Methods for improving the production of biological products by reducing the levels of endogenous proteins

Abstract

The present disclosure provides methods, cells or cell lines, and compositions for increasing the amount of a product, e.g., a protein, produced by a cell or cell line by reducing the levels of a nonessential endogenous protein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and benefits from U.S. Provisional Application USSN 62 / 697,480 (filed on July 13, 2018), the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present disclosure relates to methods and compositions for modifying host cell expression to increase the production of a product (eg, a polypeptide) expressed in cultured cells. Background Art

[0004] Cell products (e.g., recombinant therapeutic proteins) are typically expressed in cell expression systems (e.g., mammalian cell expression systems). Hundreds of market-approved biopharmaceuticals have been expressed in mammalian cell lines. However, the high costs associated with their production have led to an increase in global health costs.

[0005] Therefore, there is a need to develop and produce methods for producing products (eg, polypeptides) expressed in cultured cells. Summary of the invention

[0006] The present disclosure is based in part on the discovery that reducing the level of one or more endogenous proteins (e.g., non-essential, redundant and / or secreted endogenous proteins) in a cell (e.g., a producer cell) capable of producing a product (e.g., a recombinant polypeptide) increases the yield of the product.

[0007] In one aspect, the invention relates to a method of producing a product (e.g., a recombinant polypeptide) in a cell (e.g., a production cell), the method comprising:

[0008] reducing the level of an endogenous protein in the cell (e.g., a production cell),

[0009] Thus, the product is produced.

[0010] In another aspect, the invention relates to a method of producing a product (e.g., a recombinant polypeptide) in a cell (e.g., a production cell), the method comprising:

[0011] providing cells (e.g., producer cells),

[0012] reducing the level of an endogenous protein in the cell (e.g., a production cell), and

[0013] culturing the cells under conditions suitable for production of the product (e.g., a recombinant polypeptide),

[0014] Thus, the product is produced.

[0015] In another aspect, the present invention relates to a cell (e.g., a production cell) capable of producing a product (e.g., a recombinant polypeptide), wherein the cell (e.g., a production cell) comprises a deletion, substitution or insertion mutation of the gene encoding the endogenous protein or a copy of a regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein, for example, wherein the mutation reduces the expression level of the endogenous protein.

[0016] In another aspect, the invention relates to a cell (e.g., a production cell) capable of producing a product (e.g., a recombinant polypeptide), wherein the cell comprises an siRNA, which is capable of binding to a nucleic acid (e.g., an mRNA encoding an endogenous protein or a regulatory element (e.g., a promoter or enhancer) operably linked thereto).

[0017] In another aspect, the invention relates to a product (eg, a recombinant polypeptide) produced by a method or cell described herein.

[0018] In another aspect, the invention relates to a pharmaceutical composition comprising a product (eg, a recombinant polypeptide) described herein.

[0019] In another aspect, the present invention relates to an siRNA capable of binding to a nucleic acid (eg, an mRNA encoding an endogenous protein selected from Table E5).

[0020] In embodiments, the product (eg, polypeptide) is one of the polypeptides provided in Tables 1 and 2, or a variant thereof.

[0021] In an embodiment, the product is a polypeptide. In an embodiment, the polypeptide is an antibody (e.g., a monoclonal antibody, e.g., an IgG1 antibody) or a polynucleotide encoding the antibody. In an embodiment, one or more cysteines in the polynucleotide encoding the antibody are replaced by another amino acid. In certain embodiments, one or more cysteines are replaced by one or more serine residues.

[0022] In one aspect, the disclosure features a method for producing a product described herein in a cell (e.g., a recombinant host cell, such as a production cell). In one embodiment, the product is a polypeptide, such as a recombinant polypeptide.

[0023] Examples of products that can be produced using any of the methods or compositions described herein include recombinant products, or products in which at least a portion (portion / moiety) is a result of genetic engineering. Recombinant products described herein can be used for diagnostic or therapeutic purposes. In one embodiment, the product includes a polypeptide, such as an antibody molecule (e.g., a bispecific or multiformat antibody molecule), a fusion protein, or a protein conjugate. The methods and compositions described herein may be particularly useful for products that are difficult to produce (e.g., difficult to produce in large quantities or with sufficient quality for commercial or therapeutic use) (e.g., next-generation biological products (e.g., fusion proteins, bispecific or multiformat antibody molecules, multiproteins, and glycosylated proteins)). In one embodiment, for example, the cell described herein for producing the product expresses the product. In one embodiment, the cell includes an exogenous nucleic acid encoding a product described herein (e.g., a polypeptide selected from Tables 1, 2, 3, or 4). Other examples of products are described in the section entitled "Product".

[0024] Compared to cells that have not been subjected to reduced temperatures, the methods described herein can also result in an improvement in product quality. The improvement in product quality can be characterized by one or more of the following: dissociation (e.g., a reduction in the dissociation of polypeptides into less active forms); aggregation (e.g., a reduction in aggregates or aggregation); proper folding or assembly (e.g., misfolded or unfolded products; or a reduction in partially assembled or disassembled products); post-translational modification (e.g., an increase or decrease in glycosylation heterogeneity, a higher percentage of desired or predetermined post-translational modification); fragmentation (e.g., a reduction in fragmentation); disulfide mismatching (scrambling) (e.g., a reduction in undesirable isomers or structures due to disulfide mismatching). In one embodiment, for example, the quality of the product (e.g., recombinant polypeptide) is improved by, for example, 1 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, compared to the quality of the product produced by cells that have not been subjected to reduced temperatures.

[0025] In an embodiment, the method for producing a product described herein may be performed by one or more additional steps to enhance the stability or activity of the polypeptide. These include, but are not limited to: introducing into the production cell an improved ER processing capacity (ER expansion) or secretion; obtaining the product from the cell or the progeny of the cell, or from a culture medium conditioned by the cell or the progeny of the cell; separating the product from at least one cell or culture medium component; and / or analyzing the product (e.g., analyzing its activity or the presence of a structural part). In one embodiment, the method further comprises a step of improving ER processing capacity (or ER expansion) by introducing a nucleic acid encoding PD1, BiP, ERO or XBP1. In one embodiment, the method further comprises additional steps for improving secretion capacity or secretion rate by regulating other mechanisms involved in the SNARE mechanism or secretory pathway (e.g., by introducing a nucleic acid encoding a SNARE component).

[0026] product

[0027] Products suitable for use in the methods described herein include polypeptides, such as recombinant proteins; nucleic acid molecules, such as DNA or RNA molecules; multimeric proteins or complexes; lipid-encapsulated particles (e.g., virus-like particles), vesicles or exosomes; or other molecules. In one embodiment, the product is a polypeptide, such as a recombinant polypeptide. For example, the recombinant polypeptide can be a protein that is difficult to express or has a complex and / or non-native structure, such as a next-generation biological product (e.g., a bispecific antibody molecule, a fusion protein, or a glycosylated protein).

[0028] In any of the methods described herein, the method for producing a product further comprises introducing into a cell an exogenous nucleic acid encoding the product (eg, a polypeptide, such as a recombinant polypeptide).

[0029] In any composition, formulation, bioreactor or method described herein, the product (e.g., a recombinant polypeptide) is a therapeutic polypeptide or an antibody molecule (e.g., an antibody or an antibody fragment thereof). In one embodiment, the antibody molecule is a monoclonal antibody. In one embodiment, the antibody molecule is a bispecific antibody molecule, such as a BiTE (bispecific T cell engager), a DART (dual affinity retargeting or redirecting T cells).

[0030] In one embodiment, the product (eg, a recombinant polypeptide) is selected from Table 1, Table 2, Table 3, or Table 4.

[0031] In an embodiment, the product is stably expressed by the cell. In one embodiment, the exogenous nucleic acid encoding the product (e.g., a recombinant polypeptide) is integrated into the chromosomal genome of the cell. Alternatively, the product is transiently expressed by the cell. In one embodiment, the exogenous nucleic acid encoding the product (e.g., a recombinant polypeptide) is not integrated into the chromosomal genome of the cell.

[0032] Host cells

[0033] Provided herein are cells (eg, recombinant cells, such as producer cells) for producing the products described herein, methods of engineering such cells, and methods of using the cells.

[0034] In any composition, formulation or method described herein, the cell is a eukaryotic cell. In one embodiment, the cell is a mammalian cell, a yeast cell, an insect cell, an algae cell or a plant cell. In one embodiment, the cell is a rodent cell. In one embodiment, the cell is a CHO cell. Examples of CHO cells include but are not limited to CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHODG44, CHO DXB11, CHOZN or CHO derived cells.

[0035] In any composition, formulation or method described herein, the cell is selected from the group consisting of: HeLa, HEK293, H9, HepG2, MCF7, Jurkat, NIH3T3, PC 12, PER.C6, BHK, VERO, SP2 / 0, NS0, YB2 / 0, EB66, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, CHOK1, CHOK1SV, PotelligentCHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11 and CHOZN.

[0036] In one embodiment, the cell is a eukaryotic cell other than a mammalian cell, such as an insect, plant, yeast or algae cell. In one embodiment, the cell is a prokaryotic cell.

[0037] In any of the methods or cells described herein (eg, production cells), the cell expresses or comprises a product, eg, a recombinant product, eg, a next generation biologic selected from the group consisting of: a bispecific antibody, a fusion protein, or a glycosylated protein.

[0038] In any of the methods or cells described herein (e.g., production cells), the cell is a CHO cell selected from the group consisting of CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11, CHOZN, or a CHO-derived cell.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference as a whole. In addition, the materials, methods and examples are only illustrative and are not intended to be restrictive. Titles, subtitles or numbered or lettered elements (e.g., (a), (b), (i) etc.) are presented only for the purpose of easy reading. The use of titles or numbered or lettered elements in this document does not require that steps or elements be performed in alphabetical order, or that steps or elements are not required to be separated from each other. Other features, purposes and advantages of the present invention will become apparent through the specification and accompanying drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A Shown is a graph of cell-specific productivity of Mabs in CHO cell lines in which selected non-essential, redundant and / or secreted endogenous proteins were knocked out relative to CHO cells transfected with a vector lacking gRNA.

[0041] Figure 1B A graph showing gene indel success as measured by TIDE analysis of genomic DNA extracted from FACS sorted pools. DETAILED DESCRIPTION

[0042] The present disclosure is characterized in that it is used to obtain a method and composition for obtaining a higher yield of a product (e.g., a polypeptide) by reducing the level of an endogenous protein (e.g., a non-essential, redundant and / or secretory endogenous protein) in a cell (e.g., a production cell) that can produce the product. The methods described herein and the cells used in the methods show a quality improvement over the yield and quality obtained by the current production methods and / or cells. Compared to the cell expression systems currently used to produce recombinant products, the methods and compositions described herein can also be used with engineered cells or cell lines with improved productivity, product quality, robustness and / or culture vigor.

[0043] The method is suitable for producing next generation biological products. As these methods continue to obtain therapeutic benefits in patients, the demand for a large number of next generation biological products with high quality grades for therapeutic use and effective production methods and production cell lines will continue to increase. In addition, many next generation biological products are difficult to express and produce in conventional cell lines using conventional expression techniques known in the art. Current methods are not enough to mass produce these products, nor can they reach the high quality grades required for clinical use. The method described herein comprising reducing the level of endogenous proteins overcomes these obstacles and can be combined with other methods to increase product yield.

[0044] definition

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice and / or testing of the present invention, preferred materials and methods are described herein. In describing and claiming the present invention, the following terms will be used in a manner as defined therein, and definitions are provided below.

[0046] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0047] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "a cell" can refer to one cell or more than one cell.

[0048] As used herein, "endogenous" refers to any material derived from or produced naturally within an organism, cell, tissue or system.

[0049] As used herein, "exogenous" refers to any material introduced into or produced outside an organism, cell, tissue, or system. Thus, "exogenous nucleic acid" refers to a nucleic acid introduced into or produced outside an organism, cell, tissue, or system. In one embodiment, the sequence of the exogenous nucleic acid is not naturally produced in the organism, cell, tissue, or system into which the exogenous nucleic acid is introduced, or cannot be found in nature. In an embodiment, a non-naturally occurring product or a product containing a non-naturally occurring portion is an exogenous material relative to a host cell as described herein.

[0050] As used herein, "heterologous" refers to any material of a different species when introduced into an organism, cell, tissue or system of a species. In embodiments, heterologous materials also encompass materials comprising portions of one or more species or non-naturally occurring portions. For example, in one embodiment, a nucleic acid encodes a fusion protein, wherein a portion of the fusion protein is human, a portion of the fusion protein is bacterial, and a portion of the fusion protein is non-naturally occurring, and the nucleic acid is introduced into a human cell, the nucleic acid is a heterologous nucleic acid.

[0051] As used interchangeably herein, "peptide", "polypeptide" and "protein" refer to compounds comprising amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids constituting a protein or peptide sequence. In one embodiment, a protein may comprise more than one (e.g., two, three, four, five or more) polypeptides, each of which is associated with each other by covalent or non-covalent bonds / interactions. A polypeptide comprises any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to two short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are commonly referred to in the art as proteins, which have many types). "Polypeptide" comprises, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc.

[0052] "Recombinant product" refers to a product that can be produced by a cell or a cell-free system. The product can be a molecule, a nucleic acid, a polypeptide (e.g., an SS polypeptide) or any hybrid thereof. A recombinant product is a product of at least one nucleotide of a sequence that forms at least one component of a product or controls the production or expression of a product through genetic engineering. The genetic engineering used herein to generate a recombinant product or a construct encoding a recombinant product covers recombinant DNA expression techniques known in the art (e.g., as described in Current Protocols in Molecular Biology); site-directed, scanning or random mutagenesis; CRISPR strategies; and zinc finger nuclease (ZFN) strategies. In one embodiment, the recombinant product is a recombinant polypeptide. In one embodiment, the recombinant product is a naturally occurring product. In one embodiment, the recombinant product is a non-naturally occurring product, such as a synthetic product. In one embodiment, a portion of the recombinant product is naturally occurring, and another portion of the recombinant product is non-naturally occurring. In another embodiment, a first portion of the recombinant product is a naturally occurring molecule, and another portion of the recombinant product is another naturally occurring molecule different from the first portion.

[0053] "Recombinant polypeptide" refers to a polypeptide that can be produced by a cell as described herein. A recombinant polypeptide is a product of at least one nucleotide of a sequence encoding a polypeptide or a sequence controlling the expression of a polypeptide formed by genetic engineering or manipulation (of a cell or precursor cell). For example, at least one nucleotide is changed, for example, introduced into a cell, or it is the product of a genetically engineered rearrangement. In one embodiment, the sequence of the recombinant polypeptide is indistinguishable from a naturally occurring or non-naturally occurring isomer of the polypeptide or protein. In one embodiment, the amino acid sequence of the recombinant polypeptide is different from the sequence of a naturally occurring or non-naturally occurring isomer of the polypeptide or protein. In one embodiment, the recombinant polypeptide and the cell are from the same species. In one embodiment, the amino acid sequence of the recombinant polypeptide is identical or substantially identical to the polypeptide encoded by the endogenous genome of the cell, or differs therefrom by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. In one embodiment, the recombinant polypeptide and the cell are from the same species, for example, the recombinant polypeptide is a human polypeptide, and the cell is a human cell. In one embodiment, the recombinant polypeptide and the cell are from different species, for example, the recombinant polypeptide is a human polypeptide, and the cell is a non-human (e.g., rodent, such as CHO), other mammalian cells, insect cells, plants, fungi or bacterial cells. In one embodiment, the recombinant polypeptide is exogenous to the cell, in other words, the cell is from a first species, and the recombinant polypeptide is from a second species. In one embodiment, the polypeptide is a synthetic polypeptide. In one embodiment, the polypeptide is derived from a non-naturally occurring source. In one embodiment, the recombinant polypeptide is a human polypeptide or protein, and its amino acid sequence is not different from the natural or non-natural isomer of the human polypeptide or protein. In one embodiment, the recombinant polypeptide is different from the natural or non-natural isomer of the human polypeptide or protein at no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues. In one embodiment, the recombinant polypeptide is different from the natural isomer of the human polypeptide at no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 15% of its amino acid residues. In an embodiment where a portion of the recombinant polypeptide includes a sequence of a portion of a natural or non-natural isomer of a human polypeptide, the portion of the recombinant polypeptide differs from the corresponding portion of the natural or non-natural isomer by no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 15% of its amino acid residues.

