Device and method for continuously harvesting biological material produced by cultured cells

Abstract

The present disclosure relates to an apparatus and method for continuously harvesting biological substances produced by cultured cells. More specifically, the present disclosure relates to an apparatus and method for continuously harvesting biological substances produced by cultured cells using two sets of filters with different pore sizes.

Description

[Technical field]

[0001] The present disclosure relates to an apparatus and method for continuously harvesting biological substances produced by cultured cells. More specifically, the present disclosure relates to an apparatus and method for continuously harvesting biological substances produced by cultured cells using two sets of filters with different pore sizes. [Background technology]

[0002] Traditional perfusion culture (TPC) and enhanced perfusion culture (IPC) require continuous perfusion of fresh culture medium (supplement medium) into the culture system to maintain cell growth and production of biological substances. However, since the harvest rate is usually equal to the perfusion rate, the concentration of the target product in the harvested liquid is low and the harvest volume is large, which puts pressure on downstream purification and requires large-sized tanks to store the harvested cell culture fluid, resulting in increased equipment and land costs. In addition, long-cycle, high-flow harvesting operations are prone to clogging of hollow fiber filters, which can cause the target product to be trapped in the culture system, and the replacement of hollow fiber filters will increase production costs, operational complexity, and contamination risks.

[0003] Therefore, in order to solve the above-mentioned problems existing in the conventional culture methods, the present inventors have developed a new biomass harvesting device and method that can be applied to both the production of stable biomass and the production of unstable biomass. [Summary of the invention]

[0005] In one aspect, the present disclosure provides an apparatus for continuously harvesting biological substances produced by cultured cells (hereinafter also referred to as "the apparatus of the present disclosure"), comprising:

[0006] (a) filter 1, which is at least one filter, the filtration pore size of which does not allow the cells to pass through the filter but allows the biological substances to pass through the filter, and

[0007] (b) a filter 2, which is at least one filter, the filtration pore size of which does not allow the cells and the biological substances to pass through the filter, but keeps the filter fluid flowing,

[0008] The filter 1 and the filter 2 are connected in parallel to a fluid of a device for culturing the cells.

[0009] In one embodiment of the device of the present disclosure, filter 1 and filter 2 are in fluid communication with the device for culturing the cells by: (a) directly each, (b) via a branch pipe, or (c) a combination of (a) and (b).

[0010] In some embodiments of the device of the present disclosure, the filtration pore size of the filter 1 is about 0.08 μm to 0.5 μm, preferably about 0.1 μm to 0.4 μm, more preferably about 0.2 μm. In some embodiments of the device of the present disclosure, the molecular weight cutoff (MWCO) of the filter 2 is below 50 kD.

[0011] In some embodiments of the apparatus of the present disclosure, the apparatus of the present disclosure further comprises a device for adjusting the filtration rate of filter 1 and / or a device for adjusting the filtration rate of filter 2.

[0012] In some embodiments of the apparatus of the present disclosure, one or both of the filter 1 and the filter 2 are alternating tangential flow (ATF) filters or tangential flow filtration (TTF) filters. In further embodiments of the apparatus of the present disclosure, one or both of the filter 1 and the filter 2 are hollow fiber filters.

[0013] In some embodiments of the device of the present disclosure, the number of the filters 1 is 1, 2, 3 or more, and / or the number of the filters 2 is 1, 2, 3 or more.

[0014] In another aspect, the present disclosure provides a system for producing biomass (hereinafter also referred to as "the system of the present disclosure"), comprising:

[0015] (a) an apparatus for culturing cells, which culture the cells in a perfusion mode using a basal medium and a supplementary medium; and

[0016] (b) The apparatus of the present disclosure.

[0017] In some embodiments of the systems of the present disclosure, the system further comprises an apparatus for continuously capturing biological matter from matter harvested by the apparatus of the present disclosure.

[0018] In some embodiments of the systems of the present disclosure, the system further comprises a micro-sprayer.

[0019] On the other hand, the present disclosure provides a method for continuously harvesting biomass produced by cultured cells, which is performed by the device of the present disclosure. In a further aspect, the present disclosure provides a method for producing biomass, which is performed by the system of the present disclosure. The above two methods are also referred to as "methods of the present disclosure" hereinafter.

[0020] In some embodiments of the methods of the present disclosure, the cell comprises a mammalian cell. In one embodiment of the methods of the present disclosure, the mammalian cell comprises a CHO (Chinese Hamster Ovary) cell, a hybridoma, a BHK (Baby Hamster Kidney) cell or a myeloma cell.

