjacent layers. In an embodiment, one or more spacer layers are arranged between the one or more cell immobilization layers. In an embodiment, the fixed bed is a 3D printed fixed bed.

[0329] In an embodiment, the present invention relates to a system for recovering biomolecules from a bioreactor, the system comprising:

[0330] - a bioreactor retained on a platform of a stirring device; and

[0331] - a fixing system for coupling at least a portion of the bioreactor to a stirrer.

[0332] In an embodiment, the system comprises: a bioreactor comprising a structure for cell retention / attachment and growth; a biomolecule harvesting mechanism adapted to stir the bioreactor and to move the liquid level relative to the structure; and a container comprising a cell lysis solution in fluid communication with the bioreactor. The biomolecule harvesting mechanism comprises means for vibrating or oscillating the bioreactor and / or a pump. A pump may be provided for pumping liquid so as to move the liquid level relative to the structure, and a controller may be provided for controlling the pump and possibly the vibrator.

[0333] In these or other embodiments, the bioreactor may be tilted relative to a horizontal plane to facilitate draining of liquid from the bioreactor. The biomolecule harvesting means may comprise an actuator for moving the structure for cell retention / attachment and growth relative to the bioreactor to move the position of the liquid level. A controller may be provided for controlling stirring of the bioreactor and moving the liquid level relative to the structure for cell retention / attachment and growth. The controller may be adapted to control the delivery of one or more solutions to the bioreactor.

[0334] Other aspects of the present disclosure relate to a system for harvesting cells, comprising: a bioreactor comprising a structure for cell retention / attachment and growth; a stirrer adapted to stir the bioreactor; an actuator for moving the liquid level relative to the structure for cell retention / attachment and growth; and a container comprising a solution in fluid communication with the bioreactor. In one embodiment, the stirrer comprises a vibrator. The actuator may comprise a linear actuator and / or a pump. A controller may also be provided to control the actuator and / or the stirrer.

[0335] In one embodiment, the structure for cell retention / attachment and growth includes a fixed bed, such as a 3D printed fixed bed. The structure for cell retention / attachment and growth includes a fixed bed having a plurality of cell immobilization layers, which are arranged, for example, in a stacked or helical configuration and either directly contact adjacent layers or have a spacing therebetween.

[0336] The present invention will now be described in more detail in conjunction with examples and the accompanying drawings, which are not limiting.

[0337] Description of the Drawings

[0338] One embodiment of a system for applying a mechanical action to a bioreactor is schematically shown in Figures 8 to 12 . The system can include means 18 for agitating the bioreactor 12. The means 18 can be, for example, any means for applying agitation energy to the bioreactor 12 or a fixed bed (not shown). The part of the bioreactor 12 to which energy is applied can include any of its components or parts, as long as the component or part generates vibrations sufficient to cause movement of the bioreactor and / or its contents (including but not limited to liquid medium, fixed bed material, cells, cell debris, and combinations thereof). The means 18 can include, for example, a stirrer in the form of a shaker table, a vortex device, an oscillator, or another means for applying mechanical energy to the bioreactor and / or the fixed bed material. The means 18 can be inside or outside the bioreactor 12. Preferably, the means 18 is outside the bioreactor. The mechanical action, preferably a vibration action, can be oscillatory, reciprocating, or periodic, harmonic or random.

[0339] The shaker table can include a motor, preferably a vibration motor, a transformer, and a frequency converter. The frequency can be 5 - 100 Hz, or more particularly, 20 - 100 Hz, and more particularly between 50 and 70 Hz. The amplitude can be low, such as 0.1 - 5.0 mm, or more particularly, 2 - 3 mm.

[0340] In other embodiments, a controller (e.g., a computer or a processor) can be provided to manage the system and apply a mechanical action (agitation) to the bioreactor. In other embodiments, a controller with software (e.g., a computer or a processor) can be provided, which manages the system and applies a mechanical action, moves the liquid associated with each step, performs agitation, and combines the process of moving liquid into and out of the bioreactor 12 in an automated manner or according to operator instructions. See Figure 11 . In an embodiment, a single controller uses the shaker table described herein to control the growth of cells in the bioreactor and the recovery of cells or target biomolecules from the bioreactor.

[0341] In one embodiment, a device for applying mechanical action (stirring) to a bioreactor, such as a fixed bed bioreactor, may include a platform 202 for supporting or docking the bioreactor. In one embodiment, the platform provides an interface for transferring mechanical energy to the bioreactor. In other embodiments, the platform may include a placeholder 204 for placing the base of the bioreactor or a portion thereof. The placeholder may include an opening, recess, insert, or indentation 204 for placing the base of the bioreactor or a portion thereof. In another embodiment, the platform can be replaced / interchanged with another platform having different sized openings, inserts, or indentations to accommodate different sized bioreactors or vessels. In another embodiment, a disk or annular structure may provide an interface between the platform and the bioreactor. The insert, disk, or annular structure may prevent direct contact between the bioreactor and the stirring device. In an embodiment, the disk or annular structure is made of a material including plastic. In a preferred embodiment, the insert, disk, or annular structure is made of a material including polyoxymethylene (POM).

[0342] In an embodiment, the placeholder 204( Figure 12 ) includes one or more concentric recesses to enable placement and use of different sized containers or bioreactors. In an embodiment, the placeholder is attached to the platform of the shaker table using screws. In an embodiment, the placeholder is made of polyoxymethylene (POM) and is machined from a block of plastic.

[0343] In another embodiment, a device for applying mechanical action (stirring) to a bioreactor may include a fixation system 210. The fixation system ensures uniform application of force to the bioreactor by properly fixing the bioreactor to the stirring system (i.e., the shaker table) and avoids mechanical constraints on the bioreactor, particularly the lid and seams of the bioreactor.

