Method for improving production of biological products by reducing level of endogenous proteins

Abstract

The present disclosure provides methods, cells or cell lines, and compositions for increasing the amount of products, e.g., proteins, produced by cells or cell lines by reducing the level of non-essential endogenous proteins.

Description

[0001] Cross-reference to Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 697,480, filed Jul. 13, 2018, the content of which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to methods and compositions for modifying host cell expression to increase the yield of a product (e.g., a polypeptide) expressed in cultured cells. BACKGROUND OF THE INVENTION

[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] Accordingly, there is a need to develop and produce methods for generating products (e.g., polypeptides) expressed in cultured cells. SUMMARY OF THE INVENTION

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

[0007] In one aspect, the invention relates to a method of making 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] thereby making the product.

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

[0011] providing a cell (e.g., a production cell),

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

[0013] culturing the cell under conditions suitable for producing the product (e.g., a recombinant polypeptide),

[0014] thereby making the product.

[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., the production cell) comprises a deletion, substitution, or insertion mutation of a copy of the gene encoding the endogenous protein or a regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein, e.g., wherein the mutation reduces the expression level of the endogenous protein.

[0016] 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 comprises an siRNA that 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 present invention relates to a product (e.g., a recombinant polypeptide) produced by the methods or cells described herein.

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

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

[0020] In an embodiment, the product (e.g., a 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 with another amino acid. In some embodiments, one or more cysteines are replaced with one or more serine residues.

[0022] In one aspect, the present disclosure features a method for producing a product described herein in a cell (e.g., a recombinant host cell, e.g., a production cell). In one embodiment, the product is a polypeptide, e.g., 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 / moiety is the result of genetic engineering. The 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 in sufficient quality for commercial or therapeutic use), such as next-generation biologics (e.g., fusion proteins, bispecific or multiformat antibody molecules, multimeric proteins, and glycosylated proteins). In one embodiment, for example, the cells described herein for producing the product express the product. In one embodiment, the cells include exogenous nucleic acid encoding the product described herein (e.g., a polypeptide selected from Tables 1, 2, 3, or 4). Additional examples of products are described in the section entitled "Products".

[0024] Compared to cells that are not subjected to a reduced temperature, 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 a polypeptide into a less active form); aggregation (e.g., a reduction in aggregates or aggregation); proper folding or assembly (e.g., a reduction in misfolded or unfolded products; or 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 modifications); fragmentation (e.g., a reduction in fragmentation); disulfide scrambling (e.g., a reduction in undesired isomers or structures due to disulfide scrambling). In one embodiment, for example, compared to the quality of a product produced by cells that are not subjected to a reduced temperature, the quality of the product (e.g., a recombinant polypeptide) is improved by, for example, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold.

[0025] In an embodiment, the method for producing a product described herein can be carried out 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 improvement in ER processing capacity (ER expansion) or secretion; obtaining the product from the cell or the progeny of the cell, or from the 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 structural moieties). In one embodiment, the method further comprises the 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 an additional step for improving the secretion capacity or secretion rate by modulating the SNARE mechanism or other mechanisms involved in the secretion 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; polyproteins 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 a protein having a complex and / or non-natural structure, such as next-generation biologics (e.g., bispecific antibody molecules, fusion proteins or glycosylated proteins).

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

[0029] In any of the compositions, formulations, bioreactors or methods 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), DART (dual-affinity retargeting or redirecting T cell).

[0030] In one embodiment, the product (e.g., 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 cell

[0033] Provided herein are cells (e.g., recombinant cells, e.g., production cells) for producing the products described herein, methods of engineering such cells, and methods of using the cells.

[0034] In any of the compositions, formulations, or methods described herein, the cell is a eukaryotic cell. In one embodiment, the cell is a mammalian cell, a yeast cell, an insect cell, an algal 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 of the compositions, formulations, or methods described herein, the cell is selected from the group consisting of: HeLa, HEK293, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, 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 algal cell. In one embodiment, the cell is a prokaryotic cell.

[0037] In any of the methods or cells (e.g., production cells) described herein, the cell expresses or comprises a product, such as a recombinant product, such as a next-generation biologic selected from the group consisting of: bispecific antibodies, fusion proteins, or glycosylated proteins.

[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 meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in 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 in their entirety. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, subheadings, or numbered or lettered elements (e.g., (a), (b), (i), etc.) are presented only for ease of reading. The use of headings or numbered or lettered elements in this document does not require steps or elements to be in alphabetical order or to be discrete from one another. Other features, objects, and advantages of the present invention will become apparent from the specification, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A A graph showing the cell-specific productivity of Mab in a CHO cell line, where a selected non-essential, redundant, and / or secreted endogenous protein was knocked out relative to CHO cells transfected with a vector lacking the gRNA.

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

[0042] The present disclosure features methods and compositions for obtaining higher yields of a product by reducing the levels of endogenous proteins (e.g., non-essential, redundant, and / or secreted endogenous proteins) in cells (e.g., production cells) capable of producing the product (e.g., polypeptide). The methods described herein and the cells used in the methods exhibit quality improvements in yield and quality compared to those obtained by current production methods and / or cells. The methods and compositions described herein can also be used with engineered cells or cell lines having improved productivity, product quality, robustness, and / or culture viability compared to current cell expression systems used for producing recombinant products.

[0043] The methods are suitable for use in the production of next-generation biologic products. As these methods continue to gain therapeutic utility in patients, the need for the efficient development of large quantities of next-generation biologic products of high quality grade for therapeutic use, as well as efficient production methods and production cell lines, will continue to increase. In addition, many next-generation biologic products are difficult to express and produce in conventional cell lines using conventional expression techniques known in the art. Current methods are not sufficient to produce these products in large quantities and do not achieve the high quality grade required for clinical use. The methods described herein, including methods for reducing the level of endogenous proteins, overcome these obstacles and can be combined with other methods to increase product yield.

[0044] Definitions

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice and / or testing of the present invention, the preferred materials and methods are described herein. When describing and claiming the present invention, the following terms will be used in accordance with the definitions provided as follows, as defined in the manner in which they are used.

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

[0047] The article "a / an" is used herein to refer to one or more than one (i.e., at least one) of the grammatical objects 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 that is from or naturally produced inside an organism, cell, tissue, or system.

[0049] As used herein, "exogenous" refers to any material that is introduced into or produced outside an organism, cell, tissue, or system. Thus, "exogenous nucleic acid" refers to a nucleic acid that is 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 or cannot be found in nature inside the organism, cell, tissue, or system into which the exogenous nucleic acid is introduced. In an embodiment, a product that is not naturally occurring or contains a non-naturally occurring portion is an exogenous material relative to the host cell 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 particular species. In embodiments, heterologous materials also encompass materials that include portions or non-naturally occurring portions of one or more species. For example, in one embodiment, a nucleic acid encodes a fusion protein, where 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 containing 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 that make up the protein or peptide sequence. In one embodiment, a protein can comprise more than one (e.g., two, three, four, five, or more) polypeptides, where each polypeptide associates with one another by covalent or non-covalent bonds / interactions. A polypeptide encompasses any peptide or protein that includes two or more amino acids linked to one another by peptide bonds or by means other than peptide bonds. As used herein, the terms refer to both 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, of which there are many types). "Polypeptide" includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc.