[0021] In some embodiments of the disclosed method, the biological substance is selected from a receptor, an enzyme, a fusion protein, a blood protein, a multifunctional protein, a viral or bacterial protein, and an immunoglobulin. In one embodiment of the disclosed method, the blood protein is from the coagulation cascade. In one embodiment of the disclosed method, the multifunctional protein is erythropoietin. In one embodiment of the disclosed method, a viral or bacterial protein is used in a vaccine. In one embodiment of the disclosed method, the immunoglobulin is an antibody or a multispecific antibody. In one embodiment of the disclosed method, the antibody is IgG or IgM. In one embodiment of the disclosed method, the multispecific antibody is a bispecific antibody.

Brief Description of the Drawings

[0023] Figure 1a is a schematic diagram of a culture system according to at least one embodiment of the present disclosure. Figure 1b is a schematic diagram of a culture system according to at least one embodiment of the present disclosure.

[0024] Figure 2 The viable cell density (10 6 A coordinate graph with the vertical axis (m / mL) and the treatment time (days) as the horizontal axis.

[0025] Figure 3 The graph is a coordinate graph of Process 1 and Process 2, with cell viability (%) as the vertical axis and treatment time (days) as the horizontal axis.

[0026] Figure 4 This is a coordinate graph of Process 1 and Process 2 with the average cell diameter (μm) as the vertical axis and the processing time (days) as the horizontal axis.

[0027] Figure 5 This is a graph of Process 1 and Process 2, with the glucose concentration (g / L) in the culture as the vertical axis and the treatment time (days) as the horizontal axis.

[0028] Figure 6 The graph is a graph of Process 1 and Process 2 with the lactic acid concentration (g / L) in the culture as the vertical axis and the treatment time (days) as the horizontal axis.

[0029] Figure 7 It is a coordinate graph of Process 1 and Process 2 with the recombinant protein titer (g / L) in the harvest as the vertical axis and the processing time (days) as the horizontal axis.

[0030] Figure 8 This is a coordinate graph with the cumulative volume productivity (Pv) (g / L) of Process 1 and Process 2 as the vertical axis and the processing time (days) as the horizontal axis.

[0031] Fig. 9The graph is a graph of Process 1 and Process 2 with the recombinant protein retention rate (Titer Retention Rate) (%) as the vertical axis and the processing time (days) as the horizontal axis.

[0032] Fig.10 is a graph comparing the peak viable cell density achieved in Process A and Process B.

[0033] Fig.11 is a graph comparing the cell viability achieved in process A and process B.

[0034] Fig.12 is a graph comparing the average cell diameters achieved in Process A and Process B.

[0035] Fig.13 is a graph comparing the glucose concentrations in the culture achieved in Process A and Process B.

[0036] Fig.14 is a graph comparing the lactate concentrations in the culture achieved in Process A and Process B.

[0037] Fig.15 is a graph comparing the recombinant protein titers in the harvests achieved in process A and process B.

[0038] Fig.16 is a graph comparing the cumulative volumetric productivity (Pv) achieved in Process A and Process B.

[0039] Fig.17 It is a coordinate graph comparing the recombinant protein retention rate (Titer Retention Rate) achieved in process A and process B. [Specific implementation method]

[0041] I. Definition

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. All patents, applications, published applications, and other publications cited herein are incorporated by reference in their entirety. If the definitions set forth in this section differ or conflict with the definitions set forth in the patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth in this section shall prevail.

[0043] As used herein, the singular forms "a," "an," and "the" include plural forms unless otherwise indicated. For example, "a" biological agent includes one or more biological agents.

[0044] As used herein, a "bioreactor" is a system that can contain a cell culture, which in turn includes cells and a cell culture medium. In some embodiments, it provides a sterile barrier, such as an air filter, to prevent other cells from contaminating the desired cells. In some embodiments, it maintains a favorable environment for the cells by providing appropriate culture conditions such as mixing, temperature, pH, oxygen concentration, etc.

[0045] "Cell culture" or "culturing" refers to the growth and propagation of cells outside of a multicellular organism or tissue. "Cell culture" includes a liquid containing a cell culture medium, cells and a biological substance, which liquid is the result of a process of culturing cells that produce biological substances in a cell culture medium in a reactor. Suitable culture conditions for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate.

[0046] "Cell" refers to a cell that produces a biological substance of interest, such as a cell that can express a gene encoding a product. For example, a cell that can express a gene encoding a product can be prepared by transfecting a cell with a plasmid containing a gene encoding a cell product and a gene encoding a suitable selection marker. In principle, cells that can be used to produce a product are all cells with the ability to produce a biological product known to those skilled in the art. The cell can be an animal cell, particularly a mammalian cell. Examples of mammalian cells include CHO (Chinese hamster ovary) cells, hybridomas, BHK (baby hamster kidney) cells, myeloma cells, human cells, such as HEK-293 cells, human lymphoblastoid cells, E1 immortalized HER cells, mouse cells, such as NS0 cells.