[0054] Oxygenation level and oxygenated level are used interchangeably and, as used herein, refer to the level of dissolved oxygen in a volume of liquid or gas (e.g., in a culture). Oxygenation level can be described in units of dissolved oxygen tension (DOT), such as as a percentage of air saturation (e.g., a percentage of oxygen level in air).

[0055] As used herein, a production culture refers to a mixture of a growth medium and cells (e.g., at least one cell), such as recombinant cells, such as recombinant host cells expressing a product (e.g., a protein (e.g., a recombinant polypeptide), a cytokine (e.g., an enzyme and a metabolite)). In some embodiments, the production culture passes through one or more culture stages (e.g., consecutive and / or overlapping culture stages) that include cell growth / division characteristics. In some embodiments, the processing of the production culture is changed as described herein as the production culture passes through different stages. As used herein, the production stage refers to the production culture stage that begins with inoculation and ends when a specific standard (e.g., the end of a process or time period, such as 10-30 days, such as 15-20 days) is reached or when a specific cell viability standard is reached (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20 or 10% viability, such as less than 50% viability). The production stage can include an exponential growth phase in which cells grow and / or divide into exponential growth. The production phase can be included in a post-growth phase after the exponential growth phase, wherein cell growth and / or division is in a linear phase of a growth curve, e.g., the rate of cell growth and / or division is reduced relative to the exponential growth phase. In some embodiments, completion of the production phase produces a production mixture from the production culture. In some embodiments, separation of cells (e.g., harvesting) from the production supernatant and further separation or purification steps (e.g., post-harvest) are stages after the end of the production phase.

[0056] As used herein, a production mixture refers to a mixture of a product (e.g., a recombinant polypeptide), a growth medium, and cells (e.g., at least one cell, e.g., a recombinant cell, e.g., a recombinant host cell capable of expressing a product). A production cell is a recombinant host cell capable of expressing a product (e.g., a recombinant polypeptide).

[0057] As used herein, operably coupled describes the relationship between containers. In one embodiment, substratum or production culture can be transmitted between operably coupled containers. In one embodiment, the flow of culture between operably coupled containers is carried out in a controlled manner, for example, using pumps, filters, sensors, devices for maintaining or changing culture conditions and / or devices for monitoring liquid flow.

[0058] As used herein, operably connected describes the relationship between a first nucleic acid sequence (e.g., a promoter or enhancer sequence) and a second nucleic acid sequence (e.g., a sequence encoding a protein), wherein the first nucleic acid sequence can, for example, affect the second nucleic acid sequence by affecting transcription, epigenetic modification and / or chromosome topology. In certain embodiments, operably connected refers to comprising two nucleic acid sequences on the same nucleic acid molecule. In another embodiment, operably connected can further refer to two nucleic acid sequences being adjacent to each other on the same nucleic acid molecule, for example, within 1000, 500, 100, 50 or 10 base pairs or directly adjacent to each other. In one embodiment, a promoter or enhancer sequence operably connected to a sequence encoding a protein can, for example, promote the transcription of a sequence encoding a protein in a cell or cell-free system that can be transcribed.

[0059] In some embodiments, the methods of the present disclosure include or the bioreactors of the present disclosure are capable of maintaining the production culture, cells of the production culture, or supernatant from the production culture at different temperatures throughout the different stages. As used herein, T 培养 It is the temperature from the start of inoculation to the end of the production phase, e.g., the temperature at which cells of a production culture are grown in a growth medium to produce a production culture in which protein is produced. In some embodiments, T 培养 is 30°C to 38°C. As used herein, T 生产 is the first temperature to which the production culture is cooled after the post-exponential growth phase. In some embodiments, T 生产 Less than 35°C, 34°C, 33°C, 32°C, 31°C or 30°C, such as 30°C-33°C. As used herein, T 收获 T is the second temperature to which the production culture is cooled prior to harvesting (e.g., separating cells from the supernatant). 收获 In one embodiment, T 收获 is 12°C-18°C, for example 15°C. In one embodiment, T 收获 Less than 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C or 24°C. In one embodiment, T 收获 It is 15℃±3℃.

[0060] As used herein, the term, Cas9 molecule or Cas9 polypeptide refers to a molecule or polypeptide that can interact with a guide RNA (gRNA) molecule and synergize with a gRNA molecule to home or locate to a site including a target domain and a PAM sequence. As used herein, the terms, Cas9 molecule and Cas9 polypeptide include naturally occurring Cas9 molecules and engineered, altered or modified Cas9 molecules or Cas9 polypeptides (which differ from a reference sequence (e.g., the most similar naturally occurring Cas9 molecule or sequence) by, for example, at least one amino acid residue). Exemplary Cas9 molecules or Cas9 polypeptide sequences can be found in WO2015 / 157070, which is incorporated by reference as a whole. Cas9 molecules or Cas9 polypeptides include Cas9 molecules with DNA shearing and cutting activity.

[0061] The disclosure of each patent, patent application and publication cited herein is hereby incorporated by reference in its entirety. Although the present invention has been disclosed with reference to specific aspects, it is apparent that other technical personnel in the art can design other aspects and variations of the present invention without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such aspects and equivalent variations.

[0062] Reduces levels of endogenous proteins

[0063] The present disclosure relates in part to a method for manufacturing a product (e.g., a recombinant polypeptide) in a cell (e.g., a production cell), wherein the method includes reducing the level of one or more endogenous proteins in the cell (e.g., a production cell). Without wishing to be bound by theory, it is believed that one or more production cell properties (e.g., specific product production rate (qP), growth rate, product yield, and / or viable cell concentration) can be improved by reducing the level of one or more endogenous proteins. Endogenous proteins may consume gaps and / or resources (e.g., translation, post-translational processing, and secretion gaps / resources) in the endoplasmic reticulum or Golgi apparatus, thereby reducing the ability of production cells to produce products (e.g., recombinant polypeptides). In an artificial environment (e.g., bioreactor and cell culture) of biomanufacturing, endogenous proteins may also be mutually redundant in terms of the function of the cell (e.g., production cell) or unnecessary for cell viability or growth.

[0064] In some embodiments, reducing the level of one or more endogenous proteins in the cell (e.g., a production cell) comprises reducing the level of the one or more endogenous proteins by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% relative to a similar cell in which the level of the one or more endogenous proteins is not reduced. In some embodiments, reducing the level of one or more endogenous proteins in the cell (e.g., a production cell) comprises substantially eliminating all endogenous proteins (e.g., all of the one or more endogenous proteins) from the cell (e.g., a production cell). The level of the one or more endogenous proteins can be determined, for example, by Porter, AJ et al. (2010), "Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: realizing the potential in bioreactors," Biotechnology Progress ( Biotechnol Prog )》26(5):1446-1454.

[0065] In some embodiments, reducing the level of one or more endogenous proteins in a cell (e.g., a production cell) can improve qP. As used herein, qP refers to the specific product production rate. In some embodiments, the unit of qP is product per unit time per unit production cell (e.g., grams or viable cell count). In some embodiments, the qP is improved, for example, by at least 1.05-fold, 1.06-fold, 1.07-fold, 1.08-fold, 1.09-fold, 11-fold, 1.12-fold, 1.13-fold, 1.14-fold, 1.15-fold, 1.16-fold, 1.17-fold, 1.18-fold, 1.19-fold, 1.2-fold, 1.25-fold, 1.3-fold, 1.35-fold, 1.4-fold, 1.45-fold, 1.5-fold, 1.55-fold, 1.6-fold, 1.65-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.85-fold, 1.9-fold, 1.95-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a similar cell (e.g., a producer cell) in which the level of the endogenous protein(s) is not reduced. In some embodiments, qP is improved, e.g., by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70% relative to a similar cell (e.g., a producer cell) that has not reduced levels of one or more endogenous proteins. qP can be assessed, e.g., by quantification of the secreted protein product by ELISA or quantitative chromatography (e.g., Protein A HPLC).

[0066] In some embodiments, reducing the level of one or more endogenous proteins in a cell (eg, a producer cell) can improve cell growth, eg, the rate of cell growth and / or division. In some embodiments, cell growth is improved, for example, at least 1.05-fold, 1.06-fold, 1.07-fold, 1.08-fold, 1.09-fold, 1.1-fold, 1.12-fold, 1.13-fold, 1.14-fold, 1.15-fold, 1.16-fold, 1.17-fold, 1.18-fold, 1.19-fold, 1.2-fold, 1.25-fold, 1.3-fold, 1.35-fold, 1.4-fold, 1.45-fold, 1.5-fold, 1.55-fold, 1.6-fold, 1.65-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.85-fold, 1.9-fold, 1.95-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a similar cell (e.g., a producer cell) in which the level of one or more endogenous proteins is not reduced. In some embodiments, cell growth is improved, e.g., by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70% relative to similar cells (e.g., producer cells) whose levels of one or more endogenous proteins are not reduced. Cell growth can be assessed, for example, by measuring the concentration of viable cells over time using an automated trypan blue exclusion assay (e.g., using a Beckman coulter Vi-CELLXR).

[0067] In some embodiments, reducing the level of one or more endogenous proteins in a cell (eg, a producer cell) can improve product yield, eg, the amount of product produced by the culture. In some embodiments, cell growth is improved by at least 1.05-fold, 1.06-fold, 1.07-fold, 1.08-fold, 1.09-fold, 1.1-fold, 1.12-fold, 1.13-fold, 1.14-fold, 1.15-fold, 1.16-fold, 1.17-fold, 1.18-fold, 1.19-fold, 1.2-fold, 1.25-fold, 1.3-fold, 1.35-fold, 1.4-fold, 1.45-fold, 1.5-fold, 1.55-fold, 1.6-fold, 1.65-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.85-fold, 1.9-fold, 1.95-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a similar culture of similar cells (e.g., producer cells) that do not have reduced levels of one or more endogenous proteins. In some embodiments, product yield is improved by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70% relative to a similar culture of similar cells (e.g., producer cells) that do not have reduced levels of one or more endogenous proteins. Product yield can be assessed, for example, by quantification of the secreted protein product by ELISA or quantitative chromatography (e.g., Protein A HPLC).

[0068] In some embodiments, reducing the level of one or more endogenous proteins in a cell (e.g., a production cell) can improve qP and cell growth. In some embodiments, reducing the level of one or more endogenous proteins in a cell (e.g., a production cell) can improve qP and product yield. In some embodiments, reducing the level of one or more endogenous proteins in a cell (e.g., a production cell) can improve product yield and cell growth. In some embodiments, reducing the level of one or more endogenous proteins in a cell (e.g., a production cell) can improve qP, product yield, and cell growth.

[0069] Endogenous proteins

[0070] Endogenous proteins whose levels may be reduced in the methods described herein include those that are non-essential (e.g., for cell growth and / or viability, such as in a biomanufacturing environment), those that are functionally redundant with other endogenous proteins whose levels may not be reduced, and / or secreted proteins. Based on the disclosure herein and the state of the art, a skilled artisan will be able to identify which endogenous proteins of a cell (e.g., a CHO cell) are non-essential, redundant, and / or secreted proteins with minimal experimentation.

[0071] In some embodiments, the endogenous protein whose level may be reduced is a protein that is expressed at least at a very high level (e.g., as determined by the methods of Example 1). In some embodiments, the endogenous protein whose level may be reduced is a protein that is expressed at least at a high level (e.g., as determined by the methods of Example 1). In some embodiments, the endogenous protein whose level may be reduced is a protein that is expressed at least at a moderate level (e.g., as determined by the methods of Example 1).

[0072] In some embodiments, the endogenous protein whose level may be reduced is a protein associated with a redundant, non-essential or secreted function in, for example, a protein function database (e.g., the PANTHER database of Example 1). The endogenous protein whose level may be reduced is associated with a functional keyword listed in Table E1 or a substantially similar functional association. In some embodiments, the endogenous protein whose level may be reduced is a protein associated with immune function, disease resistance, cell communication and / or secretion.

[0073] In some embodiments, the endogenous protein whose level may be reduced is a protein that is expressed at least at a very high level (e.g., as determined by the method of Example 1) and is associated with, for example, a redundant, non-essential or secreted function in a protein function database (e.g., the PANTHER database of Example 1) (e.g., associated with a keyword of Table E1). In some embodiments, the endogenous protein whose level may be reduced is a protein that is expressed at least at a high level (e.g., as determined by the method of Example 1) and is associated with, for example, a redundant, non-essential or secreted function in a protein function database (e.g., the PANTHER database of Example 1) (e.g., associated with a keyword of Table E1). In some embodiments, the endogenous protein whose level may be reduced is a protein that is expressed at least at a medium level (e.g., as determined by the method of Example 1) and is associated with, for example, a redundant, non-essential or secreted function in a protein function database (e.g., the PANTHER database of Example 1) (e.g., associated with a keyword of Table E1).

[0074] In some embodiments, the endogenous protein whose level may be reduced is not a protein associated with an essential function (e.g., a function that is essential or very necessary for cell growth, viability, or product production). In some embodiments, the endogenous protein whose level may be reduced is not a protein associated with an essential function in, for example, a protein function database (e.g., the PANTHER database of Example 1). In some embodiments, the endogenous protein whose level may be reduced is not associated with a functional keyword listed in Table E2 or a substantially similar functional association. In some embodiments, the endogenous protein whose level may be reduced is not associated with cellular metabolism, mitochondrial function, cell structure, DNA replication, cell division, checkpoint inhibition / passage, transcription, translation, post-translational modification, protein folding, or heat shock response.

[0075] Exemplary endogenous proteins (and / or genes encoding such endogenous proteins) whose levels may be reduced are listed in Table E5.

[0076] In some embodiments, the methods disclosed herein include reducing the level of more than one endogenous protein. In some embodiments, the methods disclosed herein include reducing one, two, three, four, five, six, seven, eight, nine, ten or more (e.g., twenty or more) endogenous proteins (and optionally, up to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 endogenous proteins). In some embodiments, the methods disclosed herein include reducing the level of two or three endogenous proteins (e.g., a combination of endogenous proteins selected from the combinations of Table E6). In some embodiments, the methods disclosed herein include reducing the level of two or three endogenous proteins (e.g., a combination of endogenous proteins selected from those listed in Table E5).

[0077] Methods for reducing levels of endogenous proteins

[0078] The present disclosure relates in part to a method for manufacturing a product (e.g., a recombinant polypeptide) in a cell (e.g., a production cell), wherein the method includes reducing the level of one or more endogenous proteins in the cell (e.g., a production cell). In some embodiments, reducing the level of an endogenous protein in the cell (e.g., a production cell) includes eliminating (e.g., knocking out) a copy of the gene encoding the endogenous protein. In some embodiments, reducing the level of an endogenous protein in the cell (e.g., a production cell) includes eliminating (e.g., knocking out) all (e.g., two) copies of the gene encoding the endogenous protein from the genome of the cell. In some embodiments, reducing the level of an endogenous protein in the cell (e.g., a production cell) includes eliminating (e.g., knocking out) a regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein. In some embodiments, reducing the level of an endogenous protein in the cell (e.g., a production cell) includes eliminating (e.g., knocking out) all (e.g., two) copies of a regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein.

[0079] In some embodiments, elimination (e.g., knockout) includes introducing a deletion, substitution, or insertion mutation into the gene encoding the endogenous protein or the regulatory nucleic acid sequence (e.g., promoter or enhancer) operably coupled to the gene encoding the endogenous protein, for example, wherein the mutation reduces the expression level of the endogenous protein. In some embodiments, elimination (e.g., knockout) includes introducing a deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more base pairs into the gene encoding the endogenous protein or the regulatory nucleic acid sequence (e.g., promoter or enhancer) operably coupled to the gene encoding the endogenous protein. In some embodiments, elimination (e.g., knockout) comprises replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more base pairs with different base pairs (e.g., transition or transversion mutations) in said gene encoding said endogenous protein or in said regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to said gene encoding said endogenous protein.