[0052] A "recombinant product" refers to a product that can be produced by a cell or a cell-free system. The product can be a molecule, nucleic acid, polypeptide (e.g., an SS polypeptide), or any hybrid thereof. A recombinant product is a product formed by genetic engineering of at least one component of the product or at least one nucleotide of a sequence that controls the production or expression of the product. The genetic engineering used herein to generate recombinant products or constructs encoding recombinant products encompasses 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 while another portion of the recombinant product is non-naturally occurring. In another embodiment, the first portion of the recombinant product is a naturally occurring molecule while 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 the cells described herein. A recombinant polypeptide is a product of genetic engineering or manipulation of (a cell or a precursor cell) to form at least one nucleotide of a sequence encoding the polypeptide or at least one nucleotide of a sequence controlling the expression of the polypeptide. For example, altering at least one nucleotide, such as introducing it into a cell, or it is a product of genetic engineering rearrangement. In one embodiment, the sequence of the recombinant polypeptide is indistinguishable from the natural or non-natural occurring isomers of the polypeptide or protein. In one embodiment, the amino acid sequence of the recombinant polypeptide is different from the sequence of the natural or non-natural occurring isomers 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 the same as or substantially the same as, or differs 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% from the polypeptide encoded by the endogenous genome of the cell. 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, e.g., CHO), other mammalian cell, insect cell, plant, fungal or bacterial cell. 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-natural occurring source. In one embodiment, the recombinant polypeptide is a human polypeptide or protein, and its amino acid sequence is indistinguishable from the natural or non-natural occurring isomers of the human polypeptide or protein. In one embodiment, the recombinant polypeptide differs from the natural or non-natural occurring isomers of the human polypeptide or protein by no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues. In one embodiment, the recombinant polypeptide differs from the natural occurring isomers of the human polypeptide by no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or 15% of its amino acid residues. In embodiments where a portion of the recombinant polypeptide comprises a sequence portion derived from a natural or non-natural occurring isomer of a human polypeptide, the portion of the recombinant polypeptide differs from the corresponding portion of the natural or non-natural occurring 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] The terms "oxygenation level" and "level of oxygenation" are used interchangeably and, as used herein, refer to the level of dissolved oxygen in a given volume of liquid or gas (e.g., in a culture). The oxygenation level can be described in units of dissolved oxygen tension (DOT), e.g., as a percentage of air saturation (e.g., a percentage of the oxygen level in air).

[0055] As used herein, a production culture refers to a mixture of growth medium and cells (e.g., at least one cell), such as recombinant cells, e.g., recombinant host cells that express 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 undergoes one or more culture phases including cell growth / division characteristics (e.g., sequential and / or overlapping culture phases). In some embodiments, the treatment of the production culture changes as described herein as the production culture progresses through different phases. As used herein, a production phase refers to a production culture phase that begins with inoculation and ends when a specific criterion is reached (e.g., the end of a process or time period, e.g., 10 - 30 days, e.g., 15 - 20 days) or when a specific cell viability criterion is reached (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20 or 10% viability, e.g., less than 50% viability). The production phase can include an exponential growth phase in which cells grow and / or divide into exponential growth. The production phase can include a post - growth phase after the exponential growth phase, in which cell growth and / or division is in the linear phase of the growth curve, e.g., the rate of cell growth and / or division has decreased relative to the exponential growth phase. In some embodiments, the completion of the production phase is when the production culture has produced a production mixture. In some embodiments, the separation of cells (e.g., harvesting) from the production supernatant and further separation or purification steps (e.g., post - harvest) are in a phase 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), growth medium, and cells (e.g., at least one cell, e.g., a recombinant cell, e.g., a recombinant host cell capable of expressing the 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, a culture medium or a production culture can be transferred between operably coupled containers. In one embodiment, the flow of the culture between operably coupled containers occurs in a controlled manner, e.g., using pumps, filters, sensors, devices for maintaining or changing culture conditions, and / or devices for monitoring liquid flow.

[0058] As used herein, operably linked 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), where the first nucleic acid sequence can affect the second nucleic acid sequence, for example, by influencing transcription, epigenetic modification, and / or chromosomal topology. In some embodiments, operably linked means that two nucleic acid sequences are contained on the same nucleic acid molecule. In another embodiment, operably linked can further mean that two nucleic acid sequences are adjacent to each other on the same nucleic acid molecule, for example, within 1000, 500, 100, 50, or 10 base pairs of each other or directly adjacent to each other. In one embodiment, a promoter or enhancer sequence operably linked to a sequence encoding a protein can promote the transcription of the sequence encoding the protein, for example, in a cell capable of transcription or a cell-free system.

[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, the cells of the production culture, or the supernatant from the production culture at different temperatures during different stages. As used herein, T 培养 is the temperature from the start of inoculation to the end of the production stage, for example, growing the cells of the production culture in a growth medium to produce the production culture (where a protein is produced). In some embodiments, T 培养 is from 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 is less than 35 °C, 34 °C, 33 °C, 32 °C, 31 °C, or 30 °C, for example, 30 °C - 33 °C. As used herein, T 收获 is the second temperature to which the production culture is cooled before harvest (e.g., separating the cells from the supernatant). In some embodiments, T 收获 is 29 °C, 28 °C, 27 °C, 26 °C, 25 °C, 24 °C, 23 °C, 22 °C, 21 °C, 20 °C, 19 °C, 18 °C, 17 °C, 16 °C, 15 °C, 14 °C, 13 °C, 12 °C, 11 °C, 10 °C, 9 °C, 8 °C, 7 °C, 6 °C, 5 °C, or 4 °C. In one embodiment, T 收获 is from 12 °C to 18 °C, for example, 15 °C. In one embodiment, T 收获 is 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 is 15 °C ± 3 °C.

[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 cooperate with the gRNA molecule to home or localize to a site comprising 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 at least one amino acid residue). Exemplary Cas9 molecule or Cas9 polypeptide sequences can be found in WO2015 / 157070, which is hereby incorporated by reference in its entirety. The Cas9 molecule or Cas9 polypeptide includes a Cas9 molecule having DNA cleavage 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 invention has been disclosed with reference to specific aspects, it will be apparent to other skilled artisans in the art that other aspects and variations of the invention can be devised without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

[0062] Reduce the level of endogenous protein

[0063] The present disclosure relates in part to methods of making 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 improving one or more production cell properties (e.g., specific product production rate (qP), growth rate, product yield, and / or viable cell concentration) can be achieved by reducing the level of one or more endogenous proteins. Endogenous proteins may consume space and / or resources (e.g., translation, post-translational processing, and secretion space / resources) in the endoplasmic reticulum or Golgi apparatus, thereby reducing the ability of the production cell to produce a product (e.g., a recombinant polypeptide). In the artificial environment of biomanufacturing (e.g., bioreactors and cell cultures), endogenous proteins may also be functionally redundant to the cell (e.g., a 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 its one or more endogenous proteins has not been 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 assessed, for example, by the method described in Porter, A. J. et al. (2010), " Biotechnological Advances ( Biotechnol Prog )" 26(5):1446-1454.

[0065] In some embodiments, reducing the levels 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 formation rate. In some embodiments, qP is expressed in units of product per unit time per unit of production cell (e.g., grams or viable cell count). In some embodiments, qP is improved, e.g., 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 higher than the qP of a similar cell (e.g., a production cell) in which the levels of one or more endogenous proteins have not been reduced. In some embodiments, qP is improved, e.g., 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% higher than a similar cell (e.g., a production cell) in which the levels of one or more endogenous proteins have not been reduced. qP can be evaluated, for example, by quantifying a secreted protein product by ELISA or quantitative chromatography (e.g., Protein A HPLC).

[0066] In some embodiments, reducing the levels of one or more endogenous proteins in a cell (e.g., a production cell) can improve cell growth, such as the rate of cell growth and / or division. In some embodiments, cell growth is improved, such as 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 the cell growth of a similar cell (e.g., a production cell) in which the levels of one or more endogenous proteins have not been reduced. In some embodiments, cell growth is improved, such as 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% compared to a similar cell (e.g., a production cell) in which the levels of one or more endogenous proteins have not been reduced. Cell growth can be evaluated, for example, by making a time measurement of the viable cell concentration using an automated trypan blue exclusion assay (e.g., using a Beckman coulter Vi-CELLXR).

[0067] In some embodiments, reducing the levels of one or more endogenous proteins in a cell (e.g., a production cell) can improve product yield, e.g., the amount of product produced in a culture. In some embodiments, the product yield of a cell culture (e.g., a production cell culture) in which the levels of one or more endogenous proteins are reduced 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., production cells) in which the levels of one or more endogenous proteins are not reduced. In some embodiments, the 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% compared to a similar culture of similar cells (e.g., production cells) in which the levels of one or more endogenous proteins are not reduced. Product yield can be evaluated, for example, by quantifying secreted protein products, e.g., by ELISA or quantitative chromatography (e.g., Protein A HPLC).

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

[0069] Endogenous protein

[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, e.g., in a biomanufacturing context) and / or secreted proteins that are functionally redundant with other endogenous proteins whose levels may not be reduced. Based on the disclosure herein and the state of the art, one of ordinary skill in the art will be able to identify, with a minimum of experimentation, which endogenous proteins of a cell (e.g., a CHO cell) are non-essential, redundant, and / or secreted proteins.