[0047] As used herein, the term "cell culturing medium" (also referred to as "culture medium" or "cell culture media") refers to any nutrient solution used to grow cells (e.g., animal or mammalian cells), and typically provides at least one or more of the following components: an energy source (usually in the form of a carbohydrate such as glucose); all essential amino acids or one or more of all twenty naturally occurring amino acids; vitamins and / or other organic compounds, usually required in low concentrations; lipids or free fatty acids; trace elements, such as inorganic compounds or naturally occurring elements, usually required in very low concentrations (usually in the micromolar range).

[0048] "Basic cell culture medium" refers to a cell culture medium that is typically used to start cell culture and is complete enough to support cell culture. Commercially available basal culture media can be used, including but not limited to CD OptiCHO AGT (Invitrogen), CDCHO AGT (Invitrogen), Dynamis AGT medium (Invitrogen), SFM4CHO ADCF (Hyclone), HyCell CHO medium (Hyclone), CDM4MAB (Hyclone), DPM Hyclone ActiPro (Hyclone), Advanced CHO Supplemented Batch Medium (Sigma).

[0049] "Feeder cell medium" or "feeder medium" refers to a cell culture medium that is typically used for cell culture during the period of exponential growth ("growth phase") and is sufficiently complete to support cell culture during this phase. The growth cell medium may also contain one or more selective agents that confer resistance or viability to a selectable marker incorporated into the host cell line. Such selective agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfenimide, methotrexate, cell culture medium without glutamine, cell culture medium lacking glycine, hypoxanthine and thymidine or thymidine alone. Commercially available supplement media can be used, including but not limited to CHO CDEfficient Feed A (Invitrogen), CHO CD Efficient Feed B (Invitrogen), CHO CDEfficient Feed C (Invitrogen), Sheff-CHO PLUS PF ACF (FM012) (Kerry), CHO CDEfficient Feed A+ (Invitrogen), CHO CD Efficient Feed B+ (Invitrogen), CHO CDEfficient Feed C+ (Invitrogen), DPM-Cell Boost 7a (Hyclone), DPM-Cell Boost 7b (Hyclone) or FAA01A (Hyclone).

[0050] In certain embodiments, the cell culture medium is serum-free and / or free of animal-derived products or components. In certain embodiments, the cell culture medium is chemically defined, wherein all chemical components are known. Commercially available culture media can be utilized, and supplemental components or ingredients, including optional components, can be added at appropriate concentrations or amounts as needed or desired, as known and practiced by those skilled in the art using conventional techniques.

[0051] In the context of the present disclosure, the terms "product", "biologic" and "biological substance" are interchangeable. Thus, the product that the cell can produce (e.g. by expressing a product encoding a (recombinant) gene) is a (recombinant) protein, in particular a receptor, an enzyme, a fusion protein, a blood protein, for example a protein from the coagulation cascade, a multifunctional protein (e.g. erythropoietin), a viral or bacterial protein such as for vaccines; an immunoglobulin, for example an antibody, such as IgG or IgM, a multispecific antibody, for example a bispecific antibody, etc. The cell preferably produces a protein, more preferably an antibody.

[0052] The term "antibody" includes glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or antigen-binding regions thereof that specifically compete with intact antibodies for binding, unless otherwise indicated, including human, humanized, chimeric, multispecific, monoclonal, polyclonal and oligomers or antigen-binding fragments thereof. Also included are proteins with antigen-binding fragments or regions, such as Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maximal antibodies, single-chain antibody molecules, complementary determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a target polypeptide. The term "antibody" includes, but is not limited to, those prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody.

[0053] Examples of antibodies include, but are not limited to, antibodies that recognize any one or combination of proteins, including, but not limited to, the above proteins and / or the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor, IL-10 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-2 receptor, IL-4 receptor, IL-5 receptor, IL-8 receptor, IL-10 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-4 receptor, IL-5 receptor, IL-8 receptor, IL-10 receptor, IL-2 ... -6 receptor, IL-13 receptor, IL-18 receptor subunit, FGL2, PDGF-β and its analogs (see U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF, TGF-β2, TGF-β1, EGF receptor (see U.S. Pat. No. 6,235,883), VEGF receptor, hepatocyte growth factor, osteoprotegerin ligand, interferon gamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1 and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.13(1):19-25), C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (gene product expressed in association with lung cancer), HER-2, HER-3, tumor-associated glycoprotein TAG-72, SK-1 antigen, tumor-associated epitopes present at elevated levels in the serum of patients with colon cancer and / or pancreatic cancer, cancer-associated epitopes or proteins expressed on breast cancer, colon cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer and / or renal cancer cells and / or melanoma, glioma or neuroblastoma cells (necrotic core of tumor), integrin α4β7, integrin VLA-4, B2 integrin, TRAIL receptors 1, 2, 3 and 4, RANK, RANK ligand, TNF-α, adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukocyte integrin adhesin, platelet glycoprotein gp IIb / IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (an inhibitor of factor VIIa tissue factor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (a cytotoxic T lymphocyte-associated antigen), Fc-gamma-1 receptor, HLA-DR 10β, HLA-DR antigen, sclerostin, L-selectin, respiratory syncytial virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcus mutans, and Staphlycoccus aureus. Specific examples of known antibodies that can be produced using the methods of the present disclosure include, but are not limited to, adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab, barbicumab, basiliximab, belimumab, briakinumab, canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab, eculizumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, labetuzumab, mapa, and sera. tumumab, matuzumab, mepolizumab, motuzumab, muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab, ogavuzumab, palivizumab, panitumumab, pemtumomab, pertuzumab, ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizomab, zalumab, and zanolimumab.