[0080] For example, the insertion mutation that destroys the reading frame of the gene encoding the endogenous protein or the substitution mutation that changes the start codon of the gene encoding the endogenous protein are all examples of the elimination (for example, knocking out) that reduces the level of endogenous protein.As another example, eliminating (for example, knocking out) can include inserting a marker (for example, antibiotic resistance, auxotrophic mitigation or fluorescent marker encoding gene) in a manner that a missing promoter or promoter is operably connected to the part of the gene encoding the endogenous protein.In certain embodiments, eliminating (for example, knocking out) causes the complete loss of the endogenous protein produced by a copy of the gene encoding the endogenous protein.In other embodiments, eliminating (for example, knocking out) causes the lower level production of the endogenous protein (for example, functional endogenous protein).In other embodiments, eliminating (for example, knocking out) causes the production of the variant of the endogenous protein, and the variant is one or more of the following: brachymemma, low function, non-functional, misfolded and / or easy to degrade.

[0081] Elimination (e.g., knockout) of a gene encoding an endogenous protein or a copy of a regulatory element operably linked to the gene can be achieved by any gene editing system known in the art. Exemplary gene editing systems include clustered regularly interspaced short palindromic repeats (CRISPR) systems, zinc finger nucleases (ZFNs), and nucleases (TALENs) based on transcription activator-like effectors. ZFNs, TALENs, and CRISPR-based methods are described, for example, in Gaj et al., Trends Biotechnol, 31.7 (2013): 397-405; CRISPR methods for gene editing are described, for example, in Guan et al., Application of CRISPR-Cas system in gene therapy: Pre-clinical progress in animal model, DNA Repair, July 30, 2016 [epub ahead of print]; Zheng et al., Precise gene deletion and replacement using the CRISPR / Cas9 system in human cells, BioTechniques, Vol. 57, No. 3, September 2014, pp. 115-124. Those skilled in the art will be aware of these and other options for editing the genome (eg, knocking out a gene) in a cell (eg, a producer cell).

[0082] In some embodiments, eliminating (e.g., knocking out) a copy of a gene encoding an endogenous protein or a regulatory element operably linked to the gene comprises using a CRISPR-Cas9 molecule (e.g., a Cas9 molecule) in combination with an RNA (e.g., a gRNA, sgRNA, and / or tracrRNA) specific for the endogenous protein encoding gene or a regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein.

[0083] In some embodiments, reducing the level of an endogenous protein in the cell (e.g., a production cell) includes reducing the level of an mRNA transcript encoding the endogenous protein in the cell (e.g., knocking out). In some embodiments, reducing the level of an mRNA transcript encoding the endogenous protein (e.g., knocking out) includes using siRNA, which is, for example, capable of binding to a nucleic acid (e.g., an mRNA encoding a regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably linked thereto). One skilled in the art will know the tools and techniques for designing siRNA and delivering it to cells. Such tools and / or techniques include, but are not limited to, those taught by Dharmacon Horizon siDesign tools, InvivoGen siRNA Wizard, GenScript siRNA Construct Services, IDT CustomDicer-Substrate siRNA, Sigma-Aldrich siRNA Design Service, or www.rnaiweb.com / RNAi / siRNA_Design / . In some embodiments, reducing the level of (e.g., knocking out) an mRNA transcript encoding the endogenous protein results in at least a 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% reduction in the level of an mRNA transcript encoding the endogenous protein in the cell.

[0084] product

[0085] Provided herein are methods, cells or cell lines, and compositions for producing high yields of products and / or improving product quality (e.g., increasing the level of a product (e.g., a recombinant polypeptide produced)) by reducing the level of an endogenous protein in a cell (e.g., a production cell). The products described herein include polypeptides, such as recombinant proteins, multimeric proteins, or complexes; lipid-encapsulated particles (e.g., virus-like particles), vesicles, or exosomes; or other molecules; or other molecules. In one embodiment, the product is a polypeptide, such as a recombinant polypeptide. In one embodiment, the product is an exosome. For example, the recombinant polypeptide can be a protein that is difficult to express or a protein with a complex and / or non-native structure, such as a next-generation biologic (e.g., a bispecific antibody molecule, a fusion protein, or a glycosylated protein).

[0086] In an embodiment, the cell or cell line generated by the methods or compositions described herein produces a product (e.g., a recombinant polypeptide) that can be used to treat a medical condition, disorder, or disease. Examples of medical conditions, disorders, or diseases include, but are not limited to, metabolic diseases or disorders (e.g., metabolic enzyme deficiency), endocrine disorders (e.g., hormone deficiency), hemostatic disorders, thrombosis, hematopoietic disorders, pulmonary disorders, gastrointestinal disorders, autoimmune diseases, immune disorders (e.g., immunodeficiency), infertility, transplantation, cancer, and infectious diseases.

[0087] In an embodiment, the product is an exogenous protein, such as a protein that is not naturally expressed by the cell. In one embodiment, the protein is from one species and the cell is from a different species. In another embodiment, the protein is a non-naturally occurring protein.

[0088] In other embodiments, the product is a protein endogenously expressed by the cell. In one embodiment, the product is a protein endogenously expressed by the cell at an endogenous or native level. The present methods and compositions described herein are used to increase the production and quality of endogenous products (e.g., naturally occurring products produced naturally by the cell). In another embodiment, an exogenous nucleic acid encoding the product (e.g., protein) is introduced into the cell and expressed by the cell. In another embodiment, an exogenous nucleic acid that increases the expression of a product endogenously expressed by the cell is introduced into the cell. For example, the exogenous nucleic acid includes a sequence that activates a promoter that controls the expression of an endogenous product of the cell.

[0089] The recombinant product can be a therapeutic product or a diagnostic product, for example, can be used for drug screening. The therapeutic or diagnostic product can include but is not limited to antibody molecules, such as antibodies or antibody fragments, fusion proteins, hormones, cytokines, growth factors, enzymes, glycoproteins, lipoproteins, reporter proteins, therapeutic peptides or any of these structural and / or functional fragments or hybrids. In other embodiments, the therapeutic or diagnostic product is a synthetic polypeptide, such as wherein the entire polypeptide or a portion thereof is not derived from any naturally occurring polypeptide (e.g., the above-mentioned naturally occurring polypeptide) or does not have any sequence or structural similarity thereto.

[0090] In one embodiment, the recombinant product is an antibody molecule. In one embodiment, the recombinant product is a therapeutic antibody molecule. In another embodiment, the recombinant product is a diagnostic antibody molecule, such as a monoclonal antibody that can be used for imaging techniques or diagnostic tests.

[0091] As used herein, an antibody molecule is a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. In one embodiment, the antibody molecule is a full-length antibody or an antibody fragment. Antibodies and polymorphic proteins can be polyclonal or monoclonal, multi-chain or single-chain or complete immunoglobulins, and can be derived from natural sources or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. In one embodiment, the antibody is a monoclonal antibody. The antibody can be a human or humanized antibody. In one embodiment, the antibody is an IgA, IgG, IgD or IgE antibody. In one embodiment, the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

[0092] "Antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen binding domain (e.g., an antigen-determining variable region of an intact antibody) sufficient to confer recognition and specific binding of the antibody fragment to a target (e.g., an antigen). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies (e.g., sdAb (VL or VH)), Camelidae VHH domains, and multispecific antibodies formed by antibody fragments (e.g., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), and isolated CDRs or other epitope-binding fragments of antibodies. Antigen binding fragments may also be incorporated into single domain antibodies, large antibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23: 1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides, such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0093] In embodiments, the polypeptide is, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alfa, daptomycin, YH-16, chorionic gonadotropin alfa, filgrastim, cetrorelix, interleukin-2, aldesleukin, tesileukin, denileukin-toxin conjugate, interferon alpha-n3 (injection), interferon alpha-nl, DL-8234, interferon, Suntory (gamma-la), interferon gamma, thymosin alpha 1, tasonamine, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif, etortermin alfa , teriparatide, calcitonin, etanercept, glutelin 250 (bovine), drocoquinol α, collagenase, caperitide, recombinant human epidermal growth factor, DWP401, darbepoetin α, epoetin ω, epoetin β, epoetin α, desirudin, lepirudin, bivalirudin, nonacog α, mononine, epoetin α (activated), recombinant factor VIII + VWF, recombinate, recombinant factor VIII, factor VIII (recombinant), Alphnmate, octocog α, factor VIII, palifermin, indikinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplasm, monteplase, follicle stimulating hormone α, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, imiglucerase, galactase, leucotropin, moraspgrastim, triptorelin acetate, histrelin (Hydron), deserelin, histrelin, nafarelin, leuprorelin (ATRIGEL), leuprorelin (DUROS), goserelin, Eutropin, somatotropin, mecasermin, envavirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhaled), insulin lispro, insulin detemir, insulin (RapidMist), mecasermin-linfilpep, anakinra, simoleukin, 99mTc-anakinra Western peptide, myelopid, Betaseron, Glatiramer acetate, Gepon, Sargramostim, Oprelvekin, human leukocyte-derived interferon alpha, Bilive, insulin (recombinant), recombinant human insulin, insulin aspart, mecasermin, Roferon-A, interferon-alpha 2, Alfaferone, interferon alfacon-1, interferon alpha, Avonex recombinant human luteinizing hormone, streptodorn alpha, trefermin, ziconotide, tartrelin, dibotermin alpha, atosiban, becaplermin, eptifibatide, Zemaira, CTC-111, Shanvac-B, octreotide, lanreotide, ancistim, galactosidase β, galactosidase alpha,laronidase, copper acetate peptide, rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant human parathyroid hormone (PTH) 1-84, epoetin delta, transgenic antithrombin III, Granditropin, Vitrase, recombinant insulin, interferon-α, GEM-21S, vapreotide, idoximetra, omapatrilat, recombinant serum albumin, certolizumab pegol, glutaric acid, human recombinant C1 esterase inhibitor, laronidase, recombinant human growth hormone, enfuvirtide , VGV-1, interferon (α), lucinatan, actidil, icatibant, icaraltide, omeganam, Aurograb, pexiganam acetate, ADI-PEG-20, LDI-200, degarelix, bexinleukin, Favld, MDX-1379, ISAtx-247, liraglutide, teriparatide, tefacolin, AA4500, T4N5 liposome lotion, catumaxomab, DWP413, ART-123, Chrysalin, desmoteplase, andeplase, choriofollicle stimulating hormone α, TH- 9507, Teduglutide, Diamyd, DWP-412, growth hormone, recombinant G-CSF, insulin, insulin (Technosphere), insulin (AERx), RGN-303, DiaPep277, interferon beta, interferon alpha-n3, Belatacept, transdermal insulin patch, AMG-531, MBP-8298, Xerecept, Opebacon, AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, Lusupeptide, MP 52, Sipuluse-T, CTP-37, Insegia, Vitspon, human thrombin, thrombin, TransMID, Afiplase, Puricere, Terlipressin, EUR-1008M, recombinant FGF-1, BDM-E, Rotigatide, ETC-216, P-113, MBI-594AN, Duramycin, SCV-07, OPI-45, Endostatin, Angiostatin, ABT-510, Bowman-Berkeley inhibitor, XMP-629, 99mTc-Hynic-Annexin V, Kahalide F, CTCE-9908, Tevrek, Ozerik, Romidepsin, BAY-504798, Interleukin-4, PRX-321, Pepscan, Apo interleukin, rhlactoferrin, TRU-015, IL-21, ATN-161, Cilengitide, Albuferon, Biphasix, IRX-2, Omega interferon, PCK-3145, CAP-232, Pasireotide, huN901-DMI, SB-249553, Oncovax-CL, OncoVax-P, BLP-25,CerVax-16, MART-1, gp100, tyrosinase, nanofibrin, rAAT, CGRP, penacept, thymosin β4, propidecin, GTP-200, ramuplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (eligen), esarelin, caprelin, Cardeva, vilafumin, 131I-TM-601, KK-220, T-10, ularitide, dilestat, hemotide, Chrysalin, rNAPc2, recombinant factor VI11 (PEGylated liposomes), bFGF, PEGylated recombinant staphylococcal kinase variant, V-10153, SonoLysis Prolyse, NeuroVax, CZEN-002, rGLP-1, BIM-51077, LY-548806, exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, avorelin, ACM-9604, linaclotide acetate, CETi-1, Hemospan, VAL, rapid-acting insulin (injection, Viadel), insulin (eligen), recombinant methionyl human leptin, pitrakinra, Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10, talactoferrin, rEV-131, rEV-131, recombinant human insulin, RPI-78M, oprelleukin, CYT-99007 CTLA4-Ig, DTY-001, Valast, Interferon α-n3, IRX-3, RDP-58, Tauferon, Bile Salt-Stimulated Lipase, Merspase, Alkaline Phosphatase, EP-2104R, Melantan-II, Bremenotriptyline, ATL-104, Recombinant Human Microplasmin, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, Dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, ALTU-135, recombinant neuraminidase, Vacc-5q, Vacc-4x, Tat toxoid, YSPSL, CHS-13340, PTH (l-34) (Novasome), Ostabolin-C, PTH analogs, MBRI-93.02, MTB72F, MVA-Ag85A, FARA04, BA-210, recombinant plague F1V, AG-702, OxSODrol, rBetV1, Der-pl / Der-p2 / Der-p7, PR1 peptide antigen, mutant ras vaccine, HPV-16 E7 lipopeptide vaccine, labyrinth protein, WT1-peptide, IDD-5, CDX-110, Pentrys,Norelin, CytoFab, P-9808, VT-111, Alocartide, Tiberium, Lupintravir, Ralticur, rGRF, HA, α-galactosidase A, ACE-011, ALTU-140, CGX-1160, Angiotensin, D-4F, ETC-642, APP-018, rhMBL, SCV-07, DRF-7295, ABT-828, ErbB2-specific immunotoxin, DT3SSIL-3, TST-10088, PRO-1762, Combotox, Cholecystokinin-B / Gastrin Receptor Binding Peptide, lllIn-hEGF, AE-37, Trastuzumab-DM1, Antagonist G , IL-12, PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647, L-19-based RA, Re-188-P-2045, AMG-386, DC / 1540 / KLH, VX-001, AVE-9633, AC-9301, NY-ESO-1 (peptide), NA17.A2 peptide, CBP-501, recombinant human lactoferrin, FX-06, AP-214, WAP-8294A, ACP-HIP, SUN-11031, peptide YY[3-36], FGLL, atacicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 , F-18-CCR1, AT-1100, JPD-003, PTH (7-34) (Novasome), duramycin, CAB-2, CTCE-0214, glycoPEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, factor XIII, aminocontin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, tevirix, EP-51216, hGH, OGP-1, sifuvirtide, TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin, r(m)CRP , liver-selective insulin, Subolin, L19-IL-2 fusion protein, elastin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist, AL-108, AL-208, nerve growth factor antagonist, SLV-317, CGX-1007, INNO-105, teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion, EP-1043, gpEl, gpE2, MF-59, hPTH (l-34), 768974, SYN-101, PGN-0052, avicumin, BIM-23190,Multi-epitope tyrosinase peptides, Encastin, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular-targeted TNF, desmopressin, Onercept, and TP-9201.

[0094] In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE TM ), Rituximab (RITUXAN TM / MAB THERA TM ), ENBREL TM ), bevacizumab (AVASTIN TM ), HERCEPTIN TM ), Pegfilgrastim (NEULASTA TM ) or any other suitable polypeptide comprising biosimilars and improved biopharmaceuticals.

[0095] Other suitable polypeptides are those listed below and in Table 1 of US2016 / 0097074:

[0096] Table 1

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] In an embodiment, the polypeptide is a hormone, a blood coagulation / coagulation factor, a cytokine / growth factor, an antibody molecule, a fusion protein, a protein vaccine or a peptide, as shown in Table 2.