[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 method 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 method of Example 1). 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).

[0072] In some embodiments, the endogenous protein whose level may be reduced is a protein associated with functions such as redundancy, non-essentiality, or secretion in a protein function database (e.g., the PANTHER database of Example 1). The endogenous protein whose level may be reduced is associated with the functional keywords listed in Table E1 or a functionally associated substantially similar function. 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 functions such as redundancy, non-essentiality, or secretion in a protein function database (e.g., the PANTHER database of Example 1) (e.g., associated with the keywords 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 functions such as redundancy, non-essentiality, or secretion in a protein function database (e.g., the PANTHER database of Example 1) (e.g., associated with the keywords 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 functions such as redundancy, non-essentiality, or secretion in a protein function database (e.g., the PANTHER database of Example 1) (e.g., associated with the keywords 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 critical or highly 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 or substantially similar functional association listed in Table E2. In some embodiments, the endogenous protein whose level may be reduced is not associated with cell metabolism, mitochondrial function, cell structure, DNA replication, cell division, checkpoint inhibition / passage, transcription, translation, post-translational modification, protein folding, or the heat shock response.

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

[0076] In some embodiments, the methods disclosed herein include reducing the levels of more than one endogenous protein. In some embodiments, the methods disclosed herein include reducing the levels of 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 levels 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 levels of two or three endogenous proteins (e.g., a combination of endogenous proteins selected from those listed in Table E5).

[0077] Methods for reducing the levels of endogenous proteins

[0078] The present disclosure relates in part to methods of making 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., the production cell). In some embodiments, reducing the level of an endogenous protein in the cell (e.g., the 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., the 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., the 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., the 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, eliminating (e.g., knocking out) includes introducing a deletion, substitution, or insertion mutation into the gene encoding the endogenous protein or the regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein, such as wherein the mutation reduces the expression level of the endogenous protein. In some embodiments, eliminating (e.g., knocking out) includes introducing a deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more base pairs in the gene encoding the endogenous protein or the regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein. In some embodiments, eliminating (e.g., knocking out) includes substituting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more base pairs for different base pairs (e.g., a transition or transversion mutation) in the gene encoding the endogenous protein or the regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably coupled to the gene encoding the endogenous protein.

[0080] For example, an insertion mutation that disrupts the reading frame of the gene encoding the endogenous protein or a substitution mutation that alters the start codon of the gene encoding the endogenous protein are examples of ablation (e.g., knockout) that reduces the level of the endogenous protein. As another example, ablation (e.g., knockout) can include inserting a marker (e.g., an antibiotic resistance, auxotrophic rescue, or fluorescent marker-encoding gene) in a manner that deletes the promoter or a portion of the promoter operably linked to the gene encoding the endogenous protein. In some embodiments, ablation (e.g., knockout) results in a complete loss of production of the endogenous protein from the copy of the gene encoding the endogenous protein. In other embodiments, ablation (e.g., knockout) results in a lower level of production of the endogenous protein (e.g., a functional endogenous protein). In other embodiments, ablation (e.g., knockout) results in the production of a variant of the endogenous protein that is one or more of the following: truncated, hypofunctional, non-functional, misfolded, and / or prone to degradation.

[0081] Elimination (e.g., knockout) of a gene encoding an endogenous protein or a copy of a regulatory element operably linked to said gene can be achieved by any gene editing system known in the art. Exemplary gene editing systems include the clustered regularly interspaced short palindromic repeats (CRISPR) system, zinc finger nucleases (ZFNs), and transcription activator-like effector-based nucleases (TALENs). ZFNs, TALENs, and CRISPR-based methods are described, for example, in Gaj et al., Trends Biotechnol., 31.7 (2013):397-405; the CRISPR method of gene editing, 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 [e-pub 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 know these and other options for editing the genome (e.g., knocking out a gene) in a cell (e.g., a production cell).

[0082] In some embodiments, elimination (e.g., knockout) of a gene encoding an endogenous protein or a copy of a regulatory element operably linked to said gene comprises using a combination of a CRISPR-Cas9 molecule (e.g., a Cas9 molecule) and an RNA (e.g., a gRNA, sgRNA, and / or tracrRNA) that is specific for the endogenous protein-encoding gene or a regulatory nucleic acid sequence (e.g., a promoter or enhancer) operably conjugated 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 the mRNA transcript encoding the endogenous protein in the cell (e.g., knockout). In some embodiments, reducing the level of the mRNA transcript encoding the endogenous protein (e.g., knockout) includes using siRNA, which is capable of binding to a nucleic acid (e.g., the mRNA encoding the endogenous protein or a regulatory nucleic acid sequence operably linked thereto (e.g., a promoter or enhancer)). Those 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: the Dharmacon Horizon siDesign tool, the InvivoGen siRNA Wizard, the GenScript siRNA Construct Services, the IDT CustomDicer-Substrate siRNA, the Sigma-Aldrich siRNA Design Service, or those taught by www.rnaiweb.com / RNAi / siRNA_Design / . In some embodiments, reducing the level of the mRNA transcript encoding the endogenous protein (e.g., knockout) results in a reduction of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% in the level of the 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 a product 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, polyproteins, 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 has a complex and / or non-native structure, such as next-generation biologics (e.g., bispecific antibody molecules, fusion proteins, or glycosylated proteins).

[0086] In embodiments, the cells or cell lines generated by the methods or compositions described herein produce 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 deficiencies), endocrine disorders (e.g., hormone deficiencies), hemostatic disorders, thrombosis, hematopoietic disorders, pulmonary conditions, gastrointestinal conditions, autoimmune diseases, immune disorders (e.g., immunodeficiencies), infertility, transplantation, cancer, and infectious diseases.

[0087] In embodiments, the product is an exogenous protein, e.g., a protein that the cell does not naturally express. 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 natural level. The methods and compositions described herein are used to increase the production and quality of endogenous products (e.g., naturally occurring products naturally produced by the cell). In another embodiment, an exogenous nucleic acid encoding the product (e.g., a 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 controlling the expression of an endogenous product of the cell.