[0344] In an embodiment, the securing system secures the bioreactor to the agitation device. The securing system can minimize movement of the bioreactor, including but not limited to lateral and vertical movement of the bioreactor. The securing system can also help maintain the integrity of the bioreactor and minimize, reduce, or prevent mechanical damage that agitation may cause to the bioreactor. In one embodiment, the securing system can include fasteners for directly or indirectly securing and / or coupling at least a portion of the bioreactor to the agitation device. In one embodiment, the fastener is a strap or tie 212. As used herein, the term "strap" is defined as a flexible material for fastening or securing one object to another object. In one embodiment, a strap is used to fasten the bioreactor to the agitation device. In other embodiments, the securing system can include one or more straps. The one or more straps can be single-use or reusable. The one or more straps can be adjustable, bendable, flexible, stretchable, or a combination thereof. The one or more straps can be detachable. The one or more straps can be made of materials including plastics, polymers, and material blends. The one or more straps can be made of materials including one or more of polymers, copolymers, mixtures of polymers, and mixtures of one or more polymers and non-polymeric substances. In an embodiment, the strap can be made of materials including one or more of rubber, nylon, neoprene, polypropylene, polyester, polyethylene, and polyethylene terephthalate (PET).

[0345] In an embodiment, the strap can be a webbed material. In an embodiment, the strap can be coated. In an embodiment, the strap can be coated with thermoplastic polyurethane (TPU) or polyvinyl chloride (PVC) or a combination thereof. In a preferred embodiment, the one or more straps used in the securing system include BioThane Also known as BioThane Beta 520. The length, thickness, and width of the one or more straps can be readily determined by those skilled in the art.

[0346] In addition to securing the bioreactor to the agitation system, the straps also provide a way to control the tightness or the amount of tension used when securing the bioreactor to the agitation system 18 and distribute the pressure of the straps evenly over the bioreactor. In a preferred embodiment, the securing system includes two straps. In an embodiment, each strap has a first end and a second end.

[0347] In one embodiment, the fixation system includes a bridge structure 214. The bridge structure is used to uniformly apply the force of the strap on the bioreactor so as to be properly fixed to the shaker table. The bridge structure is further used to avoid mechanical limitations on certain elements of the bioreactor, particularly the lid. The bridge structure is used to protect the protruding or sensitive areas of the bioreactor from damage while not hindering access to it. In an embodiment, the bridge can also provide access to ports, caps, sensors, samplers, and other hardware, pipes, devices, and / or manifolds that may be connected to the bioreactor. In an embodiment, the bridge structure extends above and / or across the bioreactor lid. The bridge can be rigid or flexible. In an embodiment, the bridge is rigid. The bridge can be detachable, disposable, single-use, and / or reusable. In an embodiment, the bridge is plastic. In an embodiment, the bridge is metal. In an embodiment, the bridge is made of a material including aluminum.

[0348] In an embodiment, the bridge includes a top portion 214A and a pendant portion 214B. As used herein, the top portion is referred to as the arachnid portion. In an embodiment, the arachnid portion includes two or more straps 214A that intersect at a common connection point at the center and top of the bridge. In an embodiment, each strap includes two legs 214B that hang down from the arachnid portion. In a preferred embodiment, these straps are perpendicular to each other and intersect, the lengths of the straps and legs are the same, and the bridge is symmetric. In an embodiment, the straps and pendant portions can have any width, length, and depth, and such a bridge structure can fit any size bioreactor or container. In an embodiment, the pendant portion can extend to and contact a part or platform of the bioreactor for connection therewith. In an embodiment, the pendant portion contacts and is supported by a part or platform of the bioreactor. In an embodiment, the bridge further includes an annular portion 214C to which one or more legs of the bridge are connected. The annular portion includes a flat surface adapted to mount the bridge to a part or platform of the bioreactor. The size of the annular portion is suitable for a part of the bioreactor so as to mount the bridge to the bioreactor. In a preferred embodiment, the size of the annular portion is suitable for surrounding and mounting to the lid or a part of the lid of the bioreactor. In an embodiment, the annular portion of the bridge rests on the annular portion of the bioreactor lid. The annular portion can extend completely or partially around the lid of the bioreactor or container (see Figure 13) This allows the bridge to be placed on the lid of the bioreactor without removing the pipes, probes, manifolds, or other facilities attached to the bioreactor or container. In an embodiment, the annular portion is coupled to the lid of the bioreactor. In an embodiment, the annular portion rests on a part of the lid of the bioreactor. In an embodiment, the annular portion is positioned around the base of the bioreactor. The annular portion can be coupled to a part of the bioreactor using clamping or other mounting means. Alternatively, the annular portion can interface with a receiving portion 220 or gasket mounted on the bioreactor, the receiving portion or gasket being configured to receive the bridge or couple the bridge structure to the platform of the bioreactor or agitator. In an embodiment, the annular receiving portion or gasket is mounted on the bioreactor lid or a part thereof, and the annular portion of the bridge is coupled to or rests on it. In an embodiment, the annular portion of the bridge further includes a lower portion (220) or edge adapted to enclose a part of the lid. This ensures the fit of the bridge to the container or bioreactor lid. In an embodiment, the annular receiving portion provides an interface between the bioreactor and the metal bridge. In an embodiment, the annular receiving portion prevents direct contact between the bioreactor and the bridge. In an embodiment, the annular receiving portion is made of a material including plastic. In a preferred embodiment, the annular receiving portion is made of a material including polyoxymethylene (POM). The annular receiving portion and the bridge can be coupled to the bioreactor, to each other, to a part thereof, to the platform, or a combination thereof using any technique known in the art (including but not limited to clips, latches, locking latches, and screws). In an embodiment, such coupling is releasable.

[0349] In an embodiment, the fixation system and the agitation system and components can be made of low particle shedding materials. In an embodiment, a plastic interface can be present in the fixation system to reduce particle shedding during the use of the agitation system.

[0350] In other embodiments, the fixation system includes a strap guide 226 for positioning and guiding the placement of the strap during installation of the strap into the fixation system. In another embodiment, the strap guide is used to evenly distribute the tension provided by the strap onto the bioreactor.

[0351] In another embodiment, the strap guide serves as an interface between the strap and the spider portion. In an embodiment, the strap guide is made of a material including plastic. In a preferred embodiment, the strap guide is made of a material including polyoxymethylene (POM). In an embodiment, the strap guide includes a recess for receiving and stabilizing the position of the strap. In an embodiment, the strap guide is attached to the spider portion. In an embodiment, the strap guide is attached to the spider portion using screws, adhesive welding, or any method known in the art for joining any two components having similar or different compositions and capable of forming a low particle shedding structure.