[0103] Exemplary recombinant products that can be produced using the methods described herein include, but are not limited to, those provided in the table below.

[0104] Table 2. Exemplary products

[0105]

[0106]

[0107] In an embodiment, the protein is a multispecific protein, such as a bispecific antibody as shown in Table 3.

[0108] Table 3: Bispecific formats

[0109]

[0110]

[0111]

[0112]

[0113] In an embodiment, the product is a polypeptide listed in Table 4.

[0114] Table 4

[0115]

[0116] Table 4

[0117]

[0118] Table 4

[0119]

[0120] Table 4

[0121]

[0122] In some embodiments, the polypeptide is an antigen expressed by a cancer cell. In some embodiments, the recombinant or therapeutic polypeptide is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the recombinant or therapeutic polypeptide is selected from HER2, CD20, 9-O-acetyl-GD3, βhCG, A33 antigen, CA19-9 marker, CA-125 marker, calreticulin, carbonic anhydrase IX (MN / CAIX), CCR5, CCR8, CD19, CD22, CD25, CD27, CD30, CD33, CD38, CD44v6, CD63, CD70, CC123, CD138, carcinoembryonic antigen (CEA; CD66e), desmoglein 4, E-cadherin neo-epitope, endosialin, ephrin A2 (EphA2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), ErbB2, fetal acetylcholine receptor, fibroblast activation antigen (FAP), fucosyl GM1, GD2, GD3, GM2, ganglioside GD3, Globo H, glycoprotein 100, HER2 / neu, HER3, HER4, insulin-like growth factor receptor 1, Lewis-Y, LG, Ly-6, melanoma-specific chondroitin sulfate proteoglycan (MCSCP), mesothelin, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC5b, MUC7, MUC16, type II Miller inhibitor (MIS) receptor, plasma cell antigen, poly SA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-α precursor, or a combination thereof.

[0123] In some embodiments, the polypeptide is an activating receptor and is selected from 2B4 (CD244), α4β1 integrin, β2 integrin, CD2, CD16, CD27, CD38, CD96, CD100, CD160, CD137, CEACAM1 (CD66), CRTAM, CS1 (CD319), DNAM-1 (CD226), GITR (TNFRSF18), an activated form of KIR, NKG2C, NKG2D, NKG2E, one or more natural cytotoxicity receptors, NTB-A, PEN-5, and combinations thereof, optionally wherein the β2 integrin comprises CD11a-CD18, CD11b-CD18, or CD11c-CD18, optionally wherein the activated form of KIR comprises K1R2DS1, KIR2DS4, or KIR-S, and optionally wherein the natural cytotoxicity receptor comprises NKp30, NKp44, NKp46, or NKp80.

[0124] In some embodiments, the polypeptide is an inhibitory receptor and is selected from KIR, ILT2 / LIR-1 / CD85j, an inhibitory form of KIR, KLRG1, LAIR-1, NKG2A, NKR-P1A, Siglec-3, Siglec-7, Siglec-9, and combinations thereof, optionally wherein the inhibitory form of KIR comprises KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, or KIR-L.

[0125] In some embodiments, the polypeptide is an activating receptor and is selected from CD3, CD2 (LFA2, OX34), CD5, CD27 (TNFRSF7), CD28, CD30 (TNFRSF8), CD40L, CD84 (SLAMF5), CD137 (4-1BB), CD226, CD229 (Ly9, SLAMF3), CD244 (2B4, SLAMF4), CD319 (CRACC, BLAME), CD352 (Lyl08, NTBA, SLAMF6), CRT AM (CD355), DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LTβR (TNFRSF3), OX40 (CD134), NKG2D, SLAM (CD150, SLAMF1), TCRα, TCRβ, TCRδγ, TIM1 (HAVCR, KIM1), and combinations thereof.

[0126] In some embodiments, the polypeptide is an inhibitory receptor and is selected from PD-1 (CD279), 2B4 (CD244, SLAMF4), B71 (CD80), B7H1 (CD274, PD-L1), BTLA (CD272), CD 160 (BY55, NK28), CD352 (Lyl08, NTBA, SLAMF6), CD358 (DR6), CTLA-4 (CD 152), LAG3, LAIR1, PD-1H (VISTA), TIGIT (VSIG9, VSTM3), TIM2 (TIMD2), TIM3 (HAVCR2, KIM3) and combinations thereof.

[0127] Other exemplary proteins include, but are not limited to, any of the proteins described in Tables 1-10 of Leader et al., "Protein therapeutics: a summary and pharmacological classification," Nature Reviews Drug Discovery, 2008, 7:21-39, incorporated herein by reference; or any conjugate, variant, analog, or functional fragment of a recombinant polypeptide described herein.

[0128] Other recombinant protein products include non-antibody scaffolds or alternative protein scaffolds, such as, but not limited to, DARPins, affibodies, and adnectins. Such non-antibody scaffolds or alternative protein scaffolds can be engineered to recognize or bind to one or two or more (e.g., 1, 2, 3, 4, or 5 or more) different targets or antigens.

[0129] In one embodiment, the vector comprising a nucleic acid sequence encoding a product described herein (e.g., a polypeptide, such as a recombinant polypeptide) further comprises a nucleic acid sequence encoding a selection marker. In one embodiment, the selectable marker comprises glutamine synthetase (GS); dihydrofolate reductase (DHFR), such as an enzyme that confers resistance to methotrexate (MTX); proline or antibiotic markers, such as enzymes that confer resistance to antibiotics (e.g., hygromycin, neomycin (G418), bleomycin, puromycin, or blasticidin). In another embodiment, the selection marker comprises a Selexis selection system (e.g., SUREtechnology Platform commercially available from Selexis SA). TM and Selexis Genetic Elements TM ) or Catalant Biologies Cell line development technology or is compatible with it.

[0130] In one embodiment, the carrier comprising the nucleic acid sequence encoding the recombinant product as described herein comprises a selection marker, which can be used to identify one or more cells comprising the nucleic acid encoding the recombinant product as described herein. In another embodiment, as described herein, the selection marker can be used to identify one or more cells comprising the nucleic acid sequence encoding the recombinant product being integrated into the genome. The identification of one or more cells having integrated the nucleic acid sequence encoding the recombinant protein can be used for the selection and engineering of cells or cell lines stably expressing the product.

[0131] In one embodiment, the product differs from a polypeptide from Tables 1-4 at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues. In another embodiment, the product differs from a polypeptide from Tables 2 or 3 at no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% of its amino acid residues. Methods for determining percent identity are discussed above.

[0132] Other recombinant products include non-antibody scaffolds or alternative protein scaffolds, such as but not limited to: DARPins, affibodies, and antibody-mimicking protein drugs.

[0133] Other exemplary therapeutic or diagnostic proteins include, but are not limited to, any protein described in Tables 1-10 of Leader et al., "Protein therapeutics: a summary and pharmacological classification," Nature Reviews Drug Discovery, 2008, 7:21-39, and Walsh, "Biopharmaceutical benchmarks 2014," Nature Biotechnology, 2014, 32:992-1000, each of which is incorporated herein by reference; or any conjugate, variant, analog, or functional fragment of a recombinant polypeptide described herein.

[0134] Production Application

[0135] Methods and cells or cell lines disclosed herein can be used to produce various products, evaluate various cell lines, or evaluate the production of various cell lines for bioreactors or processing vessels or tanks, or more generally used with any supply source. Devices, facilities and methods described herein are suitable for cultivating any desired cell line, including prokaryotic and / or eukaryotic cell lines. In addition, in an embodiment, the device, facilities and methods are suitable for cultivating suspended cells or anchorage-dependent (adhesive) cells, and are suitable for being configured to be used for the production of drugs and biopharmaceutical products (e.g., polypeptide products or cells and / or viruses, such as those used in cell and / or virotherapy) production operations.

[0136] As mentioned, in an embodiment, the apparatus, facilities and methods allow for the production of eukaryotic cells, e.g., mammalian cells or lower eukaryotic cells (such as, for example, yeast cells or filamentous fungal cells), or prokaryotic cells (e.g., Gram-positive or Gram-negative cells) and / or eukaryotic or prokaryotic cell products (e.g., proteins, peptides, antibiotics, amino acids synthesized on a large scale by eukaryotic cells). Unless otherwise specified herein, the apparatus, facilities and methods can include any desired volume or production capacity, including but not limited to bench-scale, pilot-scale and full production scale capabilities.

[0137] In addition and unless otherwise indicated, the devices, facilities and methods may include any one or more suitable reactors, including but not limited to stirred tanks, airlift, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds and / or spouted bed bioreactors. As used herein, a "reactor" or "bioreactor" may include a fermentor or fermentation unit or any other reaction vessel, and the terms "reactor" and "bioreactor" may be used interchangeably with "fermentor". For example, in some aspects, a bioreactor unit may carry out one or more or all of the following: supply of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inflow and outflow of fermentation or cell culture media, separation of gas phases and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring) and / or cleaning / disinfection. Exemplary reactor unit (for example, fermentation unit) can contain multiple reactors in the unit, for example, the unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 or more bioreactors in each unit, and / or facilities can contain multiple units with one or more reactors in the facility. In various embodiments, the bioreactor can be suitable for batch, semi-batch feeding, batch feeding, perfusion and / or continuous fermentation process. Any suitable reactor diameter can be used. In an embodiment, the bioreactor can have a volume of about 100mL and about 50,000L. Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 6 In some embodiments, the reactor can have a volume of 100, 200, 300, 400, 500, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, and / or 50,000 liters. In addition, suitable reactors can be multi-use, single-use, disposable, or non-disposable, and can be formed of any suitable material, including metal alloys (e.g., stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel), plastics, and / or glass. In some embodiments, suitable reactors can be round (e.g., cylindrical).In some embodiments, suitable reactors can be square (e.g., rectangular). In some cases, square reactors provide advantages over circular reactors, such as ease of use (e.g., loading and setup by a technician), greater mixing and uniformity of reactor contents, and lower floor space.

[0138] In an embodiment and unless otherwise specified herein, the device, facilities and methods described herein for use with the method for manufacturing a preparation may also include any suitable unit operation and / or equipment not otherwise mentioned, such as operations and / or equipment for separating, purifying and separating such products. Any suitable facilities and environment may be used, such as traditional component facilities, modular removable temporary facilities, or any other suitable construction, facilities and / or layout. For example, in some embodiments, a modular clean room may be used. In addition and unless otherwise specified, the device, system and method described herein may be accommodated and / or carried out in a single location or facility, or may alternatively be accommodated and / or carried out in separate or multiple locations and / or facilities.

[0139] By way of non-limiting example and not limitation, U.S. Publication Nos. 2013 / 0280797; 2012 / 0077429; 2011 / 0280797; 2009 / 0305626; and U.S. Patent Nos. 8,298,054; 7,629,167; and 5,656,491 (which are hereby incorporated by reference in their entireties) describe exemplary facilities, devices and / or systems that may be suitable.

[0140] Bioreactor setup and conditions

[0141] In one aspect, a bioreactor of the present disclosure or a bioreactor utilized by a method of the present disclosure is a single use bioreactor.

[0142] A single-use bioreactor may comprise a bioprocess container, a housing, at least one agitator, at least one sparger, at least one gas filter inlet for one or more of the spargers and headspace coverage, at least one fill port, at least one harvest port, at least one sample port, and at least one probe.

[0143] In one embodiment, the present disclosure relates to a bioreactor comprising a bioprocess container. The bioprocess container is made of a liquid-impermeable and flexible shape-conforming material. For example, the bioprocess container can be made of a flexible film (e.g., a multilayer film). For example, in one embodiment, the film comprises a polyethylene polymer, such as a low-density polyethylene that has been modified to form a hydrophilic surface. The hydrophilic surface is used to contact the cell culture in the bioreactor and improve wettability. In one embodiment, the polyethylene polymer is modified by being subjected to irradiation, light or plasma induction, or oxidation.

[0144] The bioprocess vessel may have a top, a bottom, and at least one sidewall therebetween. The bioprocess chamber may define a hollow housing for containing a culture medium. The hollow housing may have any suitable volume, for example, 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters , 550 litres, 600 litres, 650 litres, 700 litres, 750 litres, 800 litres, 850 litres, 900 litres, 950 litres, 1000 litres, 1500 litres, 2000 litres, 2500 litres, 3000 litres, 3500 litres, 4000 litres, 4500 litres, 5000 litres, 6000 litres, 7000 litres, 8000 litres, 9000 litres, 10,000 litres, 15,000 litres, 20,000 litres and / or 50,000 litres.

[0145] The bioreactor may include at least one inlet for feeding material into the hollow housing of the bioprocess vessel. A mixing device including a rotatable shaft coupled to at least one agitator may extend into the hollow housing of the bioprocess vessel. In one embodiment, the rotatable shaft may be retractable. For example, the rotatable shaft may include at least one impeller made of a hydrophilic polymer material that may be retracted or folded toward the rotatable shaft.

[0146] The bioreactor may also include at least one baffle configured to extend adjacent to the sidewall of the bioprocess container in the longitudinal direction. The baffle may have a shape extending radially inward from the sidewall, and its extension is sufficient to affect the flow of fluid in the hollow shell during mixing of the culture medium by the mixing device. The baffle may be retractable and / or foldable. For example, in one embodiment, the baffle may define an inflatable fluid sac so that the baffle can be inflated and deflated. The baffle may be integral with the bioprocess container, which means that the baffle is formed as a flexible shape forming material. Alternatively, the baffle may be separated from the bioprocess container. The baffle may be configured to be placed inside the hollow shell or may be placed outside the hollow shell. When placed outside the hollow shell, the sidewall of the bioprocess container is consistent with the shape of the baffle. For example, in one embodiment, the baffle may be removably attached to the outer metal shell. The bioprocess container may be placed in a metal shell to be consistent with the shape of the baffle. In one embodiment, the bioreactor may include about two to about six baffles spaced apart around the circumference of the hollow shell of the bioprocess container.

[0147] In one embodiment, the bioprocess container has a diameter and the one or more baffles extend radially inwardly a distance of about 3% to about 20%, such as about 5% to about 15%, of the diameter of the bioprocess container.

[0148] The bioreactor may further include at least one distributor. For example, the distributor may include a ballast distributor including an air tube having a longitudinal portion and a lateral portion. The longitudinal portion may extend vertically into the hollow shell of the bioprocess container. On the other hand, the lateral portion may be located at the end of the longitudinal portion below the agitator. The lateral portion may define a plurality of holes to release gas into the culture medium contained in the bioprocess container. In one embodiment, the plurality of holes are drilled. The lateral portion may have any suitable shape. In one embodiment, the lateral portion may be configured to engage a rotatable shaft of the mixing device to stabilize the shaft. The rotatable shaft may extend through the lateral portion or may be accommodated in a shaft receiving member formed as the lateral portion.

[0149] In one embodiment, the bioreactor comprises a first subsurface distributor and a second surface distributor. The plurality of holes in the subsurface distributor may be larger or smaller than the plurality of holes on the surface distributor. In one embodiment, the plurality of holes are drilled.

[0150] In one embodiment, the bioreactor may include at least one supply line extending into the hollow housing to supply fluid to the bioprocess container. The supply line may include a subsurface fluid outlet positioned adjacent to the agitator. The fluid outlet may be associated with a fluid control device that only allows fluid to flow out of the fluid outlet and prevents fluid from flowing in the opposite direction. For example, the fluid control device may include a one-way valve.

[0151] In another embodiment, the bioreactor can include a supply line located at the top of the bioprocess container. The supply line can include a fluid discharge port located above a volume of culture medium resident in the bioprocess container. The fluid discharge port can be positioned so that the fluid flowing through the fluid discharge port is in direct contact with the culture medium contained in the bioprocess container. In one embodiment, when rotating, the agitator can form a circumference, and the fluid discharge port on the surface of the supply line can be located above the circumference of the agitator so that the fluid flowing through the fluid discharge port contacts the culture medium in the circumference.