[0089] The recombinant product can be a therapeutic or diagnostic product, e.g., useful for drug screening. The therapeutic or diagnostic product can include but is not limited to antibody molecules, e.g., antibodies or antibody fragments, fusion proteins, hormones, cytokines, growth factors, enzymes, glycoproteins, lipoproteins, reporter proteins, therapeutic peptides, or structural and / or functional fragments or hybrids of any of these. In other embodiments, the therapeutic or diagnostic product is a synthetic polypeptide, e.g., where the entire polypeptide or a portion thereof is not derived from any naturally occurring polypeptide (e.g., the aforementioned naturally occurring polypeptides) or has no 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, e.g., a monoclonal antibody useful 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 multi-form proteins can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer 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 a full-length antibody or a recombinant variant thereof, and refers to an antigen-binding domain (e.g., the antigen-determining variable region of a full-length antibody) sufficient to confer the ability to recognize and specifically bind 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)), camelid VHH domains, and multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment including two Fab fragments linked by a disulfide bridge through a hinge region), as well as isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, large antibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23:1126-1136, 2005). Antigen-binding fragments can also be grafted into scaffolds based on polypeptides (e.g., type III fibronectin (Fn3)) (see U.S. Patent 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), agalsidase alfa, daptomycin, YH-16, chorionic gonadotropin alfa, filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleukin, denileukin-diftitox, interferon alfa-n3 (injectable), interferon alfa-nl, DL-8234, interferon, Suntory (γ-la), interferon gamma, thymosin alfa1, tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif, eterotide alfa, teriparatide, calcitonin, etanercept, glutathion Hb250 (bovine), clostridiopeptidase alfa, collagenase, captoprilatide, recombinant human epidermal growth factor, DWP401, darbepoetin alfa, epoetin omega, epoetin beta, epoetin alfa, desirudin, lepirudin, bivalirudin, nonacog alfa, Mononine, hexaferon alfa (activated), recombinant factor VIII+VWF, Recombinate, recombinant factor VIII, factor VIII (recombinant), Alphanate, octocog alfa, factor VIII, palifermin, Indikinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alfa, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, imiglucerase, sulfatase, Leucotropin, molgramostim, triptorelin acetate, histrelin (Hydron), deslorelin, histrelin, nafarelin, leuprorelin (ATRIGEL), leuprorelin (DUROS), goserelin, Eutropin, somatropin, mecasermin, enfuvirtide, Org-33408, insulin glargine, insulin lispro, insulin (inhaled), insulin lispro protamine, insulin detemir, insulin (RapidMist), mecasermin-rinfabate, anakinra, simtuzumab, 99mTc-acesulfame, myelopid, Betaseron, glatiramer acetate, Gepon, sargramostim, oprelvekin, human leukocyte-derived alpha interferon, Bilive, insulin (recombinant), recombinant human insulin, insulin aspart, mecasermin, Roferon-A, interferon-α2, Alfaferone, interferon alfacon-1, interferon alfa, Avonex recombinant human luteinizing hormone, streptodornase alfa, treflan, ziconotide, tatserelin, dibotermin alfa, atosiban, becaplermin, eptifibatide, Zemaira, CTC-111, Shanvac-B, octreotide, lanreotide, anastellin, beta-galactosidase, alpha-galactosidase,Laronidase, Cuprizone acetate, 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, Idursulfase, Omaveloxolone, Recombinant serum albumin, Cimzia, Carboxypeptidase G2, Human recombinant C1 esterase inhibitor, Lanoteplase, Recombinant human growth hormone, Enfuvirtide, VGV-1, Interferon (α), Lusianthridin, Apetider, Icatibant, Ecallantide, Omiganan, Aurograb, Perampanel acetate, ADI-PEG-20, LDI-200, Degarelix, Besifloxacin, Favld, MDX-1379, ISAtx-247, Liraglutide, Teriparatide, Tefacitinib, AA4500, T4N5 Liposomal Lotion, Catumaxomab, DWP413, ART-123, Chrysalin, Desmoteplase, Anistreplase, Choriogonadotropin alfa, TH-9507, Teduglutide, Diamyd, DWP-412, Growth hormone, Recombinant G-CSF, Insulin, Insulin (Technosphere), Insulin (AERx), RGN-303, DiaPep277, Interferon beta, Interferon α-n3, Belatacept, Transdermal insulin patch, AMG-531, MBP-8298, Xerecept, Opaganib, AIDSVAX, GV-1001, LymphoScan, Bombesin, Lipoxysan, Lulupetide, MP52, Ciluprase-T, CTP-37, Insegia, Vitespen, Human thrombin, Thrombin, TransMID, Afibrinase, Puricase, Terlipressin, EUR-1008M, Recombinant FGF-1, BDM-E, Rotigaptide, ETC-216, P-113, MBI-594AN, Duramycin, SCV-07, OPI-45, Endostatin, Angiostatin, ABT-510, Bowman-Birk inhibitor, XMP-629, 99mTc-Hynic-Annexin V, Kahalide F, CTCE-9908, Tiverek, Ozeremide, Romidepsin, BAY-504798, Interleukin 4, PRX-321, Pepscan, Alferon, Recombinant human lactoferrin (rhlactoferrin), TRU-015, IL-21, ATN-161, Cetuximab, Albuferon, Biphasix, IRX-2, Interferon ω, PCK-3145, CAP-232, Pasireotide, huN901-DMI, SB-249553, Oncovax-CL, OncoVax-P, BLP-25,CerVax-16, MART-1, gp100, tyrosinase, nemifitide, rAAT, CGRP, pegcetacoplan, thymosin β4, piltidepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (eligen), exenatide, camorilin, Cardeva, vilafermin, 131I-TM-601, KK-220, T-10, ulinastatin, delisert, heamorphin, Chrysalin, rNAPc2, recombinant factor VII (PEGylated liposome), bFGF, PEGylated recombinant staphylokinase 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 (injectable, Viadel), insulin (eligen), recombinant methionyl human leptin, pitumastatin, Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10, talactoferrin, rEV-131, rEV-131, recombinant human insulin, RPI-78M, oprelvekin, CYT-99007 CTLA4-Ig, DTY-001, varast, interferon α-n3, IRX-3, RDP-58, Tauferon, bile salt-stimulated lipase, Merispase, alkaline phosphatase, EP-2104R, Melanotan-II, bumetanide, 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(1-34) (Novasome), Ostabolin-C, PTH analogue, MBRI-93.02, MTB72F, MVA-Ag85A, FARA04, BA-210, recombinant Yersinia pestis F1V, AG-702, OxSODrol, rBetV1, Der-pl / Der-p2 / Der-p7, PR1 peptide antigen, mutant ras vaccine, HPV-16E7 lipopeptide vaccine, laminin, WT1-peptide, IDD-5, CDX-110, Pentrys, Norelin,CytoFab, P-9808, VT-111, elotuzumab, teplizumab, lopinavir, raltegravir, 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, abatacept, 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, glycosylated PEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, factor XIII, aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, teverelkin, EP-51216, hGH, OGP-1, T-20, TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin, r(m)CRP, liver-selective insulin, Subalin, 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(1-34), 768974, SYN-101, PGN-0052, aviculmin, BIM-23190, multi-epitope tyrosinase peptide, enocitabine,APC-8024, GI-5005, ACC-001, TTS-CD3, vascular-targeted TNF, desmopressin, anakinra, and TP-9201.

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

[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 embodiments, the polypeptide is a hormone, blood clotting / coagulation factor, cytokine / growth factor, antibody molecule, fusion protein, protein vaccine, or 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 embodiments, the protein is a multispecific protein, such as the bispecific antibodies shown in Table 3.

[0108] Table 3: Bispecific Formats

[0109]

[0110]

[0111]

[0112]

[0113] In the embodiment, the product is the 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 cancer cells. 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 / CA IX), CCR5, CCR8, CD19, CD22, CD25, CD27, CD30, CD33, CD38, CD44v6, CD63, CD70, CC123, CD138, carcinoembryonic antigen (CEA; CD66e), desmoglein 4, novel epitope of E-cadherin, 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 mullerian 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), the activating 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 activating 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, inhibitory forms of KIR, KLRG1, LAIR-1, NKG2A, NKR-P1A, Siglec-3, Siglec-7, Siglec-9, and combinations thereof, optionally wherein the inhibitory forms of KIR include 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, 0X34), 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), CRTAM (CD355), DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LTβR (TNFRSF3), 0X40 (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), CD160 (BY55, NK28), CD352 (Lyl08, NTBA, SLAMF6), CD358 (DR6), CTLA-4 (CD152), 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 Table 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, analogue, or functional fragment of the recombinant polypeptides 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, e.g., a recombinant polypeptide) further comprises a nucleic acid sequence encoding a selectable marker. In one embodiment, the selectable marker includes glutamine synthetase (GS); dihydrofolate reductase (DHFR), e.g., an enzyme conferring resistance to methotrexate (MTX); proline or an antibiotic marker, e.g., an enzyme conferring resistance to an antibiotic (e.g., hygromycin, neomycin (G418), bleomycin, puromycin, or blasticidin). In another embodiment, the selectable marker includes the Selexis selection system (e.g., the SUREtechnology Platform TM and Selexis GeneticElements TM ) or Catalant Biologies cell line development technology or is compatible therewith.

[0130] In one embodiment, the vector comprising a nucleic acid sequence encoding a recombinant product described herein includes a selectable marker that can be used to identify one or more cells comprising the nucleic acid encoding the recombinant product described herein. In another embodiment, as described herein, the selectable marker can be used to identify one or more cells comprising integration of the nucleic acid sequence encoding the recombinant product into the genome. Identification of one or more cells that have integrated the nucleic acid sequence encoding the recombinant protein can be used for selection and engineering of cells or cell lines that stably express the product.

[0131] In one embodiment, the product differs from the polypeptides 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 the polypeptides from Table 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 were discussed above.

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

[0133] Other exemplary therapeutic or diagnostic proteins include, but are not limited to, any of the proteins described in Table 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 incorporated herein by reference); or any conjugates, variants, analogs, or functional fragments of the recombinant polypeptides described herein.

[0134] Production applications

[0135] The 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 in bioreactors or processing vessels or tanks, or more generally with any source. The devices, facilities, and methods described herein are suitable for culturing any desired cell line, including prokaryotic and / or eukaryotic cell lines. Additionally, in embodiments, the devices, facilities, and methods are suitable for culturing suspension cells or adherent-dependent (adherent) cells and are suitable for production operations configured for the production of pharmaceutical and biopharmaceutical products (e.g., polypeptide products or cells and / or viruses, such as those used in cell and / or virus therapies).