[0352] In an embodiment, the bridge further includes a push pin for restricting displacement and / or breakage of the lid of the container or bioreactor. As Figure 9 and Figure 13 shown, the push pin 230 can extend from the arachnoid part and be fixed to the arachnoid part using fixing methods known in the art. The push pin and the bridge can be composed of the same or different materials. In an embodiment, the push pin and the bridge can be connected or molded together (if composed of plastic). In an embodiment, the push pin can be fixed to the bridge with screws or clamped. In an embodiment, the push pin can be welded or glued to the bridge. The push pin can be adjustable, detachable, replaceable, and / or flexible, and can be made of plastic, metal, rubber, or a combination thereof. The push pin can further include an end cap, sleeve, or cap made of a material such as rubber, which is adapted to or used for restricting displacement and breakage of the lid of the container or bioreactor. When such a push pin is installed together with the bridge, it will rest on or slightly above the lid surface.

[0353] In an embodiment, the fixing system includes an anchoring device capable of placing and fixing the strap to the stirring system. In an embodiment, the anchoring device is anchored to the top or platform of the stirring system using any technique known in the art. In an embodiment, the anchoring device is connected to the stirring system by bolts, welding, or a combination thereof. In an embodiment, the anchoring device is composed of metal, plastic, or a combination thereof. In an embodiment, the fixing system can include any number of anchoring devices. In an embodiment, the anchoring device is installed on the stirring system. In an embodiment, the anchoring device is installed on the stirring system for holding and connecting the strap of the fixing system to the stirring system. In an embodiment, the anchoring device is installed on the platform of the stirring device. In an embodiment, the anchoring device is a hook, and these hooks are installed on the platform or top of the stirring system. In an embodiment, there are four hooks 222. In an embodiment, the hooks are placed at or near the corners of the platform or top of the stirring system, or at the corners of an imaginary square on the top of the stirring device.

[0354] In an embodiment, the anchoring device or hook is positioned to align with the axis of the bridge and is capable of positioning and securing the bioreactor with the strap. In an embodiment, the first end of the strap is directly fastened to the anchoring device. In an embodiment, both ends of each strap in the fixation system are directly mounted or fastened to the anchoring device. In an embodiment, one or more straps are coupled, fastened, or held to the anchoring device in a releasable manner. In an embodiment, the strap of the fixation system is coupled to the anchoring device by wrapping the strap around the anchoring device. In an embodiment, one strap wraps around two anchoring devices. In a preferred embodiment, the fixation system includes two straps, and each strap wraps around two anchoring devices or is coupled to two anchoring devices. In another embodiment, the fixation system includes means for adjusting one or more straps. In an embodiment, the means for adjusting the strap is releasable. In an embodiment, the adjusting means is retractable. In an embodiment, the adjustment includes changing the length of the strap. The adjustable strap ensures that the bioreactor is held on the agitation system, and the fixation system applies an appropriate amount of tension to the bioreactor. In addition, the adjustable strap enables the agitation device to be adapted to different sizes of containers or bioreactors.

[0355] The means for adjusting one or more straps can include any technique or device known in the art, including stretching the strap. In another embodiment, the adjusting means can include one or more of a clamp, a ratchet, a buckle, a latch, and combinations thereof. In an embodiment, the adjusting means includes a ratchet. In a preferred embodiment, the fixation system includes two straps, and each strap includes a ratchet device for adjusting the length of the strap. The ratchet device or other adjusting means can further facilitate the closing of the two ends of the strap. Figure 8 Embodiments of the agitation system 18 and the fixation system 210 are shown respectively. In Figure 8 the embodiment shown, the fixation system includes two straps 212A and 212B. In an embodiment, each strap includes ratchet devices 224A and 224B. The ratchet device and its assembly and implementation are well known in the art, and the ratchet device used in the fixation system described herein is a ratchet tensioner system, and preferably, the ratchet tensioner system is made of stainless steel.

[0356] In an embodiment, the first end of the first strap is inserted into the first end of the first ratchet, and the second end of the first strap is inserted into the second end of the first ratchet. Preferably, each strap has a ratchet. Preferably, the fixation system includes two straps, and each strap further includes a ratchet for adjusting the length of the strap.

[0357] As Figure 8 and Figure 9As shown, in an embodiment, the fixation system includes: one end of a first strap fastened to a first hook, and a second end of the first strap fastened to a first end of a ratchet; a first end of a first extension strap fastened to a second hook in a diagonal position to the first hook, a second end of the first strap coupled to a second end of the ratchet, and the first strap starting from the first hook, extending along a part of the bridge and in a first set of recesses (also called strap guides) of the bridge to the first end of the ratchet, wherein the second end of the ratchet is fastened to the second end of the first extension strap, and wherein the ratchet is adjustable. As Figure 8 and Figure 9 Further shown, the fixation system may further include: a first end of a second strap fastened to a third hook, and a second end of the second strap fastened to a first end of a second ratchet; a first end of a second extension strap fastened to a fourth hook in a diagonal position to the third hook, a second end of the second strap coupled to a second end of the second ratchet, and the second strap starting from the first hook, extending along a part of the bridge and in a second set of recesses (strap guides) of the bridge to the first end of the second ratchet, and wherein the second end of the second ratchet is fastened to the second end of the second extension strap, and wherein the second ratchet is adjustable.

[0358] Figure 10 An embodiment of a system for applying mechanical energy to a bioreactor 12 or other process-related vessel is shown, the system including a stirring device 18 and a fixation system 210.

[0359] In an embodiment, the bioreactor includes a structured fixed bed, possibly further including a helically wound structured fixed bed. In an embodiment, the bioreactor includes a cell culture, a cell culture harvest, or a process solution from one or more steps of cell harvesting. In an embodiment, the bioreactor includes one or more of cells, cell debris, and target biomolecules. In an embodiment, one or more of the cells, cell debris, and target biomolecules are captured in the fixed bed, attached to the fixed bed, or aggregated with cells or cell debris or other biomolecules. In an embodiment, the aluminum bridge 214 includes an arachnid portion 214A and four pendant leg structures 214B, and further includes an annular structure portion that is mounted on top of a POM gasket 220 for receiving the annular portion of the bridge. The POM gasket and the annular portion of the bridge are mounted and optionally fastened to a part of the bioreactor cap. The POM strap guide 226 is mounted on the arachnid portion of the bridge, the strap guide including recesses for positioning and supporting the strap.