[0054] In some embodiments, the products produced by the cells, such as proteins or vaccines, can be used as active ingredients in pharmaceutical preparations. Non-limiting examples of products include: anti-hTNFα (adalimumab (Humira TM )), fusion proteins targeting VEGF (Aflibercept (EYLEA TM )), Erythropoietin alpha Lymphoblastoid interferon α-n1 (Wellferon TM ), (recombinant) coagulation factor (NovoSeven TM )、Etanercept(Enbrel TM ), Herceptin TM )、Infliximab(Remicade TM )、Basiliximab(Simulect TM )、Daclizumab (Zenapaz TM ), (recombinant) coagulation factor IX (Benefix TM ), glucocerebrosidase (Cerezyme TM ), interferon β1b G-CSF( Filgrastim), interferon α-2b Recombinant insulin Interferon beta 1a Coagulation Factor VIII Tenecteplase (TNKase TM ), (recombinant) antihemophilic factor (ReFacto TM ), TNFα receptor Follicle-stimulating hormone Mab abcixmab Mab ritiximab Tissue plasminogen activator Human Growth Hormone ( GenoTropin TM). In addition, the definition of the term "antibody construct" includes monovalent, bivalent and multivalent / multivalent constructs, thus, bispecific constructs that specifically bind only to two antigenic structures, and multispecific / multispecific constructs that bind to more than two, for example three, four or more antigenic specific structures through different binding domains. In addition, the definition of the term "antibody construct" includes molecules consisting of only one polypeptide chain and molecules consisting of more than one polypeptide chain, which chains can be the same (homodimers, homotrimers or homooligomers) or different (heterodimers, heterotrimers or heteropolymers). Examples of the above antibodies and their variants or derivatives are recorded in Harlow and Lane's Antibodies a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Dubel's Antibody Engineering, Springer, 2nd ed. 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0055] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly connected by amide bonds (also called peptide bonds). The term "polypeptide" refers to any chain consisting of two or more amino acids, and is not limited to a specific length of the product. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to two or more amino acid chains. "Polypeptide" can be used to replace any of these terms or to be used interchangeably with them. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolysis or modification by non-natural amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. The polypeptide can be produced in any way, including by chemical synthesis. The size of the polypeptides of the invention can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides that do not have a defined three-dimensional structure but can adopt a large number of different conformations are referred to as unfolded.

[0056] The term "microsparger" generally refers to a sparger configured to provide oxygen and / or other gases to a cell culture within a bioreactor tank. An aerator or microsparger can be coupled to an oxygen or other gas source and can direct the gas to the cell culture so that the gas is bubbled in the cell culture, thereby aerating the cell culture. In some examples, a microsparger can be used in conjunction with a drill pipe sparger.

[0057] Biological preparations prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography (SEC), etc. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, biologically bound antibodies, ligands, receptors, or antigens can be used. For example, for affinity chromatography purification of biological preparations (e.g., immunoconjugates) disclosed herein, a matrix with protein A or protein G can be used. As described in the Examples, biological substances, such as immunoconjugates, can be separated by sequential protein A or G affinity chromatography and size exclusion chromatography. The purity of biological preparations (e.g., immunoconjugates) can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and similar methods.

[0058] In the present disclosure, the term "harvest rate" refers to the rate at which the culture product passes through the filter 1 (which is at least one filter whose filtration pore size does not allow the cells to pass through the filter but allows biological substances to pass through the filter).