[0136] As mentioned, in embodiments, the apparatus, facility, and method allow for the production of eukaryotic cells, such as 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 cell products of eukaryotic or prokaryotic cells (e.g., proteins, peptides, antibiotics, amino acids that are synthesized on a large scale by eukaryotic cells). Unless otherwise specified herein, the apparatus, facility, and method 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 specified, the devices, facilities and methods may include any one or more suitable reactors, including but not limited to stirred tanks, air-lift, fibrous, microfibrous, hollow fiber, ceramic matrix, fluidized bed, fixed bed and / or spouted bed bioreactors. As used herein, "reactor" or "bioreactor" may include a fermenter or fermentation unit or any other reaction vessel, and the terms "reactor" and "bioreactor" may be used interchangeably with "fermenter". For example, in some aspects, a bioreactor unit may perform 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 medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring) and / or cleaning / disinfection. An exemplary reactor unit (e.g., a fermentation unit) may contain multiple reactors within the unit. For example, the unit may 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 the facility may contain multiple units having one or more reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-batch feed, fed-batch, perfusion and / or continuous fermentation processes. Any suitable reactor diameter may be used. In an embodiment, the bioreactor may have a volume of about 100 mL and about 50,000 L. Non-limiting examples include volumes of 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, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters and / or 50,000 liters. Additionally, suitable reactors may be multi-purpose, single-purpose, disposable or non-disposable, and may 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 may be circular (e.g., cylindrical).In some embodiments, a suitable reactor can be square (e.g., rectangular). In some cases, a square reactor offers advantages over a circular reactor, such as ease of use (e.g., loading and setup by a technician), greater mixing and homogeneity of the reactor contents, and a lower footprint.

[0138] In embodiments and unless otherwise stated herein, the devices, facilities, and methods described herein for use with methods of manufacturing formulations may also include any suitable unit operations and / or equipment not otherwise mentioned, such as operations and / or equipment for separating, purifying, and isolating such products. Any suitable facilities and environments may be used, such as conventional modular facilities, modular movable temporary facilities, or any other suitable construction, facility, and / or layout. For example, in some embodiments, a modular cleanroom may be used. Additionally and unless otherwise stated, the devices, systems, and methods described herein may be housed and / or performed in a single location or facility, or alternatively may be housed and / or performed at 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 entirety) describe exemplary facilities, equipment, and / or systems that may be suitable.

[0140] Bioreactor Setup and Conditions

[0141] In one aspect, the bioreactors of the present disclosure or bioreactors utilized by the methods of the present disclosure are single-use bioreactors.

[0142] A single-use bioreactor may include a bioprocess vessel, a housing, at least one agitator, at least one distributor, at least one gas filter inlet for one or more distributors and headspace covering, 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 vessel. The bioprocess vessel is made of a liquid-impermeable and flexible conformable material. For example, the bioprocess vessel can be made of a flexible membrane (e.g., a multilayer membrane). For example, in one embodiment, the membrane comprises a polyethylene polymer, such as low-density polyethylene that has been modified to form a hydrophilic surface. The hydrophilic surface is used to contact the cell culture within the bioreactor and improve wettability. In one embodiment, the polyethylene polymer is modified by undergoing irradiation, light or plasma-induced or oxidation.

[0144] The bioprocess vessel can have a top, a bottom, and at least one sidewall therebetween. The bioprocess chamber can define a hollow enclosure for containing a culture medium. The hollow enclosure can have any suitable volume, such as 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, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters.

[0145] The bioreactor can comprise at least one inlet for supplying materials into the hollow enclosure of the bioprocess vessel. A mixing device including a rotatable shaft coupled to at least one agitator can extend into the hollow enclosure of the bioprocess vessel. In one embodiment, the rotatable shaft can be retractable. For example, the rotatable shaft can comprise at least one impeller made of a hydrophilic polymer material that can contract or fold towards the rotatable shaft.

[0146] The bioreactor may further comprise at least one baffle configured to extend longitudinally adjacent to the sidewall of the bioprocess vessel. The baffle may have a shape that extends radially inward from the sidewall by an amount sufficient to affect fluid flow in the hollow housing during mixing of the culture medium by the mixing device. The baffle may be collapsible and / or foldable. For example, in one embodiment, the baffle may define an inflatable fluid bladder such that the baffle can be inflated and deflated. The baffle may be integral with the bioprocess vessel, meaning that the baffle is formed from a flexible shape-forming material. Alternatively, the baffle may be separate from the bioprocess vessel. The baffle may be configured to be placed inside the hollow housing or may be placed outside the hollow housing. When placed outside the hollow housing, the sidewall of the bioprocess vessel conforms to the shape of the baffle. For example, in one embodiment, the baffle may be removably attached to the outer metal housing. The bioprocess vessel may be placed in the metal housing to conform to the shape of the baffle. In one embodiment, the bioreactor may comprise from about two to about six baffles spaced around the circumference of the hollow housing of the bioprocess vessel.

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

[0148] The bioreactor may further comprise at least one distributor. For example, the distributor may include a ballast distributor including a gas pipe having a longitudinal portion and a lateral portion. The longitudinal portion may extend vertically into the hollow housing of the bioprocess vessel. 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 for releasing gas into the culture medium contained in the bioprocess vessel. 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 the rotatable shaft of the mixing device to stabilize the shaft. The rotatable shaft may extend through the lateral portion or may be received within 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 in the surface distributor. In one embodiment, the plurality of holes are drilled.

[0150] In one embodiment, a bioreactor can include at least one supply line that extends into a hollow housing to supply fluid into a bioprocess vessel. The supply line can include a submerged fluid outlet positioned adjacent to an agitator. The fluid outlet can 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 can include a one-way valve.

[0151] In another embodiment, a bioreactor can include a supply line located at the top of the bioprocess vessel. The supply line can include a surface fluid drain located above a volume of culture medium residing in the bioprocess vessel. The surface fluid drain can be positioned such that fluid flowing through the fluid drain directly contacts the culture medium contained within the bioprocess vessel. In one embodiment, when rotating, the agitator can form a circumference, and the surface fluid drain of the supply line can be located above the circumference of the agitator such that fluid flowing through the fluid drain contacts the culture medium within the circumference.

[0152] The bioreactor can be placed operably associated with a load cell to indicate the mass of the culture medium contained within the hollow housing. The bottom of the bioprocess vessel can have a convex shape to facilitate drainage. For example, the bioprocess vessel can include a drain line located at the bottom of the bioprocess vessel. A fluid collection device can be positioned between the hollow housing of the bioprocess vessel and the drain line. The fluid collection device can have a shape configured to cause a vortex of fluid from the bioprocess vessel 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 illustrative purposes, for every liter of volume of the hollow housing, the drain line can have a cross-sectional area of about 0.3 mm 2 to about 0.7 mm 2 (e.g., about 0.4 mm 2 to about 0.6 mm 2 ).

[0153] In one embodiment, the bioprocess vessel can include a plurality of ports to connect to a plurality of supply lines for supplying fluid to the bioprocess vessel. Each port and the corresponding supply line can include a matching indication to assist a user in connecting the supply line to the corresponding port. For example, the matching indication can include color such that each port and the corresponding supply line are color-coded. The matching indication can also be applied to the supply line and any corresponding port and to the distributor and any corresponding connector.

[0154] In one embodiment, a bioprocess container can include a port that includes 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 can include a bioprocess container made of a liquid-impermeable and flexible conformable material. The bioprocess container can have a top, a bottom, and at least one sidewall therebetween. The bioprocess chamber can define a hollow enclosure for containing a culture medium. The bioprocess container can further include a plurality of inlets for supplying materials into the hollow enclosure. A drain line can be located at the bottom of the bioprocess container to drain fluid. A mixing device can extend into the hollow enclosure of the bioprocess container and can include a rotatable shaft coupled to at least one agitator.

[0156] The bioreactor system can further include at least one sensor operably associated with the bioprocess container to monitor at least one parameter within the hollow enclosure. The at least one sensor can 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 can be placed in communication with the at least one sensor. The controller can be configured to receive information from the at least one sensor and, based on the information, control a fluid source to vary a flow rate of fluid from the fluid source into the hollow enclosure of the bioprocess container to maintain the at least one parameter of the culture medium contained within the hollow enclosure within a preset limit.