[0360] The first strap is assembled by: passing the first end of the strap through the first end of the first ratchet 224A; looping the free end of the strap above or below the first anchoring hook 222A; positioning the strap in the first recess in the strap guide, the first recess extending parallel to the second anchoring hook diagonally positioned with respect to the first anchoring hook; looping the free end of the strap around a second anchoring hook (not shown), and bringing the end of the strap back towards the first anchoring hook by: stacking the strap in the strap guide on top of the first layer of the strap and passing the end of the strap through the second end of the first ratchet; and tensioning the first ratchet to a tension appropriate for the operation of the agitation system. The second strap is assembled by passing the first end of the second strap through the first end of the second ratchet 224B; looping the free end of the strap above or below the third anchoring hook 222B; positioning the strap in the first recess in the strap guide, the first recess extending parallel to the fourth anchoring hook 222C diagonally positioned with respect to the third anchoring hook; looping the free end of the second strap around the fourth anchoring hook, and bringing the end of the strap back towards the third anchoring hook by: stacking the strap in the strap guide on top of the first layer of the second strap and passing the end of the strap through the second end of the second ratchet; and tensioning the second ratchet to a tension appropriate for the operation of the agitation system.

[0361] In an embodiment, the ratchet 224 is pre-mounted with a strap (see Figure 14 ). In another embodiment, the fixing system is installed on the bioreactor according to the following steps:

[0362] 1. Place the bioreactor in the placeholder on the shaker table.

[0363] 2. Place the bridge on top of the bioreactor in an open orientation (the adapter notch always facing the front of the shaker table).

[0364] 3. Ensure that the center projection pin is firmly placed in a directly parallel position to the probe and to the left of the probe.

[0365] 4. Loop the first strap around the opposing hooks (on the shaker table) and connect it to the lower part of the upper strap holder above the strap guide, pressing the two layers of the strap firmly against each other. On the other opposing hook, loop the strap around the hook with the ratchet.

[0366] 5. Loop the strap under the ratchet hook, then pull the strap through, and then tension the ratchet until the strap is taut. The ratchet should be as close to the table as possible without touching it to avoid interfering with the vibration.

[0367] 6. Cross the second strap in a suitable holder and connect it in the appropriate opposing hooks like the first connected strap, but place the double layer of the strap above the upper part of the upper strap guide.

[0368] 7. Secure with the strap ratchet until tightened.

[0369] In another embodiment, the shaker table includes a holder for holding bottles, containers, or other components during the agitation process of the shaker table (see Figure 15 ). In an embodiment, the holder is secured to the shaker table using a strap or arm 236. In an embodiment, the holder is reversibly secured to the shaker table. In an embodiment, the holder is flexible, rigid, and / or semi-rigid. In an embodiment, the holder is plastic, metallic, or a combination thereof. In an embodiment, the holder is made of a material including polyoxymethylene (POM). In an embodiment, the holder includes a bracket or clamp portion (234) and a strap or arm portion (236). The strap portion secures the holder to the fixation system, and the clamp or bracket portion receives the bottle or container, thereby holding it in place and preventing it from tipping over during vibration. In an embodiment, the holder receives a bottle including a foam trap. In an embodiment, the bottle is glass or plastic or a combination thereof. In an embodiment, the foam trap is coupled to the bioreactor.

[0370] In an embodiment, the foam trap is fluidly connected 238 to the bioreactor. In an embodiment, the foam trap is fluidly connected to the bioreactor during cell culture 240 and moves with the bioreactor when placed and mounted on the shaker table (agitation system) 18 (see Figure 11 and Figure 16 ).

[0371] In another embodiment, a pump is shared between the cell culture system 240 and the agitation system 18 to pump fluid into and out of the bioreactor during cell culture growth, cell culture harvest, and target biomolecule recovery.

[0372] The agitation and fixation systems described and disclosed herein and in PCT Publication WO2022 / 254039 (the entire content of which is incorporated herein by reference) can be used for cell detachment, seed culture, cell harvest, and target biomolecule recovery. Although the disclosed embodiments illustrate a bioreactor, optionally having a fixed bed, receiving mechanical energy from the agitation system, it is contemplated that the agitation and fixation systems can be used at any time in biomolecule production, and their use is not limited to bioreactors, but includes, for example, harvest containers, and such systems can be used with any container.

[0373] Non-limiting examples: The examples provided herein are not intended to be limiting, but rather provide working examples of the invention. It is contemplated that all amounts and experimental variables, including parameters, concentrations, amounts, and conditions, can be alternatively performed at values above or below those provided.

[0374] Examples

[0375] Example 1: Method for Obtaining AAV2 According to the Embodiment of the Present Invention

[0376] AAV2 is produced using a suspension-adapted cell line in a scale-X hydro bioreactor.

[0377] The process flow diagram of the method adopted in Example 1 is shown in Figure 1 。

[0378] Inoculation

[0379] Suspension cells are inoculated from the pre-culture in SF (sequential fermentation) into the Scale-X bioreactor in batch mode at an equivalent cell density of 30,000 cells / cm 2 。

[0380] Cell growth

[0381] After several hours (between 4 and 24 h), the recirculation loop (containing the medium required for cell growth) is started, and the cells grow for 3 days (or 4 days, depending on cell growth).

[0382] Transfection

[0383] Cells are usually transfected when they reach 200 - 300,000 cells / cm 2 . The transfection mixture is prepared outside the bioreactor and pre-diluted in the medium. See details below. The bioreactor should be partially emptied to enable the addition of the transfection complex inside the bioreactor. Transfection is carried out in batch mode with a reduced volume (e.g., 800 ml), and the addition of base is stopped.

[0384] Changes in the post-transfection loop

[0385] Four hours after transfection, the recirculation loop is restarted (with fresh medium in the loop - partial medium exchange).

[0386] Production

[0387] Production is carried out in recirculation for 3 days.