[0059] [II. Apparatus and method for continuous harvesting of biological substances produced by cultured cells]

[0060] In various embodiments, the biological substances produced by the cultured cells are continuously harvested by the device of the present disclosure. Any type of filter can be used as the device of the present disclosure, as long as the filtration pore size of one of the two sets of filters (such as filter 1) does not allow the cells to pass through the filter, but allows the biological substances to pass through the filter, and the filtration pore size of the other set of filters (such as filter 2) does not allow both cells and biological substances to pass through, but keeps the filter fluid flowing, wherein filter 1 and filter 2 are connected to the fluid of the device for culturing the cells in parallel. In one embodiment, filter 1 and filter 2 (a) are each directly, (b) via a branch tube, or (c) a combination of (a) and (b) and are connected to the fluid of the device for culturing the cells. In some embodiments, the filtration pore size of filter 1 is about 0.08 μm to 0.5 μm, preferably about 0.1 μm to 0.4 μm, and more preferably about 0.2 μm. In some embodiments, the molecular weight cutoff (MWCO) of the filter 2 is 50 kD or less, 45 kD or less, 40 kD or less, 35 kD or less, 30 kD or less, 25 kD or less, 20 kD or less, etc. The approximate correspondence between the pore size expressed as pore diameter (μm) and the pore size expressed as molecular weight cutoff (kD) is as follows.

[0061]

[0062] Thanks to the diversion effect of the filter 2, the flow through the filter 1 is reduced, which can effectively reduce or even avoid the risk of clogging of the filter 1, thereby extending the service life of the filter 1.

[0063] Since part of the waste liquid in the harvest is removed by the filter 2, the volume of the harvest liquid can be effectively reduced, thereby effectively reducing the volume and storage space of the storage device.

[0064] Since part of the waste liquid in the harvest is removed by the filter 2, the harvested target biological substance can be effectively concentrated, thereby effectively reducing its downstream purification burden.

[0065] Since the filter 2 can retain some small molecular biological substances (such as PF68) that are beneficial to maintaining cell viability in the culture system, it can increase the viable cell density (VCD) in the culture system, extend the culture cycle, and increase the batch yield.

[0066] In some embodiments of the device of the present disclosure, the device of the present disclosure further comprises a device for adjusting the filtration rate of the filter 1 and / or a device for adjusting the filtration rate of the filter 2. Thanks to such a device, the flow rate ratio of the cell culture passing through the filter 1 and the filter 2 can be adjusted as needed, so that the biological substances in the harvested liquid can be concentrated to a desired degree.

[0067] The circulation of the cell culture on the filter is a flow substantially parallel to the filter surface, also referred to as a tangential flow, such as a one-way tangential flow filtration (TFF) or a cross flow. A preferred example of a cross flow is an alternating tangential flow (ATF), when ATF is used, it is found that even at very high cell densities, filter clogging will not occur (rapidly). "Alternating tangential flow" refers to a flow back and forth in the same direction as the filter surface (i.e., a direction tangential to the filter surface), and there is another flow in a direction substantially perpendicular to the filter surface. Alternating tangential flow can be achieved according to methods known to those skilled in the art (e.g., as described in U.S. Patent No. 6,544,424, the entire contents of which are incorporated herein by reference).

[0068] Non-limiting examples of filters suitable for use in the present disclosure include membrane filters, ceramic filters, and metal filters. The filter can be used in any shape. The filter can be, for example, spirally wound or tubular, or can be used in the form of a sheet. In various embodiments, the filter used is a membrane filter. In at least one embodiment, the filter is a hollow fiber filter. Filter devices containing hollow fibers are commercially available from, for example, Refine Technology.

[0069] In some embodiments of the device of the present disclosure, the number of the filters 1 is 1, 2, 3 or more, and / or the number of the filters 2 is 1, 2, 3 or more.

[0070] In another aspect, the present disclosure provides a method for continuously harvesting biomass produced by cultured cells, which is performed by the apparatus of the present disclosure as described above.

[0071] [3. Systems and methods for producing biomass]

[0072] The present disclosure provides a system for producing biomass, comprising:

[0073] (a) an apparatus for culturing cells, which culture the cells in a perfusion mode using a basal medium and a supplementary medium; and

[0074] (b) An apparatus according to the present disclosure as described above.

[0075] A "perfusion" culture process is one in which fresh medium is added to the cell culture and spent medium is removed from the bioreactor. Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of these.

[0076] In various embodiments, a cell culture is established by inoculating a bioreactor with cells expressing a biological substance of interest. The cell culture is maintained by feeding a basal medium and a feed medium.

[0077] The rate at which cell culture medium is added to the culture may affect cell viability and density.The term "viable cell density (VCD)" refers to the number of viable cells in a given volume of culture medium, as determined by standard viability assays (eg, trypan blue dye exclusion).

[0078] In at least one embodiment of the present disclosure, a microsparger is used in the method of the present disclosure. In another embodiment of the present disclosure, a microsparger is used when the required oxygen flow rate reaches about 0.5 VVM. In the present disclosure, the implementation of the microsparger mitigates cell damage caused by bubble collapse during culture.

[0079] In another aspect, the present disclosure provides a method of producing biomass, which is performed by the system of the present disclosure as described above. [Example]

[0080] The present disclosure, as generally described above, will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present disclosure.