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

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

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

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

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

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

[0163] In one embodiment, the controller can 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 sensors can be integrated into the bioprocess vessel and can be disposable along with the bioprocess vessel.

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

[0166] In one embodiment, the supply line includes a subsurface fluid outlet positioned adjacent to the agitator. The fluid outlet can 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 a fluid discharge port on a surface above a volume of culture medium residing in a bioprocess container. The fluid discharge port on the surface can be positioned such that fluid flowing through the fluid discharge port directly contacts the culture medium contained within the bioprocess container without contacting the sidewalls.

[0168] In one embodiment, a bioreactor can include a first supply line and a second supply line, the first supply line including a sub-surface fluid outlet, and the second supply line including a surface fluid discharge port. In one embodiment, a bioreactor can contain from about one to about five (e.g., from about two to about three) supply lines having surface fluid discharge ports.

[0169] In yet another embodiment, the present disclosure relates to a method for producing a single-use bioreactor. The method includes the steps of 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 enclosure for containing a culture medium. The volume of the hollow enclosure can be from about 1 - 250 milliliters, 250 milliliters to 50 liters, 50 to 800 liters, or 800 - 200,000 liters. The bioprocess container includes a plurality of inlets for supplying materials into the hollow enclosure of the bioprocess container. Each inlet has a certain diameter.

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

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

[0172] According to the present disclosure, the diameter of the inlets, the diameter of the plurality of holes in the distributor, and the cross-sectional area of the drain line are proportional to the volume of the hollow enclosure. For each liter of the volume of the hollow enclosure, the drain line can have a cross-sectional area of from about 0.3 mm 2 to about 0.7 mm 2 of cross-sectional area.

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

[0174] According to the present disclosure, the bioreactor may further include a [] cell retention chamber in fluid communication with the hollow outer shell of the bioprocess vessel. The filtrate outlet may be placed in fluid communication with the cell retention chamber. The filtrate outlet includes a biofilter that is permeable to liquid but impermeable to the biological materials 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 outer shell of the bioprocess vessel and the cell retention chamber for a perfusion process.

[0175] For example, the flow regulator may be in communication with a pressurized gas source and a vacuum source. The flow regulator may be configured to alternately apply a vacuum or air pressure to the fluid contained in the cell retention chamber to circulate the fluid back and forth between the hollow outer shell of the bioprocess vessel 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 that includes a bioprocess vessel made of a liquid-impermeable and flexible conformable material. The bioprocess vessel defines a hollow outer shell for containing a culture medium. A mixing device including a rotatable shaft coupled to at least one agitator may extend into the hollow outer shell of the bioprocess vessel. According to the present disclosure, the agitator may be retractable onto the rotatable shaft. For example, the agitator may include an impeller that includes at least one blade element. The blade element may be folded towards the rotatable shaft. In one embodiment, the rotatable shaft is coupled to a first impeller and a second impeller, and both impellers may include at least one foldable blade element. A retaining ring may be positioned on the shaft. The retaining ring may include an agitator engagement position and an agitator disengagement position to hold the agitator in an upright position (during mixing) or in a retracted and folded position, respectively.

[0178] In one embodiment, the rotatable shaft includes a metal reinforcing rod surrounded by a bushing. The metal reinforcing rod, which may be made of stainless steel, may be made of a plurality of parts attached together. The top of the reinforcing rod may include a magnetic member for magnetically engaging a motor. The bushing 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 bushing and the agitator may include a polyethylene polymer that has been modified by undergoing irradiation, light or plasma-induced or oxidation.

[0179] In one embodiment, a single-use bioreactor system according to the present disclosure includes: a single-use cell culture bioprocess vessel ("SUB"), a reusable housing in which the SUB is maintained 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 distributor system, a supply system, a harvest system, a monitoring system, one or more control systems, and a filling system.

[0180] In one embodiment, each of the cell culture contact surface and the process fluid contact 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 vessel, a housing, at least one agitator, at least one distributor, at least one gas filter inlet for one or more distributors and headspace covering, at least one filling port, at least one harvest port, at least one sample port, and at least one probe.

[0182] The single-use bioreactor of the present disclosure may also be used in conjunction with a single-use bioreactor (SUB) system. The system may include a single-use and disposable flexible bioreactor bioprocess vessel, a SUB housing configured to hold the flexible bioreactor bioprocess vessel, an agitator, a distributor, 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 biomaterials corresponding to those that can be produced in a stainless steel bioreactor of a similar size.

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

[0184] For example, in one embodiment, the top impeller and the bottom impeller are made of a polyolefin polymer (e.g., polyethylene or polypropylene). In one embodiment, low density polyethylene may be used. The low density polyethylene may be modified by exposure to irradiation, light, or plasma-induced or oxidative means to form a hydrophilic surface.

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

[0186] In some embodiments, the bioreactor of the present disclosure is the bioreactor as described in U.S. Patent No. 9,670,446, the content of which is hereby incorporated by reference in its entirety. In some embodiments, the bioreactor of the present disclosure includes a part or feature of the bioreactor described in U.S. Patent No. 9,670,446.

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

[0188] The harvest vessel can include at least one mixer, at least one gas filter inlet for headspace covering, at least one filling port having an internal J-shaped tube pointing to the vessel 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 includes a headspace gas mixture of air / O2 that maintains the DOT within a range of greater than 40% air saturation to less than or equal to 500% air saturation. In one embodiment, the bioreactor or the harvest vessel includes a redox probe, such as an on-line redox probe, such as a Mettler-Toledo on-line redox probe.

[0190] In some embodiments, a method includes a bioreactor or harvest vessel being able to provide (e.g., add) components of the thioredoxin or glutathione / glutaredoxin system or modulate (e.g., increase or decrease) their activity / abundance to provide or maintain a redox potential in a culture or culture cells or culture supernatant. The thioredoxin and glutathione / glutaredoxin systems and their components are known in the art. See, for example, Holmgren et al., Biochem Soc Trans, December 2005; 33(Pt 6):1375-7; Nordberg and Amer, Free Radic Biol Med, December 1, 2001; 31(11):1287-312; Ghezzi P, Biochem Soc Trans, December 2005; 33(Pt 6):1378-81; Ivarsson et al., Diabetes, July 2005; 54(7):2132-42; Sen, CK, Biochem Pharmacol, 55(11), 1747-1758, June 1, 1998; and May et al., J Biol Chem, September 5, 1997; 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 harvest vessel is capable of providing (e.g., adding) GILT (gamma interferon-inducible lysosomal thiol reductase) or modulating (e.g., increasing or decreasing) its activity / abundance. See, e.g., Rausch and Hastings, Mol Immunol, December 2015; 68(2 Pt A):124-8, doi:10.1016 / j.molimm.2015.06.008, published electronically June 23, 2015; and Hastings and Cresswell, Antioxid 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 harvest vessel is capable of providing to a growth culture (e.g., adding) ascorbic acid, dehydroascorbic acid, or an ascorbic acid- or dehydroascorbic acid-modified component. The redox potential modulating properties of ascorbic acid and dehydroascorbic acid are known in the art. See, e.g., Winkler et al., Free Radic Biol Med, October 1994; 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 label, such as a redox-sensitive dye or molecular probe, to a production culture). Such a redox potential indicator label can be used to monitor the redox potential of a culture or of cells within the culture. Redox potential indicator labels include, but are not limited to: 2,2'-bipyridine (Ru complex), nitrophenanthroline (Fe complex), N-phenylanthranilic acid, 1,10-phenanthroline iron(II) sulfate complex (ferroin), N-ethoxycoelenterazine, 2,2'-bipyridine (Fe complex), 5,6-dimethylphenanthroline (Fe complex), o-dianisidine, sodium diphenylamine sulfonate, diphenylbenzidine, diphenylamine, viologen, sodium 2,6-dibromophenol-indophenol, sodium 2,6-dichlorophenol-indophenol, sodium o-cresol indophenol, thionine (Lauth's violet), methylene blue, indigo tetrasulfonate, indigo trisulfonate, indigo carmine (indigo disulfonate), indigo monosulfonate, phenosafranine, safranine, neutral red, labels disclosed in any document incorporated herein, and labels disclosed in Schwarzlander M et al., Antioxid Redox Signal, May 1, 2016; 24(13):680-712 (which is hereby incorporated 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, an alkali solution needs to be added to the production culture in response to the process pH dropping below the control range. However, due to the continuously decreasing operating volume and the excessive use of the alkali solution, there is a potential risk of greater cell damage and cell death with the application of the alkali solution while harvesting the production culture. While the culture is actively growing and metabolizing, the feed needs to be applied to the production culture. However, during harvesting, due to the non-physiological nature of the feed (higher osmolarity and either high or low pH) and the microenvironment created in a poorly mixed vessel, the continuously decreasing operating volume can affect the mixing behavior of the bioreactor and can lead to greater cell damage and cell death with the application of the feed solution.