[0388] Harvest

[0389] At the end of the run, the supernatant (extracellular AAV) is harvested, the bioreactor is rinsed 2X, and lysed using a lysis buffer (Pluronic F-68, Triton X-100, salts, and benzonase). A washing step is performed to recover the void volume. For example, harvesting is carried out at 800 ml. Harvesting is carried out in batch mode. The harvest flow diagram and the following steps for harvesting AAV are shown in Figure 4 。

[0390] Harvest of extracellular AAV

[0391] At the end of production, stop the recirculation. Add 0.1% (v / v) Pluronic F-68 to the culture medium in the bioreactor and the recirculation loop, and continue to control the operation for more than 30 minutes. Empty the bioreactor vessel (void volume ~90 mL), and collect samples from the bioreactor and the recirculation loop.

[0392] 2X rinse

[0393] Add ( ~0.7 L) pre-warmed (37 °C) rinse buffer to fill the bioreactor up to ~0.8 L.

[0394] The rinse buffer consists of:

[0395] - PBS

[0396] - 2.5 mM KCl

[0397] - 1 mM MgCl2

[0398] - pH: 7.0

[0399] - 0.1% Pluronic F-68

[0400] Apply a stirring speed of 810 rpm for 5 min, then empty the bioreactor. Or apply a stirring speed of 0.5 - 2 cm / s for 5 min, then empty the bioreactor. Thus, the movement of the liquid level relative to the fixed bed structure occurs at a speed of 0.5 to 2 cm / s. This refers to the (vertical) movement speed of the liquid inside the bioreactor. Repeat the steps of filling with the rinse buffer, mixing for 5 min, and emptying once.

[0401] Cell lysis and AAV harvest (Step #1)

[0402] Add ~0.7 L of pre-warmed lysis solution to the bioreactor, mix at 810 rpm for 2 h at 37 °C to reach 0.8 L. Or add the pre-warmed lysis solution to the bioreactor and mix at 0.5 - 2 cm / s for 2 h at 37 °C. Thus, the movement of the liquid level relative to the fixed bed structure occurs at a speed of 0.5 to 2 cm / s. This refers to the (vertical) movement speed of the liquid inside the bioreactor. Perform back-and-forth circulation (once every 30 min) because the elution effect on the fixed bed helps to recover the virus. This back-and-forth circulation allows a portion of the liquid to move between the inside and outside of the fixed bed bioreactor (empty / fill steps).

[0403] Lysis buffer #1 (Triton - benzonase) consists of:

[0404] · 1% Triton (v / v)

[0405] · 20 U / mL Benzonase

[0406] · PBS containing 137 mM NaCl

[0407] · 10 mM Tris

[0408] · 2 mM MgCl2

[0409] · 0.1% (v / v) Pluronic F-68

[0410] · pH: 8.0

[0411] After incubation for 2 hours, the ionic strength of the lysis buffer was increased by adjusting the NaCl concentration up to 1 M (final concentration) using a 5 M NaCl stock solution (e.g., adding 140 mL of 5 M NaCl—removing some volume from inside the harvest vessel so that the bioreactor can be run at 0.8 L). Stirring was applied for 30 min and then the bioreactor was emptied. For example, a stirring speed of 810 rpm was applied for 30 min and then the bioreactor was emptied. A back-and-forth cycle was performed (once every 15 min) as the elution effect on the fixed bed helps in virus recovery.

[0412] Cell lysis and AAV harvest (Steps #2 and #3)

[0413] The pre-warmed lysis solution was added to the bioreactor and mixed at 810 rpm for 30 min at 37°C. Alternatively, the pre-warmed lysis solution was added to the bioreactor and stirring was started at 37°C for 30 min. A back-and-forth cycle was performed (once every 15 min) as the elution effect on the fixed bed helps in virus recovery. The steps of filling with the rinse buffer, mixing for 30 min, and emptying were repeated once.

[0414] Lysis buffer #2 (Triton-salt) consists of:

[0415] - 1% Triton (v / v)

[0416] - 1 M NaCl

[0417] - PBS

[0418] - 10 mM Tris

[0419] - 2 mM MgCl2

[0420] - 0.1% (v / v) Pluronic F-68

[0421] - pH: 8.0

[0422] Washing

[0423] Add 0.7 L of pre-warmed wash buffer to fill the bioreactor up to a maximum of ~0.8 L.

[0424] The wash buffer comprises:

[0425] - PBS

[0426] - 2.5 mM KCl

[0427] - 1 mM MgCl2

[0428] - 10 mM Tris

[0429] - pH: 8.0

[0430] - 0.1% (v / v) Pluronic F-68

[0431] Apply a stirring speed for 5 min and then empty the bioreactor. For example, apply a stirring speed of 810 rpm for 5 min and then empty the bioreactor. Repeat the steps of filling with the rinse buffer, mixing for 5 min, and emptying once.

[0432] Example 2: Protocol for Harvesting rAAV2 Produced by Transfected HEK293T Cells in a Fixed - Bed Bioreactor According to the Embodiment of the Present Invention

[0433] Introduction

[0434] Recombinant adeno-associated virus (rAAV) is a viral vector for gene therapy. It consists of an icosahedral capsid made of viral proteins that encloses a DNA fragment (i.e., the transgene). The produced viral vector is used to transfer the transgene (therapeutic gene) into the nucleus of patient cells as a therapy for life-threatening diseases.

[0435] The following protocol is used to harvest rAAV2 after transient transfection in HEK293T cells in a fixed-bed bioreactor.

[0436] The serotype 2 of known rAAV easily attaches to the surface of cells (membrane receptors for transduction) or plastic materials (walls and pipes of bioreactors), and aggregates if the ionic strength is too low. Relevant tests have been carried out to study the affinity of rAAV2 for the fixed-bed materials (PP and PET) of bioreactors. Satisfactorily, no specific affinity between rAAV2 and the fixed-bed materials was detected. Even if some rAAV2 was released into the supernatant (culture medium), most remained inside the cells (stored in the cells). Fortunately, the rAAV capsid (as well as rAAV2) has strong elasticity and physical stability; therefore, downstream processes can utilize conditions that are usually avoided, such as long-term exposure to elevated temperatures (rAAV2 is stable at 37 °C) or exposure to organic solvents. Considering the large-scale production of rAAV2 in a fixed-bed bioreactor, the most suitable method currently is chemical lysis by using detergents. The goal of harvesting is to effectively lyse the cells without affecting the integrity of the product and without promoting its aggregation or non-specific surface interactions. Figure 2 The harvesting flow chart is shown. The steps and the formulations of the solutions will be described in the protocol instructions below in this article.