[0081] Cell lines and culture conditions

[0082] Thaw a vial containing CHO-K1 host cells purchased from ATCC (ATCC No.: CCL 61). Generate 100 vials of master cell bank (MCB) and then 136 vials of working cell bank (WCB). Then thaw the WCB vial and culture it in suspension with serum-free medium. Generate 60 vials of PCB, 170 vials of MCB and 230 vials of WCB using the suspension-suitable clone CHO-K1-A4. Thaw a WCB vial of CHO-K1 host cells (CHO-K1-A4) for stable transfection.

[0083] The cDNA sequence expressing the anti-hTNFα antibody disclosed in U.S. Patent No. 6,090,382 was cloned into two vectors, which contained Blasticidin and Zeocin resistance markers, respectively. Liposomes were used for stable transfection. After transfection, the cells were transferred to selective medium (CD CHO medium containing 9 μg / mL Blasticidin and 400 μg / mL Zeocin) for cell selection. After about 2 weeks of cell selection, the cells were cloned by FACS sorting. Clones were screened by batch feeding culture in centrifuge tubes. A high-yielding clone was selected and named Clone X.

[0084] [Example 1]

[0085] In this example, using Clone X, the performance of intensified perfusion culture (IPC) processes (Process 1 and Process 2) performed using the apparatus and methods of the present disclosure (but using different harvest rates) was evaluated.

[0086]

Technology 1

[0087] Process 1 was performed in a 7L Applikon vessel using a delta V controller to control the temperature at 36.5°C, the pH range at 7.2 to 6.8, and the DO at 40% air saturation. Cells were retained using a hollow fiber filter (filter 1) (Spectrum labs) with a cutoff of 0.2 μm running in ATF flow mode with an ATF-2H system (Refine Technology). Cells and recombinant proteins were retained using a hollow fiber filter (filter 2) (Spectrum labs) with a cutoff of 50 kD (about 0.008 μm) running in ATF flow mode with an ATF-2H system (Refine Technology). The filter 1 and filter 2 were connected to the Applikon vessel fluid in parallel via a branch pipe.

[0088] The cells were cultured at 0.90-1.10 × 10 6 The culture was started with an initial VCD of 1.5 cells / mL. About 10-100 ppm of antifoam was added daily from day 0. The basal medium (Excell Advanced CHO supplemented batch medium, Sigma) was perfused at a rate of 0.4 VVD from day 1 and increased to 1.5 VVD on day 3. The supplement medium (CB7a / CB7b) was perfused from day 4 and its rate was adjusted daily to maintain the minimum glucose concentration above 2.0 g / L. From day 3 to the end of the culture, the perfusion rate was maintained at 1.5 VVD. When the required oxygen flow reached 0.5 VVM, a microsparger was used. The temperature was changed from 36.5°C to 31.0°C on day 5 and maintained at 31.0°C until the end of the culture.

[0089] In order to perform continuous concentration harvesting, the filtrate (containing recombinant protein (i.e., harvest)) passing through filter 1 is continuously collected. At the same time, the filtrate without recombinant protein passing through filter 2 is discarded to maintain a constant working volume. Before day 13, the harvest rate is equal to the perfusion rate, and after day 13, the harvest rate is reduced to one-third of the perfusion rate. During the entire culture process, the cells remain in the bioreactor without outflow.

[0090]

Technology 2

[0091] Process 2 was performed in a 7L Applikon vessel using a delta V controller to control the temperature at 36.5°C, the pH range at 7.2 to 6.8, and the DO at 40% air saturation. Cells were retained using a hollow fiber filter (filter 1) (Spectrum labs) with a cutoff of 0.2 μm running in ATF flow mode with an ATF-2H system (Refine Technology). Cells and recombinant proteins were retained using a hollow fiber filter (filter 2) (Spectrum labs) with a cutoff of 50 kD (about 0.008 μm) running in ATF flow mode with an ATF-2H system (Refine Technology). The filter 1 and filter 2 were connected to the Applikon vessel fluid in parallel via a branch pipe.

[0092] The cells were cultured at 0.90-1.10 × 10 6 The culture was started with an initial VCD of 1.5 cells / mL. About 10-100 ppm of antifoam was added daily from day 0. The basal medium (Excell Advanced CHO supplemented batch medium, Sigma) was perfused at a rate of 0.4 VVD from day 1 and increased to 1.5 VVD on day 3. The supplement medium (CB7a / CB7b) was perfused from day 4 and its rate was adjusted daily to maintain the minimum glucose concentration above 2.0 g / L. From day 3 to the end of the culture, the perfusion rate was maintained at 1.5 VVD. When the required oxygen flow reached 0.5 VVM, a microsparger was used. The temperature was changed from 36.5°C to 31.0°C on day 5 and maintained at 31.0°C until the end of the culture.