[0198] - Once the production culture has reached the target temperature to initiate the harvesting step, ensure that all process controls that may promote the oxygenation of the production culture are activated. These can include: enabling the distributed air and / or oxygen flow (if not already enabled); enabling the headspace air and / or oxygen flow (if not already enabled); and finally enabling the headspace pressure (if not already 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 may become very low or even stop. Continuous application of air and oxygen distribution at various fixed flow rates is crucial for 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 vessel (e.g., the harvesting vessel). Headspace or blanket aeration is typically enabled during the cultivation of the production culture and ensures the continuous removal of the metabolized CO2 gas from the bioreactor headspace. Continuous blanket aeration of the production culture during harvesting promotes surface oxygenation of the culture, although slower than with distributed gas, and avoids the generation of foam when the production culture is discharged from the bioreactor.

[0199] - For bioreactors capable of operating at headspace pressure and in cases where pressure is used during the cultivation of the production culture to help achieve greater solubility of gas in the liquid phase (the culture) or to maintain a positive pressure within the bioreactor to prevent environmental contaminants from crossing the aseptic boundary of the vessel, it is recommended to maintain this state during harvesting. In harvesting practices driven by pressure, as long as the gas used to pressurize the bioreactor is air or a blended mixture of air and oxygen, it will contribute to the oxygenation of the culture. However, if the harvesting is pump-driven, the headspace pressure is generally not used. In such cases, the use of headspace pressure with air or an air / oxygen mixture helps to oxygenate the culture and prevents the harvesting tube from collapsing under the suction head upstream of the pump when the culture flows 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 regarding internal bore size and wall strength to prevent constriction under suction generated upstream of the pump head at high flow rates is important for ensuring that the harvest flow rate is not impeded by tube blockages. The effects of tube blockages are not only limited to restricting the harvest flow rate but may also promote greater cell death and cell lysis and the degassing of dissolved oxygen from the culture. The effects of tube blockages have the potential to expose cells and products to a hypoxic / anoxic environment within the housing due to increased residence time in the filter housing resulting from slower flow rates, promote the release of intracellular factors as a result of greater cell death and cell lysis passing through restricted openings, and remove dissolved oxygen from the culture by degassing within the suction zone, thereby reducing the amount of dissolved oxygen brought into the filter housing by the culture. In one embodiment, the methods disclosed herein avoid these negative impacts on product stability.

[0201] The inner diameter of the harvest tube and the selection of the pump used are chosen to achieve the desired flow rate and thus the process volume flux (liters of process supernatant per filter area, L / m2) of the cell clarification step performed by a depth filter or a centrifuge coupled to the depth filter. The construction material of the harvest line needs to be rigid to prevent constriction in the suction head generated upstream of the pump head. It is recommended to use materials other than the commonly used platinum-cured silicone or C-flex in the construction of the harvest tube. The use of braided tubing can 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 blockage of the filter during the filter clarification step, and thus, minimizing the residence time of the cell culture within the filter housing is an essential step to ensure oxygenation of the production culture and ensure that the dissolved oxygen brought out of the bioreactor does not become depleted while flowing into and residing within the filter housing before the culture is discharged as supernatant. Additionally, the flow rate should be sufficient to minimize the residence time of the supernatant between the clarification step (filter housing) and the c supernatant collection container (e.g., harvest container).

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

[0204] - In some embodiments, a method includes, or a bioreactor is capable of ensuring that 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 surface of the vessel wall through an internal nozzle designed towards the vessel wall, thereby forcing the filtrate to cascade down the vessel wall; and / or the ability to fill the harvest vessel with a gas 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 vessel is a stainless-steel harvest vessel. The method of collecting the supernatant and the design of the harvest vessel are the final elements of the method for protecting the product during the harvest step. Currently, 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 vessel through a headspace port with an internal "J" tube that directs the supernatant stream onto the vessel wall, and the supernatant stream cascades down the wall from the vessel wall to be collected at the bottom of the tank. Additionally, these stainless-steel containers are filled with sterile air to pressurize them above atmospheric pressure after in-situ steam sterilization to prevent environmental contaminants from crossing the aseptic boundary of the container. It is assumed that the harvested supernatant collected in these containers is oxygenated as it cascades down the vessel wall under a pressurized air atmosphere. Further oxidation of the supernatant occurs through surface contact with 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 has been gamma irradiated and emptied. During supernatant collection, the bag fills and displaces the vacuum, and no gas headspace forms above the collected supernatant. These bags do not have agitators, so the collected supernatant cannot be mixed, and the bags are mounted in a jacketless plastic or stainless-steel housing 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 begin with a primary capture step immediately following harvest or within the 14-day retention period.

[0207] A method for ensuring that the dissolved oxygen tension (DOT) of the production culture is maintained within a range of greater than 40% air saturation to less than 500% air saturation during the harvest step is intended to "load" the production culture with dissolved oxygen, which is expected to carry over and pass through the clarification step such that when the supernatant is collected, it has sufficient dissolved oxygen to prevent the activation of cytokines that could promote product dissociation. The harvest container is designed to ensure that the harvested supernatant continues to be well oxygenated while being stored at +5°C for up to 14 days. This is achieved through the container design, which ensures that the supernatant is continuously oxygenated, for example, by surface aeration, while being collected and stored at +5°C. In one embodiment, the harvest container design includes the following features:

[0208] - The container or single-use bag has an agitator / mixer that 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 thermocycler of appropriate size to bring the temperature of the container contents from room temperature to +5°C within 4 hours and from +5°C to room temperature within a similar time.

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

[0211] - The container or single-use bag and housing are fitted with a covering gas filter (an outlet gas vent filter suitable for air and oxygen at flow rates appropriate for the operating scale and an inlet gas filter suitable for air and oxygen at flow rates appropriate for the operating scale) to allow air and oxygen to continuously flow through the headspace to fill the harvest container with a gas environment consisting of air or any given blend of air and oxygen before collecting the supernatant filtrate, to promote the oxygenation of the supernatant filtrate to >40% air saturation and ≤500% air saturation when collecting the supernatant filtrate and once collection is complete.

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

[0213] - The container or single-use bag and the housing are equipped with process sensor ports for pH, DOT, redox, and temperature. The position of the probe inside the container or bag allows monitoring with a minimum operating volume suitable for the operating scale.

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

[0215] Cells and Cell Cultures

[0216] In one aspect, the present disclosure relates to methods and compositions for engineering or manufacturing cells or cell lines for producing the products described herein (e.g., recombinant products). In another aspect, the present disclosure relates to methods and compositions for engineering or manufacturing cells or cell lines with improved (e.g., reduced) dissociation, increased productivity, and / or product quality.

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

[0218] In one embodiment, the cells are Chinese hamster ovary (CHO) cells. In one embodiment, the cells are CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN, or CHO-derived cells. The CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. The CHO FUT8 knockout cells are, for example, CHOK1SV (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 of the cells 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 one of the cells described herein that produce a product (e.g., a product described herein). In one embodiment, the cell is any one of the cells described herein that comprises an exogenous nucleic acid encoding a recombinant polypeptide (e.g., expressing a recombinant polypeptide, e.g., 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, fed-batch culture, intermittent culture or continuous culture. In one embodiment, the cell culture is an adherent 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 a recombinant polypeptide, e.g., 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 having at least two impellers), a large-scale bioreactor system and a method for large-scale culture and propagation of mammalian cells. In one embodiment, the bioreactor is the bioreactor 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 by reference in their entirety. A system and method for controlling at least one bioreactor, other cell culture-related equipment, and systems containing any combination thereof are disclosed therein. For example, the system and method for bioreactor control may include controlling multiple bioreactors and other types of culture-related equipment, such as equipment for fermentation, harvesting, microfiltration and purification (e.g., liquid chromatography skid systems), buffer preparation, medium preparation, and the like. The multiple bioreactors and other types of culture-related equipment may be located in a facility, 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 uses at least one single-use disposable technology (e.g., those described in USSN 62 / 246,478, the contents of which are incorporated herein by reference in their entirety) to provide batch and continuous manufacturing. 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 will have a water jacket through which temperature-controlled or thermostatically controlled water is passed 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 culturing (i.e., increasing the cell number or inducing the production of a desired product by the cell culture), at the end of the production phase when cooling the production culture, or when harvesting the cells. Since the optimal growth temperatures of organisms are different, the cooling temperature will be selected based on the relatively higher temperature of one or more steps prior to the cooling step. In an embodiment, the cultured cells are cooled to reduce the oxygen consumption rate of the cells such that the harvest stream remains oxygenated during the harvest process.