[0152] The bioreactor can be placed in operative association with a load cell to indicate the mass of culture medium contained within the hollow housing. The bottom of the bioprocess container can have a convex rounded shape to facilitate drainage. For example, the bioprocess container can include a drain line located at the bottom of the bioprocess container. The fluid collection device can be positioned between the hollow housing of the bioprocess container and the drain line. The fluid collection device can have a shape configured to induce a vortex of fluid from the bioprocess container into the drain line. In one embodiment, the cross-sectional area of ​​the drain line is proportional to the volume of the hollow housing. For example, for exemplary purposes, the drain line can have a cross-sectional area of ​​approximately 0.3 mm for each liter of volume of the hollow housing. 2 To about 0.7mm 2 (For example, about 0.4 mm 2 To about 0.6mm 2 )’s cross-sectional area.

[0153] In one embodiment, the bioprocess container can include multiple ports to connect to multiple supply lines for supplying fluid to the bioprocess container. Each port and corresponding supply line can include matching instructions to help users connect the supply line to the corresponding port. For example, the matching instructions can include colors so that each port and corresponding supply line are color-coded. The matching instructions can also be applied to the supply line and any corresponding port and the distributor and any corresponding connector.

[0154] In one embodiment, the bioprocess container can include a port including a universal connector. The port can have a first end and a second end. The first end can be used to form a reconnectable attachment to a corresponding supply line. Each supply line can include a fluid filter located upstream of the corresponding port.

[0155] The present disclosure also relates to a bioreactor system. The bioreactor system may include a bioprocess container made of a liquid-impermeable and flexible conformable material. The bioprocess container may have a top, a bottom, and at least one sidewall therebetween. The bioprocess chamber may define a hollow shell for containing culture medium. The bioprocess container may also include a plurality of inlets for supplying materials into the hollow shell. A drain line may be located at the bottom of the bioprocess container to drain fluid. A mixing device may extend into the hollow shell of the bioprocess container and may include a rotatable shaft coupled to at least one agitator.

[0156] The bioreactor system may further include at least one sensor operably associated with the bioprocess container to monitor at least one parameter within the hollow shell. The at least one sensor may include a pH sensor, a dissolved carbon dioxide sensor, a dissolved oxygen sensor, a redox sensor, a load cell, a temperature sensor, or a tachometer. A controller may be placed in communication with the at least one sensor. The controller may be configured to receive information from the at least one sensor and control a fluid source based on the information to change the flow rate of fluid from the fluid source into the hollow shell of the bioprocess container to maintain the at least one parameter of the culture medium contained in the hollow shell within a preset limit.

[0157] For example, in one embodiment, the bioreactor system may include a carbon dioxide gas source in fluid communication with the bioprocess container and a liquid alkali source also in fluid communication with the bioprocess container. The at least one sensor may include a pH sensor, and the controller may be configured to adjust the pH level of the culture medium within preset limits by adding an amount of carbon dioxide gas from the carbon dioxide gas source to selectively lower the pH or by adding an amount of alkali from the liquid alkali source to selectively increase the pH. In one embodiment, the system may include a first pH sensor and a second pH sensor, both in communication with the controller.

[0158] In yet another embodiment, the bioreactor system may include an oxygen source, and the at least one sensor may include a dissolved oxygen sensor. The controller may regulate the dissolved oxygen level in the culture medium within preset limits by periodically adding an amount of oxygen from the oxygen source to the culture medium based on information received from the dissolved oxygen sensor.

[0159] In yet another embodiment, the bioreactor system may include a carbon dioxide gas source, and wherein the at least one sensor includes a dissolved carbon dioxide sensor. The controller may be configured to regulate the dissolved carbon dioxide level in the culture medium within preset limits by periodically adding an amount of carbon dioxide gas from the carbon dioxide gas source to the culture medium based on information received from the dissolved carbon dioxide sensor.

[0160] In yet another embodiment, the bioreactor system may include an insulation jacket surrounding the bioprocess container. The insulation jacket may be in fluid communication with at least one of a heating fluid or a refrigeration fluid. The bioreactor system may further include a temperature sensor for sensing the temperature of the culture medium contained in the bioprocess container. The temperature sensor may communicate with the controller. The controller may be configured to receive information from the temperature sensor and control the flow of fluid entering the insulation jacket based on the information to increase or decrease the temperature of the culture medium contained in the bioprocess container to keep the culture medium within a preset temperature limit.

[0161] In another embodiment, the bioreactor system may further include a tachometer to monitor the rotational speed of a rotatable shaft coupled to the at least one agitator. The tachometer may be in communication with the controller. The controller may be in communication with a motor that rotates the shaft. The controller may be configured to control the motor in a manner that rotates the shaft at a predetermined speed based on information received from the tachometer.

[0162] The controller may include one or more microprocessors.

[0163] In one embodiment, the controller may be configured to receive information from a plurality of sensors in order to control a plurality of parameters within the bioreactor.

[0164] In one embodiment, one or more of the above-described sensors may be integrated into a bioprocess container and may be disposable with the bioprocess container.

[0165] The present disclosure also relates to a bioreactor comprising a bioprocess container made of a liquid-impermeable and flexible conformable material. The bioprocess container may have a top, a bottom, and at least one sidewall therebetween. The bioprocess chamber may define a hollow housing for containing a culture medium. At least one supply line may extend into the hollow housing to supply a fluid to the bioprocess container.

[0166] In one embodiment, the supply line comprises a subsurface fluid outlet located adjacent the agitator.The fluid outlet may be associated with a fluid control device that only allows fluid to flow out of the fluid outlet and prevents fluid from flowing in the opposite direction.

[0167] In an alternative embodiment, the supply line can include an on-surface fluid discharge port located above a volume of culture medium residing in the bioprocess container. The on-surface fluid discharge port can be positioned such that fluid flowing through the fluid discharge port is in direct contact with the culture medium contained within the bioprocess container without contacting the sidewalls.

[0168] In one embodiment, the bioreactor may include a first supply line and a second supply line, wherein the first supply line includes a subsurface fluid outlet and the second supply line includes a surface fluid discharge port. In one embodiment, the bioreactor may contain about one to about five (e.g., about two to about three) supply lines having a surface fluid discharge port.

[0169] In yet another embodiment, the present disclosure relates to a method for producing a single-use bioreactor. The method comprises the following steps: constructing a bioprocess container from a liquid-impermeable and flexible conformable material. The bioprocess container has a top, a bottom, and at least one sidewall therebetween. The bioprocess chamber defines a hollow shell for containing a culture medium. The volume of the hollow shell can be about 1-250 milliliters, 250 milliliters to 50 liters, 50 to 800 liters, or 800-200,000 liters. The bioprocess container comprises a plurality of inlets in the hollow shell for feeding materials into the bioprocess container. Each inlet has a certain diameter.

[0170] A mixing device is inserted into the hollow housing. The mixing device includes a rotatable shaft coupled to at least one agitator. At least one distributor is also inserted into the hollow housing of the bioprocess container. The distributor includes an air tube having a longitudinal portion and a lateral portion. The longitudinal portion extends vertically into the hollow housing. The lateral portion is located at an end of the longitudinal portion below the agitator. The lateral portion defines a plurality of holes to release gas into a culture medium contained in the bioprocess container. The plurality of holes have a certain diameter.

[0171] The drain line is connected to the bottom of the bioprocess vessel. The drain line has a certain cross-sectional area.

[0172] According to the present disclosure, the diameter of the inlet, the diameter of the plurality of holes on the distributor, and the cross-sectional area of ​​the drainage line are proportional to the volume of the hollow housing. For each liter of the volume of the hollow housing, the drainage line may have a cross-sectional area of ​​about 0.3 mm. 2 To about 0.7mm 2 cross-sectional area.

[0173] The present disclosure also relates to a bioreactor comprising a bioprocess container made of a liquid-impermeable and flexible conformable material. The bioprocess chamber may define a hollow housing for containing a culture medium and may include at least one inlet. A mixing device including a rotatable shaft coupled to a plurality of agitators may extend into the hollow housing of the bioprocess container.

[0174] According to the present disclosure, the bioreactor may further comprise a cell retention chamber in fluid communication with the hollow housing of the bioprocess container. A filtrate outlet may be placed in fluid communication with the cell retention chamber. The filtrate outlet comprises a biofilter that is permeable to liquid but impermeable to biological material contained in the culture medium. The filtrate outlet is used to continuously or periodically remove liquid from the cell retention chamber. The flow regulator is configured to alternate the flow of the culture medium between the hollow housing of the bioprocess container and the cell retention chamber to perform a perfusion process.

[0175] For example, the flow regulator can be in communication with a pressurized gas source and a vacuum source. The flow regulator can be configured to alternately apply vacuum or gas pressure to the fluid contained in the cell retention chamber to circulate the fluid back and forth between the hollow housing of the bioprocess container and the cell retention chamber.

[0176] In one embodiment, the flow regulator may include a reciprocating diaphragm that alternates between applying pressure and applying suction to the fluid contained in the cell retention chamber.

[0177] The present disclosure also relates to a bioreactor comprising a bioprocess container made of a liquid-tight and flexible conformable material. The bioprocess container defines a hollow housing for containing a culture medium. A mixing device comprising a rotatable shaft coupled to at least one agitator can extend into the hollow housing of the bioprocess container. According to the present disclosure, the agitator can be retracted onto the rotating shaft. For example, the agitator can include an impeller comprising at least one blade element. The blade element can be folded toward the rotatable shaft. In one embodiment, the rotatable shaft is coupled to a first impeller and a second impeller, and the two impellers can include at least one foldable blade element. A buckle can be positioned on the shaft. The buckle can include an agitator engagement position and an agitator disengagement position to respectively maintain the agitator in an upright position (during mixing) or in a retracted and folded position.

[0178] In one embodiment, the rotatable shaft includes a metal reinforcing rod surrounded by a sleeve. The metal reinforcing rod, which may be made of stainless steel, may be made of multiple parts attached together. The top of the reinforcing rod may include a magnetic member for magnetically engaging the motor. The sleeve may include a polymeric material. The agitator on the shaft may also be made of a polymeric material, such as a hydrophilic polymer. For example, the sleeve and the agitator may include a polyethylene polymer that has been modified by being subjected to irradiation, light or plasma induction, or oxidation.

[0179] In one embodiment, a single-use bioreactor system according to the present disclosure includes: a single-use cell culture bioprocess container ("SUB"), a reusable housing in which the SUB is held during operation, and a controller that controls the operation of the SUB and associated subsystems and processes. The associated subsystems include an agitation system, a baffle system, a sparger system, a supply system, a harvesting system, a monitoring system, one or more control systems, and a filling system.

[0180] In one embodiment, each of the cell culture contacting surface and the process fluid contacting surface of the SUB is preferably free of animal derived components.

[0181] According to one aspect of the present disclosure, a single-use bioreactor is provided. The single-use bioreactor may include a bioprocess container, a housing, at least one agitator, at least one sparger, at least one gas filter inlet for one or more sparger and headspace coverage, at least one filling port, at least one harvesting port, at least one sample port, and at least one probe.

[0182] The single-use bioreactors of the present disclosure may also be used with a single-use bioreactor (SUB) system. The system may include a single-use and disposable flexible bioreactor bioprocess container, a SUB housing configured to hold the flexible bioreactor bioprocess container, an agitator, a sparger, a plurality of ports, and at least one controller configured to control a plurality of parameters associated with the SUB system, such that the SUB system produces a biomaterial that corresponds to a biomaterial that can be produced in a stainless steel bioreactor of similar size.

[0183] According to the present disclosure, a rotatable shaft can be coupled to a top impeller and a bottom impeller. Both the top impeller and the bottom impeller can be made of a polymer material. For example, in one embodiment, the impeller can be 3D printed. Both the top impeller and the bottom impeller can define a hydrophilic surface. For example, the polymer material used to form the impeller can include a hydrophilic polymer, or can include a polymer that has been surface-modified to make the surface hydrophilic.

[0184] For example, in one embodiment, the top and bottom impellers are made of a polyolefin polymer (e.g., polyethylene or polypropylene). In one embodiment, low density polyethylene can be used. The low density polyethylene can be modified to form a hydrophilic surface by subjecting it to irradiation, light or plasma induction, or oxidation.

[0185] The top impeller may include a hydrofoil impeller. On the other hand, the bottom impeller may include a four-pitch blade high consistency impeller. The impeller to tank diameter ratio may be about 0.35 to about 0.55, such as about 0.44 to about 0.46. The top impeller and the bottom impeller may have a power factor (N) of about 0.1 to about 0.9.P ), and may have a flow rate number (N) of about 0.4 to about 0.9 q ).

[0186] In some embodiments, the bioreactor of the present disclosure is a bioreactor as described in US Patent No. 9,670,446, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the bioreactor of the present disclosure includes a portion or features of the bioreactor described in US Patent No. 9,670,446.

[0187] In one embodiment, the bioreactor can be operably coupled to a harvesting container (e.g., a harvesting container). The harvesting container can include a device for mixing culture and gas (e.g., aerating the culture), for example to ensure sufficient oxygenation of the culture. In one embodiment, the harvesting container includes a supernatant that has been deposited in the harvesting container (e.g., from a bioreactor); and a headspace that includes a gas (e.g., air, oxygen, or a mixture of air and oxygen). In one embodiment, the harvesting container uses surface aeration to oxygenate the culture supernatant with a headspace gas (e.g., air or oxygen or a mixture of air and oxygen). Surface aeration involves the supernatant flowing or flowing downward along the wall of the harvesting container from the J-tube inlet and from the surface of the supernatant in contact with the headspace. In some embodiments, movement or flowing results in an increase in the oxygenation level of the culture.

[0188] The harvest container can include at least one mixer, at least one gas filter inlet for headspace coverage, at least one fill port with an internal J-tube pointing toward the container wall, at least one harvest port, at least one sample port, at least one temperature probe, at least one redox probe, at least one DOT probe and at least one pH probe, at least one temperature control, at least one gas flow (e.g., at least one air flow control and at least one O2 flow control).

[0189] In one embodiment, the harvest vessel comprises a headspace gas mixture of air / O2 that maintains the DOT in a range of greater than 40% air saturation to less than or equal to 500% air saturation. In one embodiment, the bioreactor or harvest vessel comprises a redox probe, such as an online redox probe, such as a Mettler-Toledo online redox probe.

[0190] In some embodiments, a method includes a bioreactor or harvesting vessel capable of providing (e.g., adding) thioredoxin or a component of a glutathione / glutaredoxin system or modulating (e.g., increasing or decreasing) its activity / abundance to provide or maintain a redox potential in a culture or culture cells or culture supernatant. Thioredoxin and glutathione / glutaredoxin systems and their components are known in the art. See, e.g., Holmgren et al., Biochem Soc Trans, 2005 Dec;33(Pt 6):1375-7; Nordberg and Amer, Free Radic Biol Med, 2001 Dec 1;31(11):1287-312; Ghezzi P, Biochem Soc Trans, 2005 Dec;33(Pt 6):1378-81; Ivarsson et al., Diabetes, 2005 Jul;54(7):2132-42; Sen, CK, Biochem Pharmacol, 55(11), 1747-1758, 1 Jun 1998; and May et al., J Biol Chem. Biol Chem, 1997 Sep 5;272(36):22607-10, the contents of which are hereby incorporated by reference in their entirety.

[0191] In one embodiment, a method includes, or a bioreactor or harvesting container is capable of providing (e.g., adding) GILT (gamma-interferon-induced lysosomal thiol reductase) or regulating (e.g., increasing or decreasing) its activity / abundance. See, e.g., Rausch and Hastings, Mol Immunol, December 2015; 68(2Pt A): 124-8, doi: 10.1016 / j.molimm.2015.06.008, electronic publication June 23, 2015; and Hastings and Cresswell, Antioxidants and Redox Signal, August 1, 2011; 15(3): 657-668, the contents of which are hereby incorporated by reference in their entirety. In one embodiment, a method includes, or a bioreactor or harvesting container is capable of providing (e.g., adding) ascorbic acid, dehydroascorbic acid, or ascorbic acid or dehydroascorbic acid modification components to the growing culture. The redox potential modulating properties of ascorbic acid and dehydroascorbic acid are known in the art. See, for example, Winkler et al., Free Radic Biol Med, 1994 Oct; 17(4): 333-49, the contents of which are hereby incorporated by reference in their entirety.