[0437] Protocol instructions

[0438] General notes: Preheat all the solutions used to 37 °C before adding them to the bioreactor. Except for agitation and temperature, other regulations required to stop biomolecule production; maintain the temperature at 37 °C, and when the bioreactor is full, keep the agitation between 0.5 and 1 cm / s. Therefore, the movement of the liquid level relative to the fixed-bed structure occurs at a speed of 0.5 to 1 cm / s. This refers to the (vertical) movement speed of the liquid inside the bioreactor.

[0439] Step #1 - Empty the bioreactor

[0440] Between 30 minutes and 1 hour before emptying the bioreactor, add 0.1% Pluronic TM F-68 (v / v) to the culture supernatant. 0.1% (v / v) Pluronic TM F-68 already exists in some culture media. Even if Pluronic is already in the culture medium, adding Pluronic TM F-68 until it reaches 0.2% (v / v) can be useful. Pluronic TMF-68 is a non-ionic surfactant that is commonly used to control shear forces, prevent foaming in agitated cultures, and reduce cell attachment to hydrophilic surfaces. It is also known that adding it can prevent AAV from adhering to plastic pipettes and plastic containers during storage. For AAV, the commonly recommended usage concentration is between 0.01% and 0.2%. If there is no Pluronic F-68 in the transfection mixture recirculation loop, we recommend adding it.

[0441] Step #2 - Rinse the bioreactor

[0442] Use a neutral solution to eliminate residual medium (components that may interfere with the lysis step), and continue to collect the extracellular portion of rAAV2. Once the bioreactor is emptied, fill it completely with the rinse solution and then empty it again. Perform two rinse steps because it has been observed that filling / emptying the bioreactor has a positive effect on the harvest of rAAV. For this purpose, use classical PBS-MK with added Pluronic.

[0443] Rinse solution formulation:

[0444] - PBS

[0445] - 2.5 mM KCl

[0446] - 1 mM MgCl2

[0447] - 0.1% (v / v) Pluronic

[0448] - pH 7

[0449] Step #3 - Cell lysis

[0450] This is the core step of the protocol because most rAAV2 is intracellular. The contact time between the cells and the lysis solution, as well as the temperature at which lysis is performed, will affect the efficiency of lysis. Therefore, it is recommended to perform the lysis step for 2 hours at 37°C (which may vary depending on the process), while maintaining agitation at 0.5 - 1 cm / s. Thus, the movement of the liquid level relative to the fixed bed structure occurs at a speed of 0.5 to 1 cm / s. This refers to the (vertical) movement speed of the liquid inside the bioreactor. Agitation can be turned on / off periodically to disrupt the flow. It is also possible to perform a back-and-forth circulation (emptying / filling steps) because the elution effect on the fixed bed helps in the recovery of the virus. Alternatively, the lysis step can be performed 2X, each time for 1 (or 2) hour.

[0451] According to scientific literature, the lysis step may take from 30 min to several hours (up to 6 hours) (e.g., 4 hours), and the raw lysate is sampled periodically (e.g., after 1, 2, 3, 4 hours).

[0452] The lysis buffer formulation is as follows:

[0453] - 1% (v / v) Tween 20 or Triton X-100

[0454] - 1M NaCl

[0455] - 10mM Tris-pH8

[0456] - 0.1% Pluronic (v / v)

[0457] - 20 - 50U / mL endonuclease (DNase) and 1 - 2mM MgCl2

[0458] Tween 20 or Triton X-100 is used because the detergent disrupts the cell membrane. The use of the detergent is related to the concentration and time. It has been reported that the concentration of Triton X-100 is between 0.1% and 0.5%, and the concentration of Tween 20 is between 0.1% and 1% (v / v). Zwittergent 3 - 14 (Calbiochem) also seems promising. Due to the European REACH regulations tending to ban the use of Triton and its derivatives, it is beneficial to use Tween 20 or zwitterionic detergents. Triton is known to be more effective than Tween. A higher concentration of Tween is recommended (the concentration of Triton or zwitterionic detergent required for sufficient virus release is lower compared to Tween 20. This is related to the inherent property of the detergent - the critical micelle concentration).

[0459] NaCl is added because sufficient ionic strength must be maintained to avoid rAAV2 aggregation and binding to other cell components released during the lysis process. Using a high salt concentration during the lysis step reduces rAAV2 aggregation and binding to other cell components, and reduces the surface interaction between rAAV2 and cells or cell debris that may still be adsorbed on the surface of the bioreactor.

[0460] A buffer of 10mM Tris-pH8 is used because it has been observed that an alkaline solution plus an appropriate amount of detergent such as Triton X-100 or Tween 20 is sufficient to lyse the cells encapsulating rAAV and release the virus particles. According to different processes, the pH reported for chemical cell lysis protocols is usually between 8 - 9. An acidic pH (3 - 4) also seems promising for harvesting virus particles, but it may affect the integrity of the viral proteins.

[0461] The role of 0.1% Pluronic (v / v) is similar to its role in the rinsing solution.

[0462] Nuclease treatment during the lysis step. DNase is added for:

[0463] 1. Digest the nuclear material of the host cells during rAAV extraction

[0464] 2. Avoid forming complexes of nuclear material and AAV

[0465] 3. Reduce the viscosity of the lysate to facilitate subsequent filtration and chromatography steps.

[0466] To limit the complexes and viscosity induced by genomic DNA, it is preferred to add the enzyme simultaneously with cell lysis. However, this step may not be compatible with the optimal conditions for enzyme activity (pH and salinity, such as NaCl). In our case, if we use the widely applied Benzonase (Millipore Sigma), pH 8 does not affect the enzyme's efficacy as it is active between pH 7 - 9. However, the salt concentration is too high (1M NaCl; it needs to be reduced to 100 - 150 mM to maintain effective activity). A salt - activated nuclease (SAN - HQ; ArcticZymes) can be used. Its activity at 500 mM NaCl has been reported.