[0093] In order to perform continuous concentration harvesting, the filtrate (containing recombinant protein (i.e., harvest)) passing through filter 1 is continuously collected. At the same time, the filtrate without recombinant protein passing through filter 2 is discarded to maintain a constant working volume. Before day 13, the harvest rate is one-third of the perfusion rate, from day 13 to day 17, the harvest rate is equal to the perfusion rate, and after day 17, the harvest rate is reduced to one-third of the perfusion rate. During the entire culture process, the cells remain in the bioreactor without outflow.

[0094]

Comparison between process 1 and process 2

[0095] Figure 2 It was shown that higher peak viable cell densities were achieved in the processes with concentrated harvest (process 1 after day 13 and process 2 before day 13).

[0096] Figure 3 It was shown that in the processes with concentrated harvest (process 1 after day 13 and process 2 before day 13), the viability of the cells remained high.

[0097] Figure 4 It shows that the average diameter of cells in process 1 and process 2 is similar.

[0098] Figure 5 It was shown that the glucose concentrations in Process 1 and Process 2 were comparable.

[0099] Figure 6 It was shown that no significant lactic acid production or accumulation problems were observed in either process.

[0100] Figure 7 It was shown that the recombinant protein titer in the harvest was significantly higher in the processes with concentrated harvest (process 1 after day 13 and process 2 before day 13).

[0101] Figure 8 It is shown that the cumulative volumetric productivity (Pv) is slightly lower in the processes with concentrate harvest (process 1 after day 13 and process 2 before day 13).

[0102] Fig. 9 It was shown that the recombinant protein retention rate (Titer Retention Rate) was significantly lower in the processes with concentrated harvest (Process 1 after day 13 and Process 2 before day 13).

[0103] [Example 2]

[0104] In this example, using clone X, the performance of an IPC process performed using the prior art apparatus and method (Process A) and an IPC process performed using the apparatus and method of the present disclosure (Process B) were compared.

[0105]

Process A

[0106] Process A was performed in a 7 L Applikon vessel using a delta V controller to control the temperature at 36.5°C, the pH range between 7.2 and 6.8, and the DO at 40% of air saturation. The cells were retained using a 0.2 μm cutoff hollow fiber filter (Spectrum labs) operating in ATF flow mode with an ATF-2H system (Refine Technology).

[0107] This process was carried out in CDM4 medium (Hyclone) supplemented with 4.0 mM L-glutamine at a concentration of 0.90-1.10 × 10 6Cells / mL were added at an initial VCD of 1.5 cells / mL. About 10-100 ppm of defoamer was added daily from day 0. From day 1, basal medium (CDM4 medium, Hyclone) was perfused at a rate of 0.4 VVD, and increased to 1.0 VVD on day 2, and maintained until the end of the culture. From day 3, supplementary medium (CB7a / CB7b) was perfused at a rate of 2% of the basal medium, and gradually increased to 18% on day 14, and maintained until day 16. Oxygen was delivered using a microsparger when needed. The temperature was changed from 36.5°C to 31.0°C on day 6 and maintained at 31.0°C until the end of the culture.

[0108] The cell culture is continuously harvested by ATF. Throughout the culture process, the cells remain in the bioreactor without flowing out.

[0109]

Process B

[0110] Process B was performed in a 3L Applikon vessel using a delta V controller to control the temperature at 36.5°C, the pH range at 7.2 to 6.7, and the DO at 40% air saturation. Cells were retained using a hollow fiber filter (filter 1) (Spectrum labs) with a cutoff of 0.2 μm running in ATF flow mode with an ATF-2H system (Refine Technology). Cells and recombinant proteins were retained using a hollow fiber filter (filter 2) (Spectrum labs) with a cutoff of 50 kD (about 0.008 μm) running in ATF flow mode with an ATF-2H system (Refine Technology). The filter 1 and filter 2 were connected to the Applikon vessel fluid in parallel via a branch pipe.

[0111] This process was carried out in CDM4 medium (Hyclone) supplemented with 8.0 mM L-glutamine at 19.00-21.00 × 10 6 Cells / mL were added at an initial VCD of 1.0 VCD. About 10-100 ppm of defoamer was added daily from day 0. The basal medium (CDM4 medium, Hyclone) was perfused at a rate of 1.0 VVD from day 1 and maintained until the end of the culture. The supplement medium (CB7a / CB7b) was perfused at a rate of 3% of the basal medium from day 0 and gradually increased to 8% on day 6. From day 9 to day 12, the perfusion rate of the supplement medium was gradually reduced to 6%. The temperature was changed from 36.5°C to 31.0°C on day 2 and maintained at 31.0°C until the end of the culture.