[0192] In one embodiment, a method includes, or a bioreactor is capable of providing an intracellular reagent (e.g., adding a redox potential indicator, such as a redox sensitive dye or molecular probe, to a production culture). Such a redox potential indicator can be used to monitor the redox potential of a culture or cells within a culture. Redox potential indicator markers include, but are not limited to, 2,2'-bipyridine (Ru complex), nitrophenanthroline (Fe complex), N-phenylanthranilic acid, 1,10-phenanthroline sulfate iron (II) complex (ferritin), N-ethoxychloridin, 2,2'-bipyridine (Fe complex), 5,6-dimethylphenanthroline (Fe complex), o-dianisidine, sodium diphenylamine sulfonate, diphenylbenzidine, diphenylamine, viologen, 2,6-dibromophenol-sodium indophenol, 2,6-dichlorophenol-sodium indophenol, o-cresol sodium indophenol, thionine (Law's purple), methylene blue, indigo tetrasulfonic acid, indigo trisulfonic acid, indigo carmine (indigo disulfonic acid), indigo monosulfonic acid, phenol saffron, saffron, neutral red, markers disclosed in any document incorporated herein, and Schwarzlander M et al., Antioxidants and Redox Signaling. Signal, 2016 May 1;24(13):680-712 (which is incorporated herein by reference in its entirety).

[0193] In another embodiment, a method includes, or a bioreactor or harvest vessel capable of providing an extracellular agent, such as a metabolite, transition metal ion described herein, to a production culture.

[0194] In some embodiments, a bioreactor or bioreactors for use in the methods of the present disclosure may be configured according to the following principles:

[0195] - The functional setup of the bioreactor when harvesting the production culture is important to ensure that the culture leaving the bioreactor is adequately oxygenated and carries dissolved oxygen to subsequent processing steps. Once the cell culture has reached the target temperature to initiate the harvesting step, ensure that all processes that may adversely affect the oxygenation of the production culture are inhibited. These may include: inhibiting the distributed nitrogen flow (if enabled), inhibiting the on-demand CO2 gas flow control (if enabled), inhibiting the on-demand base control (if enabled), and inhibiting the supply application (if enabled).

[0196] -Nitrogen distribution gas flow is usually used as ballast to control dissolved oxygen tension (DOT) to a set point when the cell demand for oxygen is low, but this can maintain active flow during the production phase process. Nitrogen is also used as a carrier ballast in airlift bioreactors to maintain mixing in these containers. However, where nitrogen distribution gas is used, it must be suppressed to prevent dilution of the oxygen component of the distribution gas used to aerate the production culture during the harvesting step, because this will prevent the dissolved oxygen, DOT in the culture from reaching 100% air saturation (if a single air distribution is used) or the expected maximum DOT (if a blend of air and oxygen is used). Therefore, distributing nitrogen limits the expected maximum dissolved oxygen concentration for a given single air distribution or air-oxygen mixture distribution. In response to the process pH exceeding the control range, the CO2 gas flow is distributed to the production culture. When the CO2 gas flow distribution is enabled, it also dilutes the oxygen component of the distribution gas, which in turn limits the expected maximum dissolved oxygen concentration for a given single air distribution or air-oxygen mixture distribution. Therefore, deactivating it during the harvesting of the production culture prevents dilution of the aeration distribution gas.

[0197] - During the cultivation of the production culture, in order to strictly control the pH, it is necessary to add alkaline solutions to the production culture in response to the process pH falling below the control range. However, due to the ever-decreasing operating volumes and the excessive use of alkaline solutions, there is a potential risk of greater cell damage and cell death with the application of alkaline solutions while harvesting the production culture. Feeds must be applied to the production culture while the culture is actively growing and metabolizing. However, during harvest, due to the non-physiological properties of the feeds (higher osmolality and either high or low pH) and the microenvironment created in the poorly mixed container, the ever-decreasing operating volumes affect the mixing behavior of the bioreactor and can lead to greater cell damage and cell death with the application of feed solutions.

[0198] -Once the production culture has reached the target temperature to initiate the harvesting step, ensure that all process controls that may promote oxygenation of the production culture are activated. These may include: enabling distribution air and / or oxygen flow (if not enabled); enabling headspace air and / or oxygen flow (if not enabled); and finally enabling headspace pressure (if not enabled). Once the production culture has stopped growing or when it is cooled in preparation for harvesting, the on-demand air and / or oxygen distribution flow rates may become very low or even stopped. The continuous application of air and oxygen distribution at various continuous fixed flow rates is critical to oxygenating the production culture and ensuring that the production culture brings dissolved oxygen into the cell clarification filter housing or centrifuge and the flow path between the bioreactor and the filter housing or centrifuge or between the filter housing or centrifuge and the supernatant collection container (e.g., harvest container). Headspace or cover aeration is typically enabled during the cultivation of the production culture and ensures that the bioreactor headspace is continuously cleared of metabolic CO2 gas. Continuous blanket aeration of the production culture during harvest promotes surface oxygenation of the culture, albeit more slowly than with distributed gas, but avoids the generation of foam when the production culture is discharged from the bioreactor.

[0199] -For bioreactors capable of operating with headspace pressure, and where pressure is used during cultivation of the production culture to help achieve greater solubility of gases in the liquid phase (culture) or to maintain a positive pressure within the bioreactor to prevent entry of environmental contaminants across the sterile boundaries of the container, it is recommended to maintain this state during harvest. In practices where harvesting is driven by pressure, oxygenation of the culture will be aided as long as the gas used to pressurize the bioreactor is air or a mixed blend of air and oxygen. However, if harvesting is driven by a pump, headspace pressure is not typically used. In this case, the use of headspace pressure with the aid of air or an air / oxygen mixture is beneficial in aiding oxygenation of the culture and preventing the harvest tube from collapsing under the suction head upstream of the pump head when the culture is flowing at the required rapid rate depending on the scale of operation required during the harvesting operation.

[0200] -For single-use bioreactors, the design of the harvest line with respect to internal pore size and wall strength to prevent constriction under suction generated upstream of the pump head at high flow rates is important to ensure that the harvest flow rate is not impeded by tube clogging. The effect of tube clogging is not only to limit the harvest flow rate, but also to promote greater cell death and cell lysis and degassing of dissolved oxygen from the culture. The effect of tube clogging is likely to expose cells and products to the hypoxic / anoxic environment of the filter housing due to increased residence time in the housing due to slower flow rates, promote the release of intracellular factors due to greater cell death and cell lysis through the restricted openings, and remove dissolved oxygen from the culture by degassing in the suction zone, thereby reducing the amount of dissolved oxygen brought into the filter housing by the culture. In one embodiment, the method disclosed herein avoids these negative effects on product stability.

[0201] The inner diameter of the harvest tubing and the choice of pump used are selected to achieve the desired flow rate to achieve the process volume throughput (liters of process supernatant per filter area, L / m2) of the cell clarification step performed by the depth filter or centrifuge coupled to the depth filter. The material of construction of the harvest line needs to be rigid to prevent constriction in the tip created upstream of the pump head. It is recommended that materials other than the commonly used platinum-cured silicone or C-flex be used in the construction of the harvest tubing. The use of braided tubing may be considered as an alternative to the commonly used materials.

[0202] - In some embodiments, a method includes, or a bioreactor is capable of optimizing the depth filter area to avoid fouling and clogging of the filter during the filter clarification step, and thereby, minimizing the residence time of the cell culture within the filter housing is an essential step to ensure oxygenation of the production culture and to ensure that the dissolved oxygen carried away from the bioreactor is not depleted while flowing into and residing in the filter housing before the culture is discharged as supernatant. In addition, the flow rate should be sufficient to minimize the residence time of the supernatant between the clarification step (filter housing) and the supernatant collection container (e.g., harvest container).

[0203] The cell clarification filter (primary, secondary and tertiary) areas are optimized to ensure that the required process volume flux is met without causing a loss of flux (L / m2 / h) or a loss of filtrate quality across the filters (due to breakthrough of particulates) to an extent that would affect the performance of the secondary or 2nd filter and tertiary or 3rd filter downstream of the primary or 1st filter. In addition, the filter areas and filter types selected for optimal cell clarification must avoid higher pressure differentials between the different filter stages, which indicate filter fouling and impending filter clogging. Therefore, it can be expected that a reduction in flux across different series-coupled filters will result in an increase in the residence time within the filter housing and a greater consumption / depletion of dissolved oxygen in the supernatant within the filter housing, resulting in hypoxia in the supernatant. The establishment of a higher pressure differential between the primary and secondary filters is also problematic because it promotes higher cell lysis and the release of cytokines (which, when enabled, can undermine the stability of the product).

[0204] - In some embodiments, a method comprises, or a bioreactor capable of ensuring that the cell-free supernatant is collected in a well-aerated and mixed harvest vessel (steel or single use). The harvest vessel is designed to ensure that it promotes good surface oxygenation by: impinging the collected filtrate onto the vessel wall surface by designing internal nozzles towards the vessel wall, thereby forcing the filtrate to flow down the vessel wall; and / or the ability to fill the harvest vessel with a gaseous environment consisting of air or any given blend of air and oxygen prior to filtrate collection to promote oxygenation of the collected filtrate to greater than >40% air saturation and ≤500% air saturation.

[0205] In one embodiment, the harvest container is a stainless steel harvest container. The collection method of the supernatant and the design of the harvest container are the final elements of the method for protecting the product during the harvest step. At present, the harvested supernatant is collected in a jacketed stainless steel container with a mixer to continuously mix the collected supernatant. The harvested supernatant is discharged into the harvest container through a headspace port with an internal "J" shaped tube, and the internal "J" shaped tube guides the supernatant flow to the container wall, and the supernatant flow flows down along the wall from the container wall to collect at the bottom of the tank. In addition, these stainless steel containers have been filled with sterile air to pressurize them to above atmospheric pressure after they are subjected to in-situ steam sterilization to prevent environmental contaminants from entering across the container aseptic boundary. It is assumed that the harvested supernatant collected in these containers is oxygenated when it flows down along the container wall under a pressurized air atmosphere. Further oxidation of the supernatant is carried out by contacting the surface of the headspace above the collected supernatant.

[0206] In another embodiment, the harvest container is a single use harvest container. The single use supernatant harvest container is a bag that is gamma irradiated and evacuated. During supernatant collection, the bag fills and replaces the vacuum, and no gas headspace is formed above the collected supernatant. These bags have no agitators, so the collected supernatant cannot be mixed, and the bags are installed into unjacketed plastic or stainless steel housings that cannot heat or cool the collected supernatant. Once the bags are filled with supernatant, they are transferred to storage at +5°C for up to 14 days. The purification process can be started with the primary capture step immediately after harvest or within the 14 day retention period.

[0207] The method of ensuring that oxygenation of the production culture is maintained at a DOT in the range of greater than 40% air saturation to less than 500% air saturation during the harvesting step is intended to "load" the production culture with dissolved oxygen that is expected to be carried into and through the clarification step so that when the supernatant is collected, it has enough dissolved oxygen to prevent the activation of cytokines that may promote product dissociation. The harvest container is designed to ensure that the harvested supernatant continues to remain well oxygenated while being stored at +5°C for up to 14 days. This is achieved by a container design that ensures that the supernatant continues to be oxygenated while being collected and maintained at +5°C storage, for example by surface aeration. In one embodiment, the harvest container design includes the following features:

[0208] - The container or single use bag has an agitator / mixer which provides sufficient axial bulk mixing to oxygenate the collected supernatant by surface aeration.

[0209] - The container or single use bag housing is fitted with a thermal cycler of appropriate size to allow the temperature of the container contents to reach +5°C from room temperature within 4 hours and to heat from +5°C to room temperature within a similar time.

[0210] - The container or single use bag housing is fitted with appropriately sized air and oxygen mass flow controllers to allow headspace aeration with air, oxygen or oxygen enriched gas.

[0211] - The container or single-use bag and housing are equipped with a cover gas filter (suitable for air and oxygen at flow rates suitable for operating scale and an outlet gas vent filter suitable for air and oxygen at flow rates suitable for operating scale to allow continuous flow of air and oxygen through the headspace to fill the harvest container with a gas environment composed of air or any given blend of air and oxygen prior to supernatant filtrate collection to promote oxygenation of the supernatant filtrate to >40% air saturation and ≤500% air saturation during collection of the supernatant filtrate and once collection is completed.

[0212] - The container or single use bag and housing are equipped with a pressure sensor and safety interlock to stop the headspace gas if the pressure exceeds the safety limit of the operating scale and container design.

[0213] - The container or single use bag and housing are equipped with process sensor ports for pH, DOT, redox and temperature. The location of the probe within the container or bag allows monitoring at the lowest operational volume appropriate to the scale of operation.

[0214] - A container or single-use bag is equipped with an additional port at the top of the container, the container having a port equipped with an internal nozzle that directs the flow of liquid onto the container wall and promotes the liquid to flow down the container wall.

[0215] Cells and cell culture

[0216] On the one hand, the present disclosure relates to methods and compositions for engineering or manufacturing cells or cell lines that produce products described herein (e.g., recombinant products). On the other hand, the present disclosure relates to methods and compositions for engineering or manufacturing cells or cell lines that have improved (e.g., reduced) dissociation, increased productivity and / or product quality.

[0217] In an embodiment, the cell is a mammalian or non-mammalian cell, such as an insect cell, a yeast cell, a fungal cell, a plant cell, an archaeal cell (e.g., a cell from an archaeal species), or a bacterial cell. In one embodiment, the cell is from a human, a mouse, a rat, a Chinese hamster, a Syrian hamster, a monkey, ape, a dog, a duck, a horse, a parrot, a ferret, a fish, or a cat. In one embodiment, the cell is an animal cell. In an embodiment, the cell is a mammalian cell, such as a human cell or a rodent cell (e.g., a hamster cell, a mouse cell, or a rat cell). In one embodiment, the cell is a prokaryotic cell, such as a bacterial cell. In one embodiment, the cell is an actinomycete species, such as Mycobacterium tuberculosis).

[0218] In one embodiment, the cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN or a CHO derived cell. The CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. The CHO FUT8 knockout cell is, for example, CHOK1 SV (Lonza Biologies, Inc.).

[0219] In another embodiment, the cell is Hela, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney cells), VERO, SP2 / 0, NS0, YB2 / 0, Y0, EB66, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, CHO-K1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11 or CHOZN, or any cell derived therefrom. In one embodiment, the cell is a stem cell. In one embodiment, the cell is a differentiated form of any cell described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

[0220] In one embodiment, the cell is any of the cells described herein that produce a product (e.g., a product described herein). In one embodiment, the cell is any of the cells described herein that include an exogenous nucleic acid encoding a recombinant polypeptide (e.g., expressing a recombinant polypeptide, such as a recombinant polypeptide selected from Table 2 or 3).

[0221] In one embodiment, the cell culture (e.g., production culture) is carried out in batch culture, batch feed culture, intermittent culture or continuous culture. In one embodiment, the cell culture is an adherence culture. In one embodiment, the cell culture is a suspension culture. In one embodiment, the cell or cell culture is placed in vivo to express the recombinant polypeptide, for example, in a model organism or a human subject.

[0222] For example, the method is carried out in a large-scale bioreactor (e.g., a bioreactor with at least two impellers), a large-scale bioreactor system, and a method for large-scale cultivation and propagation of mammalian cells. In one embodiment, the bioreactor is a bioreactor as described in 2011 / 0312087.

[0223] In one embodiment, the cells are cultured in a bioreactor as disclosed in USSN 62 / 242,758, the contents of which are incorporated herein in their entirety. Disclosed therein is a system and method for controlling at least one bioreactor, other equipment associated with cell culture, and a system containing any combination of these. For example, the system and method for bioreactor control may include controlling multiple bioreactors and other types of equipment associated with culture, such as equipment for fermentation, harvesting, equipment for microfiltration and purification (e.g., liquid chromatography skid-mounted systems), buffer preparation, culture medium preparation, etc. Multiple bioreactors and other types of equipment associated with culture may be located in a factory, and the control of such equipment may be referred to as a plant-wide control system ("PWCS").