[0467] During the lysis step, it is preferred to add Benzonase between 20 - 50 U / mL and incubate for a minimum of 30 - 60 min. In addition, 1 - 2 mM MgCl2 must be added to maintain the enzyme's activity. Note that higher concentrations of Benzonase can also be used.

[0468] High ionic strength (NaCl > 150 mM) is important for avoiding viral vector aggregation, but the concentration of these monovalent salts greatly reduces the activity of Benzonase (e.g., at 150 mM NaCl, the relative activity loss is + / - 70%). To ensure an effective harvest, the following compromise can be made:

[0469] First, start the lysis step without adding NaCl (note: the PBS buffer provides 137 mM) to maintain the effective activity of Benzonase (manufacturer's manual). Since Mg 2+ is a cofactor for Benzonase, its concentration must be accurate. Therefore, we recommend adding 2 mM MgCl2, which is optimal for enzyme activity. Under these salinity conditions, Benzonase retains effective activity and the aggregation of the vector should be limited, but this should be carefully tested beforehand. After incubating for 1 (or 2) hours, increase the ionic strength of the lysis buffer by adjusting the NaCl concentration up to 1M. Under these conditions, Benzonase is inhibited, but the risk of viral vector aggregation is reduced, and / or aggregates of viral particles are dissolved (if this process is reversible). At such a salinity, the interaction of the virus with plastic materials should be reduced.

[0470] Another possibility is to perform a separate additional step using endonuclease / Benzonase immediately after lysis to limit interference between cell lysis and endonuclease activity. Since the lysis step will be carried out at high ionic strength, this additional step using endonuclease should be done with a salt-activated nuclease.

[0471] Note: If endonuclease is added, it is necessary to check whether inactivation is required before quantifying the virus titer by qPCR.

[0472] Step #4 - Wash the bioreactor after cell lysis

[0473] Two hours after cell lysis, washing is performed to remove rAAV2 trapped in the fixed bed (fibers, cell debris, extracellular matrix) and / or interacting with the bioreactor wall. Once the bioreactor is emptied, it is filled with the wash solution and then emptied again. This step is repeated once more. Washing is done twice because, as described in step #2, filling / emptying the bioreactor has a positive effect on the harvest of rAAV.

[0474] The wash solution formulation is as follows:

[0475] - PBS

[0476] - 2.5 mM KCl

[0477] - 1 mM MgCl2

[0478] - 0.1% (v / v) Pluronic

[0479] - 10 mM Tris - pH 8

[0480] Note: The pH of the wash solution is more alkaline compared to the rinse solution.

[0481] Example 3: Exemplary Process for Harvesting and Recovering Target Biomolecules from a Fixed - Bed Bioreactor According to the Embodiment of the Present Invention

[0482] Figure 3 An exemplary process for harvesting and recovering the target biomolecule with stirring at 1 cm / s (the liquid level moves at a speed of 1 cm / s relative to the fixed bed structure) is depicted and described as follows.

[0483] 1. Before cell lysis, add 0.1% of Pluronic to the SN (supernatant) (to reach a final concentration of 0.2%)

[0484] 2. Perform 2 wash steps using a wash buffer comprising 2.5 mM KCl, 1 mM MgCl2, 0.1% Pluronic, and Tris pH 7.

[0485] 3. Use a lysis solution containing 1% Triton, 1M NaCl, 0.1% Pluronic, and Tris pH 8 for two lysis steps of 30 minutes each.

[0486] 4. Use a wash buffer containing 2.5 mM KCl, 1 mM MgCl2, 0.1% Pluronic, and Tris pH 8 for two wash steps.

[0487] 5. Use a lysis solution containing 1% Triton, citric acid pH 3.0, 0.1% pluronic for one lysis step of 30 minutes.

[0488] 6. Use a wash buffer containing 2.5 mM KCl, 1 mM MgCl2, 0.1% Pluronic, and Tris pH 8 - pre - warmed at 37°C for two wash steps.

[0489] 7. Add DNase (200 U / ml) to a buffer containing 2.5 mM KCl, 1 mM MgCl2, 0.1% pluronic, and Tris pH 8 and incubate at 37°C for 30 minutes.

[0490] 8. Use a wash buffer containing 2.5 mM KCl, 1 mM MgCl2, 0.1% Pluronic, and Tris pH 8 for two wash steps.

[0491] 9. Use a lysis solution containing 1% Triton, 1M NaCl, 0.1% Pluronic, and Tris pH 8 for one lysis step of 30 minutes.

[0492] 10. Use a wash buffer containing 2.5 mM KCl, 1 mM MgCl2, 0.1% Pluronic, and Tris pH 8 for two wash steps.

[0493] Therefore, the pH of the wash buffer is 7 and the pH of the rinse buffer is 8.

[0494] Example 4: Harvesting of Extracellular AAV from a Bioreactor with a Fixed Bed (from Univercells Technologies) "scale-X hydro TM ” bioreactor) for harvesting AAV

[0495] Figure 4 An exemplary process for the harvest and recovery of AAV is depicted and described as follows.

[0496] Harvesting of extracellular AAV

[0497] At the end of production, stop the recirculation.

[0498] Add 0.1% (v / v) Pluronic F-68 to the culture medium and recirculation loop of the bioreactor, and continue to control the operation for more than 30 minutes. Empty the bioreactor vessel (void volume ~90 mL), and collect samples from the bioreactor and recirculation loop. Collect representative samples of the supernatant from the bioreactor and recirculation loop to estimate the amount of AAV present in the floating cells.

[0499] Perform 2 (or more) rinses

[0500] Add ~0.7 L of pre-warmed (37 °C) rinse buffer to fill the bioreactor up to ~0.8 L.

[0501] The rinse buffer (pH 7) consists of:

[0502] - PBS

[0503] - 2.5 mM KCl

[0504] - 1 mM MgCl2

[0505] - 0.1% Pluronic F-68

[0506] Apply a stirring speed of 810 rpm for 5 min, then empty the bioreactor. Repeat the steps of filling with rinse buffer, mixing for 5 min, and emptying once.