[0112] In order to perform continuous concentration harvesting, the filtrate (containing recombinant protein (i.e., harvest)) passing through filter 1 is continuously collected. At the same time, the filtrate without recombinant protein passing through filter 2 is discarded to maintain a constant working volume. Harvesting begins after the temperature shift on the second day, and the harvest rate is half of the perfusion rate thereafter. During the entire culture process, the cells remain in the bioreactor without outflow.

[0113]

Comparison between process A and process B

[0114] In order to directly compare the performance with Process B, data from Process A from Day 4 onwards are shown.

[0115] Fig.10 It was shown that the peak viable cell density achieved in process A was slightly higher.

[0116] Fig.11 The results showed that the cell viability was comparable in the two processes.

[0117] Fig.12 It was shown that the average cell diameters in the two processes were comparable.

[0118] Fig.13 It was shown that the glucose concentration in both processes was maintained above 1.00 g / L.

[0119] Fig.14 It was shown that no significant lactic acid production or accumulation problems were observed in either process.

[0120] Fig.15 It was shown that the recombinant protein titer in the harvest of process B with concentrated harvest was significantly higher than that in the harvest of process A without concentrated harvest.

[0121] Fig.16 It is shown that the cumulative volumetric productivity (Pv) in the two processes is comparable.

[0122] Fig.17 The results show that the recombinant protein retention rate (Titer Retention Rate) in process B with concentrated harvesting is significantly lower than the retention rate (Titer Retention Rate) in process A without concentrated harvesting.

Claims

1. An apparatus for continuously harvesting biological material produced by cultured cells, comprising: (a) filter 1, which is at least one filter, the filtration pore size of which does not allow the cells to pass through the filter but allows biological substances to pass through the filter, and (b) a filter 2, which is at least one filter, the filtration pore size of which does not allow the cells and the biological substances to pass through the filter, but keeps the filter fluid flowing, wherein the filter 1 and the filter 2 are connected to the cell culture device fluid in parallel from the outside of the cell culture device for culturing the cells, and The device further comprises a device for adjusting the filtration rate of the filter 1 and / or a device for adjusting the filtration rate of the filter 2.

2. The device of claim 1, wherein the filter 1 and the filter 2 are in fluid communication with the cell culture device by: (a) each directly, (b) via a branch pipe, or (c) A combination of (a) and (b).

3. The device according to claim 1 or 2, wherein the filter 1 has a filtration pore size of 0.08 μm to 0.5 μm.

4. The device according to claim 1 or 2, wherein the filter 1 has a filtration pore size of 0.1 μm to 0.4 μm.

5. The device according to claim 1 or 2, wherein the filter 1 has a filtration pore size of 0.2 μm.

6. The device of claim 1 or 2, wherein the molecular weight cutoff of the filter 2 is below 50 kD.

7. The apparatus of claim 1 or 2, wherein one or both of said filter 1 and said filter 2 are alternating tangential flow filters or tangential flow filtration filters.

8. The apparatus of claim 7, wherein one or both of said filter 1 and said filter 2 are hollow fiber filters.

9. The device of claim 1 or 2, wherein The number of the filters 1 is 1, 2, 3 or more, and / or The number of the filters 2 is 1, 2, 3 or more.

10. A system for producing biomass, comprising: (a) a cell culture device for culturing cells in a perfusion mode using a basal medium and a supplementary medium; and (b) The apparatus of any one of claims 1 to 9.

11. The system of claim 10, further comprising means for continuously capturing biological matter from matter harvested by the apparatus of any one of claims 1 to 9.

12. The system of claim 10 or 11, further comprising a micro-atomizer.

13. A method for continuously harvesting biological material produced by cultured cells, which is performed by the device according to any one of claims 1 to 9.

14. A method of producing biomass, performed by the system of any one of claims 10 to 12.

15. The method of claim 13 or 14, wherein the cells comprise mammalian cells.

16. The method of claim 15, wherein the mammalian cell comprises a Chinese hamster ovary cell, a hybridoma, a baby hamster kidney cell, or a myeloma cell.

17. The method of claim 13 or 14, wherein the biological substance is selected from the group consisting of: fusion proteins, viral or bacterial proteins, and immunoglobulins.

18. The method of claim 13 or 14, wherein the biological substance is a receptor.

19. The method of claim 13 or 14, wherein the biological substance is an enzyme.

20. The method of claim 13 or 14, wherein the biological substance is a blood protein.

21. The method of claim 20, wherein the blood protein is from the coagulation cascade.

22. The method of claim 13 or 14, wherein the biological substance is a multifunctional protein.

23. The method of claim 22, wherein the multifunctional protein is erythropoietin.

24. The method of claim 17, wherein the viral or bacterial protein is used in a vaccine.

25. The method of claim 17, wherein the immunoglobulin is an antibody or a multispecific antibody.

26. The method of claim 25, wherein the antibody is IgG or IgM.

27. The method of claim 25, wherein the multispecific antibody is a bispecific antibody.

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