[0224] In one embodiment, the method is performed in a manufacturing facility that provides batch and continuous manufacturing using at least one single-use disposable technology (e.g., those described in USSN 62 / 246,478, the contents of which are incorporated herein in their entirety). In one embodiment, the manufacturing facility is used to produce active pharmaceutical ingredients ("API").

[0225] Cooling can be performed using methods known in the art. For example, larger reactors typically have a water jacket, thermostatically controlled or temperature-controlled water passing through the jacket to control the culture temperature. In an embodiment, a process chilled water source is delivered to the jacket to obtain sufficient and rapid cooling. In an embodiment, a refrigeration unit is used to remove heat.

[0226] Cooling can be performed at one or more desired times during cultivation (i.e., increasing the number of cells or inducing production of a desired product by the cell culture) or when cooling the production culture at the end of the production phase or when harvesting the cells. Since the optimal growth temperature of organisms varies, the cooling temperature will be selected based on the relatively higher temperature of one or more steps before the cooling step. In an embodiment, cooling of the cultured cells is performed to reduce the oxygen consumption rate of the cells so that the harvest stream remains oxygenated during the harvest process.

[0227] For mammalian cell lines, the temperature of the culture process is typically varied between 27-35° C. (e.g., 27, 28, 29, 30, 31 or 32, 33, 34 or 35° C.). In some embodiments, the temperature is lowered to 30-33° C. after the exponential growth phase. Without wishing to be bound by theory, it is believed that lowering the temperature to 12-18° C. (e.g., 15° C.) after the production phase will reduce the specific oxygen consumption rate by 10-fold.

[0228] In one embodiment, the culture medium is serum-free.

[0229] Other suitable media and culture methods for mammalian cell lines are well known in the art, as described, for example, in US Pat. No. 5,633,162. Examples of standard cell culture media for laboratory flasks or low-density cell culture and adapted to the requirements of specific cell types are, for example, Resveratrol (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, lp. 173ff., 1963), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad., Sc. 53, p288ff. 1965) or Iscoves' modified DMEM lacking albumin, transferrin and phosphatidylcholine (Iscoves et al., J. Exp. med., 1, p. 923ff., 1978). For example, Ham's F10 or F12 medium are specifically designed for CHO cell culture. Other mediums particularly suitable for CHO cell culture are described in EP-481 791. Other suitable culture methods are known to the skilled person and may depend on the recombinant polypeptide product and the host cell used. It is within the skill of the ordinary technician to determine or optimize conditions suitable for expression and production of the product (e.g., a recombinant polypeptide) to be expressed by the cell.

[0230] The amount, level or quantity of the product produced or secreted (e.g., secreted into the culture medium) for quantitative determination includes protein quantitative determination, such as Bradford protein determination, SDS-PAGE analysis, immunoblotting (e.g., Western blot) and automation (e.g., using a UV spectrophotometer device). Other methods for measuring increased protein production are well known to those skilled in the art. For example, the increase in recombinant protein production can be determined on a small scale by measuring the concentration in tissue culture medium by ELISA (Smales et al., 2004, Biotechnology Bioengineering, 88: 474-488). It can also be quantitatively determined by ForteBio Octet, such as for high-throughput determination of recombinant monoclonal antibody (mAh) concentration in culture medium (Mason et al., 2012, Biotechnology Progress, 28: 846-855), or by protein A HPLC on a large scale (Stansfield et al., 2007, Biotechnology Bioengineering, 97: 410-424). Other methods for determining the production of a product (e.g., a recombinant polypeptide as described herein) may refer to the specific production rate (qP) of the product (particularly the recombinant polypeptide in the cell) and / or the time integral of the viable cell concentration (IVC). In one embodiment, the method for determining production comprises determining a combination of qP and IVC. Recombinant polypeptide production or productivity (which is defined as the concentration of the polypeptide in the culture medium) varies with these two parameters (qP and IVC) and is calculated according to Porter et al. (Porter et al., 2010, Biotechnology Progress, 26: 1446-1455). Methods for measuring protein production are further described in detail in the examples provided herein.

[0231] In one embodiment, the methods described herein produce cells with improved product quality. In one embodiment, the improvement in the quality of the product results in an increase, such as 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more of the quality of the product, such as compared to the amount, level or quantity of the product produced by cells not subjected to lower temperatures; or an increase in the quality of the product by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or 100 times or more.

[0232] This improvement in product quality can be exemplified, for example, by one or more of the following:

[0233] i) Increase or decrease of disulfide bond mismatch (e.g., for antibody molecule products, increase or decrease of desired isomers or structures due to increase or decrease of disulfide bond mismatch).

[0234] ii) an increase in the amount or number of non-aggregated products (or a decrease in the amount or number of aggregated products);

[0235] iii) an increase in the amount or number of properly folded or assembled products (or a decrease in the amount or number of misfolded, unfolded, partially assembled or non-assembled products), or an increase in the ratio of properly folded or assembled products to unfolded, misfolded, partially assembled or non-assembled products;

[0236] iv) an increase in the amount or quantity of the full-length product (or a decrease in fragmentation of the product);

[0237] v) increase in a desired post-translational modification (or decrease in unmodified or mismodified products);

[0238] vi) increase or decrease in glycan heterogeneity (e.g., for glycosylated products);

[0239] vii) an increase in the amount or quantity of a functional product (or a decrease in the amount or quantity of a non-functional or incompletely functional product), or an increase in the ratio of functional to non-functional or incompletely functional products; and / or

[0240] Methods for measuring the product quality (e.g., improvement of product quality) of cells or cell lines generated as described herein are known in the art. In one embodiment, methods for determining the fidelity of the primary sequence of the expressed recombinant polypeptide product are known in the art, such as mass spectrometry. The increase in the amount or concentration of the appropriately folded product (e.g., expressed recombinant polypeptide) can be determined by circular dichroism or the intrinsic fluorescence of the expressed recombinant polypeptide. The increase in the amount or concentration of the functional product can be tested using a variety of functional assays, depending on the identification of the recombinant product (e.g., recombinant polypeptide). For example, antibodies can be tested by ELISA or other immunoaffinity assays. Other methods for determining the improvement of product quality (e.g., determining aggregation, post-translational modification, disulfide bond mispairing) can be assessed by size exclusion chromatography, high performance liquid chromatography, dynamic light scattering (DLS) methods, and protein electrophoresis (PAGE).

[0241] In some embodiments, additional steps may be performed to improve the expression of the product (e.g., transcription, translation and / or secretion of the product) or the quality of the product (e.g., proper folding and / or fidelity of the primary sequence). These additional steps include introducing agents that improve product expression or product quality. In one embodiment, the agent that improves product expression or product quality can be a small molecule, a polypeptide, or a nucleic acid encoding a polypeptide that improves protein folding (e.g., a chaperone). In one embodiment, agents that aid protein folding include nucleic acids encoding chaperones, such as BiP, PD1, or ERO1 (Chakravarthi and Bulleid, 2004; Borth et al., 2005; Davis et al., 2000). Other additional steps for improving the yield and quality of the product include overexpression of transcription factors (e.g., XBP1 and ATF6 (Tigges and Fussenegger, 2006; Cain et al., 2013; Ku et al., 2008)) and lectin binding chaperones (e.g., calnexin and calreticulin (Chung et al., 2004)). The overexpression of the reagent that helps or improves protein folding, product quality and product yield as described herein can be achieved by introducing the exogenous nucleic acid encoding the reagent. In another embodiment, the reagent that improves product expression or product quality is a small molecule that can be added to cell culture to improve product expression or product quality, such as DMSO. In one embodiment, cells are maintained at a lower temperature (for example, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C lower than the temperature of normal cell growth) can improve the quality of the product by reducing or eliminating the dissociation of the product.

[0242] Any method described herein may further comprise an additional selection step for identifying cells with high productivity or producing high-quality products. For example, FAC selection can be used to select specific cells with a desired characteristic (e.g., higher expression of protein folding proteins (e.g., chaperone proteins)).

[0243] In one aspect, the present disclosure provides methods comprising steps for recovering or retrieving a recombinant polypeptide product. In embodiments where the recombinant polypeptide is secreted from a cell, the method may comprise steps for retrieving, collecting or separating the recombinant polypeptide from the cell, a cell population, or a culture medium in which the cells are cultured. In embodiments where the recombinant polypeptide is present within a cell, the purification of the recombinant polypeptide product comprises separating the recombinant polypeptide produced by the cell from any one or more of: host cell proteins, host cell nucleic acids, host cell lipids, and / or other fragments from the host cell.

[0244] In an embodiment, the process described herein provides a substantially pure protein product. As used herein, "substantially pure" refers to being substantially free of pyrolysis substances, substantially free of nucleic acids, and / or substantially free of endogenous cell proteases and components (e.g., polymerases, ribosomal proteins, and chaperone proteins) from host cells. Substantially pure protein products contain, for example, less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of contaminating endogenous proteins, nucleic acids, or other macromolecules from host cells.

[0245] Methods for recovering and purifying products (e.g., recombinant polypeptides) are well established in the art. In order to recycle the recombinant polypeptide product, physical or chemical or physicochemical methods are used. Physical or chemical or physicochemical methods can be filtration, centrifugation, ultracentrifugation, extraction, lyophilization, precipitation, chromatography, or a combination of two or more thereof. In one embodiment, chromatography includes one or more of size exclusion chromatography (or gel filtration), ion exchange chromatography (e.g., anion or cation exchange chromatography), affinity chromatography, hydrophobic interaction chromatography, and / or multimodal chromatography.

[0246] In embodiments, the reduction in temperature is combined with a reduction in temperature by lowering pH, reducing thioredoxin inhibitors (eg, metal ions). In embodiments, the combined treatment is performed in a bioreactor as described above.

[0247] Examples

[0248] The present invention is further described in detail by reference to the following examples. These examples are provided for illustrative purposes only and, unless otherwise indicated, are intended to be limiting. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all variations that become apparent due to the teachings provided herein.

[0249] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the compounds of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0250] Example 1. Background and Logo

[0251] Chinese hamster ovary (CHO) cell is the main production matrix for generating biopharmaceuticals in the world (Walsh, 2014). Although a lot of time, energy and money are used to develop these cells from adherent cell lines to stable cell lines, then overexpression and synthetic elements are utilized to manipulate cells to improve titer, growth rate and cell-specific recombinant protein production rate (qP), the matrix and genome of the cell are actually still the same as the ancestor Chinese hamster. Therefore, the behavior of the cell is usually like being present in the Chinese hamster, rather than in a completely artificial environment. This can be illustrated by multiple genes that seem to be functionally redundant and are still encoded by cell lines and subsequently expressed in protein. According to literature records, there are examples of genes that have been successfully knocked down or knocked out from the CHO genome to enhance the characteristics of cells or products. This includes knocking down Cers2 and TbclD20 to increase specific productivity (Pieper et al., 2017), Fut8 (to remove fucosylation) Bak and Bax (to gain greater resistance to apoptosis) (Grav et al., 2015), and depletion of endogenous CHO glutamine synthetase (Bebbington et al., 1992; Cockett, Bebbington, & Yarranton, 1990). With the recent genome sequence of CHO (Lewis et al., 2013; Xu et al., 2011) and the rapid development of genome editing technologies (Cong, Ran, Cox, Lin, & Barretto, 2013; Ran et al., 2013), the promise of knocking down or knocking out individual genes provides an opportunity to screen redundant elements within the CHO genetic code to a greater extent.

[0252] Combined with a set of bioinformatics tools and literature analysis, various omics-level data are used in the form of quantitative transcriptomics and proteomics to rationally identify high-abundance non-essential proteins that are predicted to compete with target mAbs for endoplasmic reticulum (ER) space as well as post-translational modifications (PTMs) and secretion mechanisms. The strategy is mainly based on the analysis of Lonza RNA-seq data from bioreactor experiments of CHO cell lines. Samples were selected and extracted at the 4th, 7th and 9th days, and RNA-seq analysis was performed for the CHOK1GS (Ensembl accession number: CHOK1GS_HDv1) genomic sequence. The number of transcripts per million (TPM) values ​​for each gene provide a measure of the amount of each gene expressed as part of the total cell transcript pool. Automatic gene ID assignment generated 77.5%, 77.7% and 77.6% of the IDs of the total transcript pool active genes on the 4th, 7th and 9th days, respectively. Manual assignment adds gene IDs to 18.4%, 17.8% and 18.3% of the transcript pool. A total of 13773, 14433, and 14660 gene IDs were generated for days 4, 7, and 9, respectively; these assigned values ​​represent 95.7%, 95.4%, and 95.8% of the IDs of the transcript pools for days 4, 7, and 9, respectively. Defined thresholds were then introduced to help classify gene transcripts into different abundance levels (based on Ramskold et al. (2009), PLoS Computational Biology, 2009 Dec;5(12):el000598, doi:10.1371 / journal.pcbi.l000598):

[0253] Unexpressed genes (exon model fragments per kilobase per million mapped reads (FPKM) < 0.3) (TPM < 0.4)

[0254] Lowly expressed genes (0.3≤FPKM<3)(0.4≤TPM<4)

[0255] Moderately expressed genes (3≤FPKM<30)(4≤TPM<40)

[0256] Highly expressed genes (30≤FPKM<100)(40≤TPM<133.33)

[0257] Very highly expressed genes (FPKM ≥ 100) (TPM ≥ 133.33)

[0258] Only the genes (879 genes) that always meet the extremely high abundance gene standard in all three test days (the 4th, 7th and 9th days) are proposed. Then, these genes are passed through the PANTHER classification system (http: / / www.pantherdb.org / ), and the Mus musculus database is used as the organism for analysis. Initially, only the genes containing the keywords that are regarded as targets because of functional redundancy are highlighted for further analysis (Table E1). After this, a list of functionally essential or highly necessary keywords (Table E2) is generated. Any gene containing these keywords is subsequently excluded from further analysis, and in the case of genes containing keywords from Tables E1 and E2 simultaneously, genes will be analyzed in depth to consider how important they are to the function of CHO cells. After the gene is screened by the standard of the definition, 76 targets (112 when all isomers are considered) are left.

[0259] Bioinformatic analysis was then performed using 4 different tools to hone in on genes that further met our criteria for being secreted proteins. The programs used were:

[0260] SignalP 4.1 - (Default settings - eukaryotic cells) - http: / / www.cbs.dtu.dk / services / SignalP /

[0261] TargetP 1.1 - (Default settings - non-plant) - http: / / www.cbs.dtu.dk / services / TargetP /

[0262] SecretomeP 2.0 - (settings changed to mammalian) - http: / / www.cbs.dtu.dk / services / SecretomeP /

[0263] TMHMM 2.0 - (default settings) - http: / / www.cbs.dtu.dk / services / TMHMM /

[0264] Additional data at various omics levels were generated using quantitative intracellular proteomic analysis of exponential and stationary phase cells of CHO cell lines. When present for a target protein, data detailing total protein biomass (a consideration of the average copy number of a protein and its molecular weight) were recorded.

[0265] Data from previously published quantitative proteomic analyses of extracellular proteins present in the culture medium of CHO cell cultures (Kumar et al., 2015, J Proteome Res, 2015 Nov 6;14(ll):4687-703, doi:10.1021 / acs.jproteome.5b00588; Park et al., 2017, Sci Rep, 2017 Mar 10;7:44246, doi:10.1038 / srep44246) were used to try and identify common redundant extracellular host cell (HC) proteins. Details of whether a target protein from the previously defined list was present and, if present, whether it was present at high, medium or low levels (criteria defined in Table E3) were recorded (NSAF - Normalized Spectral Abundance Factor, NSC - Normalized Spectral Count).