[0507] Cell lysis and harvesting of AAV (Step #1)

[0508] Add ~0.7 L of pre-warmed lysis solution to the bioreactor and mix at 810 rpm at 37 °C for 2 h to reach 0.8 L. Perform back-and-forth circulation (once every 30 min) as the elution effect on the fixed bed helps in virus recovery.

[0509] Triton-benzonase lysis buffer #1 (pH 8) consists of:

[0510] - 1% Triton (v / v)

[0511] - 20 U / mL Benzonase

[0512] - PBS containing 137 mM NaCl

[0513] - 10 mM Tris

[0514] - 2 mM MgCl2

[0515] - 0.1% (v / v) Pluronic F-68

[0516] After incubation for 2 hours, the ionic strength of the lysis buffer was increased by adjusting the NaCl concentration to up to 1 M (final concentration) using a 5 M NaCl stock solution (add 140 mL of 5 M NaCl - remove some volume from inside the harvest vessel so that the bioreactor can be run at 0.8 L). A stirring speed of 810 rpm was applied for 30 min, and then the bioreactor was emptied.

[0517] Back-and-forth cycling (once every 15 min) was carried out because the elution effect on the fixed bed contributed to virus recovery. The NaCl stock solution contained 5 M NaCl.

[0518] Cell lysis and harvesting of AAV (Steps #2 and #3)

[0519] Pre-warmed lysis solution was added to the bioreactor and mixed at 810 rpm for 30 min at 37 °C. Back-and-forth cycling (once every 15 min) was carried out because the elution effect on the fixed bed contributed to virus recovery. The step of filling with rinse buffer, mixing for 30 min, and emptying was repeated once.

[0520] Triton-salt lysis buffer #2 (pH 8) contained:

[0521] - 1% Triton (v / v)

[0522] - 1 M NaCl

[0523] - PBS

[0524] - 10 mM Tris

[0525] - 2 mM MgCl2

[0526] - 0.1% (v / v) Pluronic F-68

[0527] Washing

[0528] 0.7 L of pre-warmed wash buffer was added to fill the bioreactor up to a maximum of ~0.8 L.

[0529] The wash buffer (pH 8) contained:

[0530] - PBS

[0531] - 2.5 mM KCl

[0532] - 1 mM MgCl2

[0533] - 10 mM Tris

[0534] - 0.1% (v / v) Pluronic F-68

[0535] Apply a stirring speed of 810 rpm for 5 min, and then empty the bioreactor. Repeat the steps of filling with the rinsing buffer, mixing for 5 min, and emptying once.

[0536] Example 5: Structured Fixed - Bed Bioreactor - By Adding a Vibration Step, the AAV Recovery Rate is Increased 15％。

[0537] Run 1 Total recovery <![CDATA[ / cm 2 > / cell) Total AAV in supernatant per cell 4.05E+12 Total intracellular AAV with vibration 2.20E+14 Total intracellular AAV without vibration 1.91E+14 Total AAV with vibration 2.25E+14 9.36E+09 5.35E+04 Total AAV without vibration 1.95E+14 8.11E+09 4.63E+04 Vibration gain +15％ Intracellular / extracellular with vibration 98％

[0538] Run 2 Total <![CDATA[ / cm 2 > / cell) Total supernatant 1.13E+13 Total intracellular AAV with vibration 6.36E+13 Total intracellular AAV without vibration 3.95E+13 Total AAV with vibration 7.49E+13 3.12E+09 1.78E+04 Total AAV without vibration 5.08E+13 2.12E+09 1.21E+04 Vibration gain +48％ Intracellular / extracellular with vibration 85％

[0539] The present invention is in no way limited to the embodiments described in the examples and / or shown in the drawings. On the contrary, the method according to the present invention can be implemented in many different ways without departing from the scope of the present invention.

Claims

1. A method for obtaining one or more target biomolecules from cells cultured in a fixed-bed bioreactor, the method comprising: - Add a lysis solution to the bioreactor to lyse the cells in the bioreactor, and apply one or more mechanical actions to the bioreactor before, during, and / or after lysing the cells, and thereafter recover the one or more target biomolecules from the bioreactor.

2. The method according to claim 1, wherein, The mechanical action is selected from stirring, vibration, vortexing, or moving the solution inside the bioreactor.

3. The method according to claim 2, wherein, After the lysis of the cells, one or more washing steps are performed by filling and / or emptying the bioreactor with a washing solution and / or an inactivating solution.

4. The method according to any one of claims 2 or 3, wherein, The mechanical action is vibration applied to the bioreactor, and the vibration has a frequency in the range of 20 to 100 Hz.

5. The method according to claim 4, wherein, The vibration is performed simultaneously with moving the solution inside the bioreactor.

6. The method according to any one of claims 2 to 5, wherein, The vibration is applied by a vibration table.

7. The method according to any one of the preceding claims, wherein, The lysis solution includes at least one detergent, such as Triton X-100, Tween 20, or Tween 80.

8. The method according to any one of the preceding claims, wherein, The lysis solution further includes DNase and / or a surfactant.

9. The method according to any one of the preceding claims, wherein, The method further includes using a solution including 0.5 to 1 M NaCl during the lysis step and / or the washing step.

10. The method according to claim 9, wherein, The solution is obtained by increasing the ionic strength of the lysis solution during the lysis step.

11. The method according to any one of the preceding claims, wherein, The method further includes using a lysis solution having a pH lower than or higher than the isoelectric point of the target biomolecule.

12. A system, comprising: A stirring device (18) and a fixing system (210), the stirring device including a platform (202) adapted to receive a bioreactor; and, the fixing system includes at least one fastener (212) for holding the bioreactor to the platform (202), and a bridge structure (214) adapted to be mounted to the bioreactor and receive the at least one fastener.

13. The system according to claim 12, wherein, The fastener includes an adjustable strap, and wherein the fixing system further includes one or more strap guides for positioning and guiding the placement of the strap.

14. The system according to any one of claims 12 - 13, wherein, The fixing system includes an annular portion for engaging the lid or cover of the bioreactor.

15. The system according to any one of claims 12 - 14, wherein, The stirring device includes a vibration table, and the vibration table includes a placeholder on which the bioreactor is placed.

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