[0266] The summarized pipeline is shown below:

[0267] 1. All transcripts are extremely high during the entire culture process in the bioreactor

[0268] 2. All contain at least one highlighted keyword (Table E1)

[0269] 3. All required keywords were removed (unless they conflicted with point 2, which was evaluated separately here) (Table E2)

[0270] 4. All are predicted to be secreted proteins by various bioinformatics tools

[0271] 5. Try matching against Lonza IC proteomics

[0272] 6. Attempt to match previously published CHO EC abundance data

[0273] 7. Try to use the Uniprot data of Mus musculus to identify glycosylation sites and glycosylation sites / AA

[0274] 8. Ranking according to the defined criteria (Table E4)

[0275] Another set of a priori targets (fibronectin - Fn1 and heparan sulfate proteoglycan - HSPG2) were selected because of their perceived redundancy, as well as two negative targets that, despite matching most of the criteria defined above, appear to be very important for a properly functioning CHO cell expression system (clusterin - Clu and 14-3-3 protein epsilon - YWHAE). The final list of 20 targets for testing is shown in Table E5.

[0276] Example 2: Knockout of endogenous protein and increase in product yield

[0277] By using Cas9 nuclease to perform highly specific targeted knockout of a designated gene (e.g., a gene encoding an endogenous protein (e.g., an extracellular, secreted, and / or redundant protein), such as the genes identified in Example 1 (e.g., Table E5), and evaluating the effect on recombinant protein production. Using enhanced specificity Cas9 (eCas9 1.1) (Slaymaker et al., 2016), a 2A peptide fused to GFP can allow high-throughput isolation of cells with Cas9 nuclease activity, thereby potentially performing multiplexed genome editing (Lonowski et al., 2017, Nat Protoc, 2017 Mar;12(3):581-603, doi:10.1038 / nprot.2016.165).

[0278] By successfully knocking out genes encoding endogenous proteins (e.g., extracellular, secreted, and / or redundant proteins), a minimal CHO genome can be created that lacks redundant competing proteins (e.g., proteins that compete with the product (i.e., mRNA encoding the product) for translation and ER / Golgi resources (i.e., mRNA encoding the protein)). This will facilitate the generation of a more streamlined genome and proteome with improved growth and production properties, better suited to the high titer and growth rate requirements required for competitive production of biopharmaceuticals.

[0279] In some embodiments, knocking out functionally redundant secretory genes from the CHO genome will generate enhanced cell lines capable of enhanced growth rate and expression capacity.

[0280] For preliminary screening of the gene knockout targets identified in Table E5, each gene target was individually knocked out from the IgG4 Mab production clone GS-CHOK1SV cell line by transient transfection with a plasmid (pX458) encoding the Cas9-GFP nuclease described above and one or more gene target specific guide RNAs expressed from a constitutive U6 promoter. 48 hours after transfection, a heterogeneous pool of transfected cells was sorted for high GFP expression (representing high Cas9 nuclease activity) by FACS, where ≥0.5x 10^6 viable cells were selected and pooled for further culture, and then growth, Mab productivity, and gene knockout success were evaluated.

[0281] In detail, the GS-CHOK1V cell line for making IgG4 Mab was transiently transfected with a plasmid (pX458) containing: 1) Cas9-GFP nuclease fusion protein expressed from a CMV promoter and 2) one or more guide RNAs specific for target genes in its genome expressed from a constitutive U6 promoter. 48 hours after transfection, the transfectant pool was sorted for high GFP expression by FACS, where ≥0.5x 10^6 cells were collected and merged into a new flask. After 72 hours, in a new flask, the cell culture was centrifuged, the supernatant was removed, and the cell pellet was resuspended to a final concentration of 0.2x 10^6 viable cells / mL, followed by growth for ≥5 days, and then the Mab titer was determined by ValitaTITER (Valitacell, Dublin, Ireland) and the cell count was determined by automatic cell counting (ViCell-Beckman Coulter, High Wycombe, UK). The percent change in qP was calculated in FACS sorted pools of GS-CHOK1SV cells transfected with pX458 vector containing gRNA specific for the indicated target genes. All data points are expressed relative to control GS-CHOK1SV cells transfected with pX458 expressing the Cas9-GFP nuclease fusion protein but lacking gRNA.

[0282] Compared with control GS-CHOK1SV cells transfected with pX458 vector lacking gRNA ( Figure 1A ), targeting Cas9-GFP nuclease to genes Lambl, Mfge8, Sbsn, C1S, C1R, Nid1, Dcn, B2M, and Clu by gRNA increased the average qP of FACS-sorted pools of GS-CHOK1SV cells.

[0283] To determine the success of Cas9-GFP gene editing targeting specific genes, genomic DNA was extracted from FACS sorted cells and evaluated by TIDE analysis (Brinkman et al., 2014, Nucleic Acids Res, 2014 Dec 16;42(22):el68, doi:10.1093 / nar / gku936). The results showed that high targeted gene indel rates (ranging from ≥60% to 90%) were obtained by this method ( Figure 1B ) (strongly suggesting that the phenotypic readout (i.e., the observed increase in qP) varies with gene editing in the pool.

[0284] Example 3: Multiple gene knockout of endogenous targets and increased production of products.

[0285] The data provided in Example 2 partially demonstrate that several endogenous genes encoding non-essential, redundant and / or secreted endogenous proteins in cells can be knocked out individually to increase the yield of Mabs from CHO cell lines. Due to the high number of endogenous proteins that pass through the cell secretory apparatus, it may be necessary to remove multiple genes encoding essential, redundant and / or secreted endogenous proteins to maximize the increase in qP obtained using this strategy. To test this, in the GS-CHOK1SV cell line, the gene targets Lamb1, Mfge8, Sbsn, C1S, C1R, Nid1, Dcn, B2M and Clu associated with the highest qP increase when knocked out individually (Figure 1) were knocked out in random triplicate combinations using the techniques of Examples 1 and 2 (Table E6), but avoiding target combinations located on the same CHO chromosome (C1S and C1R, Mfge8 and Nid1, Dcn and Clu) to reduce the risk of large-scale genomic recombination. Some combinations of gene targets associated with significant increases in mean titer and qP when combined in triplicate (e.g., C1S, Dcn, and Lamb1) exhibited an average increase in titer of 25% and an average increase in qP of 28% when combined in the triple knockout pool, compared to control GS-CHOK1SV cells transfected with a pX458 vector lacking gRNA (Table E7).

[0286] Example 4: Table

[0287] Table E1 - PANTHER assigned keywords - highlighted genes requiring further analysis

[0288] Blood clotting Vesicle association Vesicle coat protein Components of membrane Complement components Amyloid Huntington's disease placenta Chemokines CD communication marrow Estrogen Cell adhesion Immunity Galectin Epithelial Alzheimer Neurons Parkinson's Cytokines Embryo Apoptosis Transmembrane receptors secretion exosomes Endoplasmic reticulum Extracellular space Extracellular matrix Extracellular region Histocompatibility MHC immunity

[0289] Table E2 - PANTHER-specified keywords - genes removed from further analysis

[0290] Ribosome Eukaryotic origin ATP ADP Actin Myosin RNP NADH Ubiquitin Peptide Mitochondria Transporter Polymerase Heat shock Splicing Nucleosome RNA / DNA binding Kinase Cell division Cytochrome C Histone Transcription translate cAMP Chaperone Protein fold Glycosylation Cell cycle Tubulin Kinesin Dynein metabolism Cytoskeleton transportation Calmodulin Ras Collagen Endoplasm Golgi apparatus

[0291] Table E3 - Abundance level criteria from Kumar et al. (2015) and Park et al. (2017)

[0292]

[0293] Table E4 – Final criteria considered for target ranking

[0294] Protein abundance classification Protein levels Kumar – High NSAF≥-8.8939 Kumar – Middle -8.8939<NSAF≥-12 Kumar – Low NSAF<-12 Park – High NSC≥1.0 Park – Medium 0.5≥NSC<1.0 Park – Low NSC<0.5

[0295] Table E5 - Target genome IDs

[0296]

[0297]

[0298] Key to Table E5

[0299]

[0300]

[0301] Table E6 Random triplicate combinations of knockout genes

[0302]

[0303] Table E7 Transfection of pX458 containing gRNAs specific for random triplicate combinations of target genes The percentage change of Mab titer, qP and growth (integral viable cell density, IVCD) of FACS sorted pools. All values ​​are relative Represented in control cells transfected with pX458 lacking gRNA.

[0304] Multiple Groups IVCD% Titer% qP% Random 1 2.77 24.14 21.70 Random 2 -3.75 -12.42 -8.78 Random 3 -4.35 5.54 10.83 Random 4 -8.60 -1.81 6.55 Random 5 -1.42 24.60 27.73 Random 6 -4.17 2.16 7.26 Random 7 -2.84 2.99 4.61 Random 8 -1.45 14.23 17.19 Random 9 1.18 13.93 12.17 Random 10 -1571 -1600 -033

[0305] Example 5: References

[0306] Bebbington, R.C., Renner, G., Thomson, S., King, D., Abrams, D., and Yarranton, G.T. (1992). High level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Nature Biotechnology, 10, 169-175.

[0307] Brown, AJ, Gibson, S., Hatton, D. and James, DC (2018). Transcriptome-Based Identification of the Optimal Reference CHO Genes for Normalisation of qPCR Data. Biotechnology Journal, 13(1), 1-7. https: / / doi.org / 10.1002 / biot.201700259

[0308] Cockett, IM, Bebbington, RC, and Yarranton, TG (1990). High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Nature Biotechnology, 8, 662-667. https: / / doi.org / 10.1038 / nm0798-822

[0309] Cong, L., Ran, F., Cox, D., Lin, S., and Barretto, R. (2013). Multiplex genome engineering using CRISPR / Cas systems. Science, 339(6121), 819-823. https: / / doi.org / 10.1126 / science.1231143.Multiplex

[0310] Grav, LM, Lee, JS, Gerling, S., Kallehauge, TB, Hansen, AH, Kol, S., ... Kildegaard, HF (2015). One-step generation of triple knockout CHO cell lines using CRISPR / Cas9 and fluorescent enrichment. Biotechnology Journal, 10(9), 1446-1456. https: / / doi.org / 10.1002 / biot.201500027

[0311] Kittler, R., Surendranath, V., Heninger, A. K., Slabicki, M., Theis, M., Putz, G., … Buchholz, F. (2007). Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nature Methods, 4(4), 337-344. https: / / doi.org / 10.1038 / nmethl025

[0312] Kumar, A., Baycin-Hizal, D., Wolozny, D., Pedersen, LE, Lewis, NE, Heffner, K., ... Betenbaugh, MJ (2015). Elucidation of the CHO super-ome (CHO-SO) by proteoinformatics. Journal of Proteome Research, 14(11), 4687-4703. https: / / doi.org / 10.1021 / acs.jproteome.5b00588

[0313] Lewis, NE, Liu, X., Li, Y., Nagarajan, H., Yerganian, G., O'Brien, E., ... Palsson, BO (2013). Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nature Biotechnology, 31(8), 759-765. https: / / doi.org / 10.1038 / nbt.2624

[0314] Park, JH, Jin, JH, Lim, MS, An, HJ, Kim, JW, and Lee, GM (2017). Proteomic analysis of host cell protein dynamics in the culture supernatants of antibody-producing CHO cells. Scientific Reports, 7 (November 2016), 1-13. https: / / doi.org / 10.1038 / srep44246

[0315] Pieper, L.A., Strotbek, M., Wenger, T., Gamer, M., Olayioye, M.A., and Hausser, A. (2017). Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and TbclD20. Metabolic Engineering, 40 (October 2016), 69-79. https: / / doi.Org / 10.1016 / j.ymben.2017.01.003

[0316] Ran, FA, Hsu, PD, Wright, J., Agarwala, V., Scott, DA, and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281-2308. https: / / doi.org / 10.1038 / nprot.2013.143

[0317] Slaymaker, IM, Gao, L., Zetsche, B., Scott, DA, Yan, WX, and Zhang, F. (2016). Rationally engineered Cas9 nucleases with improved specificity. Science, 351(6268), 84-88. https: / / doi.org / 10.1126 / science.aad5227

[0318] Walsh, G. (2014). Biopharmaceutical benchmarks 2014. Nature Biotechnology, 32(10), 992-1000.

[0319] Xu, X., Nagarajan, H., Lewis, N. E., Pan, S., Cai, Z., Liu, X., … Wang, J. (2011). The genomic sequence of the Chinese hamster ovary (CHO)-Kl cell line. Nature Biotechnology, 29(8), 735-741. https: / / doi.org / 10.1038 / nbt.1932

[0320] Equivalence

[0321] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0322] The present invention encompasses all variations, combinations and permutations of introducing one or more limitations, elements, clauses and descriptive terms from one or more listed claims into another claim. Where elements are presented as a list (e.g., in Markush group format) or as an alternative, each subgroup of elements is also disclosed, and any one or more elements may be removed from the group.

[0323] It should be understood that, in general, when the present invention or various aspects of the present invention are referred to as including specific elements and / or features, certain embodiments of the present invention or various aspects of the present invention are composed of or substantially consist of these elements and / or functions. It should also be noted that the terms "comprising" and "containing" are intended to be open and allow the inclusion of additional elements or steps. Where ranges are given, endpoints are also included. In addition, unless otherwise indicated or apparent from the context and understanding of a person of ordinary skill in the art, in different embodiments of the present invention, the values ​​expressed as ranges may assume any specific value or sub-range within the range to be one-tenth of the unit of the lower limit of the range, unless the context clearly specifies otherwise.

Claims

1. A method for producing a heterologous polypeptide product in Chinese hamster ovary (CHO) cells, the method comprising: reducing the expression level of the endogenous protein Lamb1 in the Chinese hamster ovary cell by an amount sufficient to increase the production of the heterologous polypeptide, culturing the Chinese hamster ovary cell under conditions suitable for producing the heterologous polypeptide product, Thereby producing the heterologous polypeptide product.

2. The method according to claim 1, further comprising the step of recovering the heterologous polypeptide product.

3. A method for producing Chinese hamster ovary cells, the method comprising: reducing the level of endogenous protein Lamb1 in the Chinese hamster ovary cells, The Chinese hamster ovary cells have increased production of a heterologous protein product compared to Chinese hamster ovary cells of the same type that do not have reduced levels of non-essential, redundant genes encoding endogenous proteins.

4. The method according to claim 1 or 3, wherein the expression level of two or more endogenous proteins is reduced.

5. The method of claim 4, wherein the expression levels of three endogenous proteins are reduced, wherein the endogenous proteins are: Lamb1, Mfge8 and Sbsn; B2m, C1S and Lamb1; C1S, Dcn and Lamb1; C1S, Lamb1 and Nid1; or Dcn, Lamb1 and Nid1.

6. The method of claim 1 or 3, wherein reducing the expression level of an endogenous protein in the Chinese hamster ovary cells comprises knocking out from the genome of the Chinese hamster ovary cells a copy of a regulatory nucleic acid sequence encoding the gene of the endogenous protein or operably coupled to the gene encoding the endogenous protein.

7. The method of claim 1 or 3, wherein reducing the expression level of an endogenous protein in the Chinese hamster ovary cells comprises knocking out from the genome of the Chinese hamster ovary cells all copies of the gene encoding the endogenous protein or a regulatory nucleic acid sequence operably coupled to the gene encoding the endogenous protein.

8. The method of claim 6 or 7, wherein knockout comprises using a CRISPR-Cas9 protein in combination with an RNA specific for a gene encoding the endogenous protein or a regulatory nucleic acid sequence operably coupled to the gene encoding the endogenous protein.

9. The method of claim 1 or 3, wherein the increased production of the heterologous polypeptide product is an increase in product production rate (qP) of at least 20% compared to the same type of Chinese hamster ovary cells that do not have reduced levels of endogenous protein.

10. The method of claim 4, wherein the endogenous proteins are Lamb1, Mfge8, and Sbsn; C1S, Dcn, and Lamb1; and wherein the increased production of the heterologous polypeptide product is at least a 20% increase in product titer compared to Chinese hamster ovary cells of the same type that do not have reduced levels of the endogenous proteins.

11. The method of claim 1 or 3, wherein the heterologous polypeptide is a monoclonal antibody.

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