[0227] For mammalian cell lines, the temperature variation during the culture process is typically 27 - 35 °C (e.g., 27, 28, 29, 30, 31 or 32, 33, 34 or 35 °C). In some embodiments, the temperature is reduced to 30 - 33 °C after the exponential growth phase. Without being bound by theory, it is believed that reducing the temperature to 12 - 18 °C (e.g., 15 °C) after the production phase reduces the specific oxygen consumption rate by a factor of 10.

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

[0229] Other suitable culture media and culture methods for mammalian cell lines are well known in the art, such as those described, for example, in U.S. Patent No. 5,633,162. Examples of standard cell culture media for use in laboratory flasks or low-density cell cultures and adapted to the needs of specific cell types are, for example: Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519 f., 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, l p. 173 ff, 1963), Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimum Essential Medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc. 53, p288 ff., 1965) or Iscove's Modified DMEM lacking albumin, transferrin and lecithin (Iscoves et al., J. Exp. med., 1, p. 923 ff., 1978). For example, Ham's F10 or F12 medium is specifically designed for CHO cell culture. Other media particularly suitable for CHO cell culture are described in EP-481 791. Other suitable culture methods are known to the person skilled in the art and may depend on the recombinant polypeptide product and the host cell used. Determining or optimizing the conditions suitable for the expression and production of the product to be expressed by the cells (e.g., a recombinant polypeptide) is within the skill of the ordinary artisan.

[0230] Assays for quantifying the amount, level or quantity of a product produced or secreted (e.g., secreted into a culture medium) include protein quantification assays such as Bradford protein assay, SDS-PAGE analysis, immunoblotting (e.g., Western blotting), and automated devices (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, an 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 determined by ForteBio Octet quantification, e.g., for high-throughput determination of the concentration of recombinant monoclonal antibody (mAb) 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) can refer to the specific production rate (qP) of the product (especially the recombinant polypeptide in cells) and / or the time integral of viable cell concentration (IVC). In one embodiment, the method for determining production includes determining a combination of qP and IVC. The production or productivity of the recombinant polypeptide, which is defined as the concentration of the polypeptide in the culture medium, varies with these two parameters (qP and IVC), as 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, for example, compared to the amount, level or quantity of the product produced by cells not subjected to a lower temperature, the improvement in product quality results in an increase, e.g., a 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 greater increase in product quality; or a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase in product quality.

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

[0233] i) An increase or decrease in disulfide bond mispairing (e.g., for an antibody molecule product, an increase or decrease in the desired isomers or structures due to an increase or decrease in disulfide bond mispairing).

[0234] ii) An increase in the amount or quantity of non-aggregated product (or a decrease in the amount or quantity of aggregated product);

[0235] iii) An increase in the amount or quantity of properly folded or assembled product (or a decrease in the amount or quantity of misfolded, unfolded, partially assembled, or unassembled product), or an increase in the ratio of properly folded or assembled product to unfolded, misfolded, partially assembled, or unassembled product;

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

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

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

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

[0240] Methods for measuring the quality of products (e.g., improvement in 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 an expressed recombinant polypeptide product are known in the art, such as mass spectrometry. An increase in the amount or concentration of properly folded product (e.g., the expressed recombinant polypeptide) can be determined by circular dichroism or by assessing the intrinsic fluorescence of the expressed recombinant polypeptide. A variety of functional assays can be used to test for an increase in the amount or concentration of functional product, depending on the identity of the recombinant product (e.g., recombinant polypeptide). For example, an antibody can be tested by ELISA or other immunoaffinity assays. Other methods for determining an improvement in product quality (e.g., determining aggregation, post-translational modifications, disulfide bond mispairing) can be evaluated by size exclusion chromatography, high performance liquid chromatography, dynamic light scattering (DLS) methods, and protein electrophoresis (PAGE).

[0241] In some embodiments, additional steps can 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 reagents that improve product expression or product quality. In one embodiment, the reagent 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 protein). In one embodiment, the reagents that assist in protein folding include nucleic acids encoding chaperone proteins 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 chaperone proteins (e.g., calnexin and calreticulin (Chung et al., 2004)). Overexpression of the reagents that assist or improve protein folding, product quality, and product yield described herein can be achieved by introducing exogenous nucleic acids encoding the reagents. In another embodiment, the reagent that improves product expression or product quality is a small molecule such as DMSO that can be added to the cell culture to increase product expression or product quality. In one embodiment, maintaining the cells at a lower temperature (e.g., a temperature 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 at which the cells normally grow) can improve the quality of the product by reducing or eliminating dissociation of the product.

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

[0243] In one aspect, the present disclosure provides a method comprising steps for recovering or retrieving a recombinant polypeptide product. In embodiments where the recombinant polypeptide is secreted from the cell, the method can include steps for retrieving, collecting, or separating the recombinant polypeptide from the cell, cell population, or culture medium of the cultured cells. In embodiments where the recombinant polypeptide is intracellular, purification of the recombinant polypeptide product includes separating the recombinant polypeptide produced by the cell from any one or more of the following: host cell proteins, host cell nucleic acids, host cell lipids, and / or other debris from the host cell.

[0244] In an embodiment, the processes described herein provide a substantially pure protein product. As used herein, "substantially pure" means substantially free of pyrolysis products, substantially free of nucleic acids, and / or substantially free of endogenous cellular proteases and components from the host cell (e.g., polymerases, ribosomal proteins, and chaperone proteins). The substantially pure protein product contains, 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 the host cell.

[0245] Methods for recovering and purifying products (e.g., recombinant polypeptides) are well established in the art. To recover the recombinant polypeptide product, physical or chemical or physicochemical methods are used. The physical or chemical or physicochemical methods can be filtration, centrifugation, ultracentrifugation, extraction, lyophilization, precipitation, chromatography, or a combination of two or more of these methods. 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 an embodiment, the reduction in temperature is combined with a reduction in temperature by lowering the pH and reducing thioredoxin inhibitors (e.g., metal ions). In an embodiment, the combined treatment is carried out in a bioreactor as described above.

[0247] Examples

[0248] The present invention is described in further detail by reference to the following examples. These examples are provided for illustrative purposes only and are intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations that become apparent as a result of the teachings provided herein.

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

[0250] Example 1. Background and Identification of Non-Essential, Redundant, and / or Secreted Endogenous Protein Candidates for Depletion

[0251] Chinese hamster ovary (CHO) cells are the world's major production matrix for generating biopharmaceuticals (Walsh, 2014). Despite spending a great deal of time, effort, and money evolving these cells from adherent cell lines to stable cell lines and then manipulating the cells using overexpression and synthetic elements to increase titers, growth rates, and the cell-specific recombinant protein production rate (qP), the cellular matrix and genome are actually still the same as those of the ancestral Chinese hamster. Thus, the cells often behave as if they were in a Chinese hamster rather than in a completely artificial environment. This can be illustrated by multiple genes that still seem to be functionally redundant and are encoded by the cell line and subsequently expressed as proteins. There are documented instances of genes that have been successfully knocked down or knocked out of the CHO genome to enhance cell or product characteristics. 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 removing endogenous CHO glutamine synthetase (Bebbington et al., 1992; Cockett, Bebbington, and Yarranton, 1990). Along with the recent genomic sequences of CHO (Lewis et al., 2013; Xu et al., 2011) and the rapid development of genome editing technologies (Cong, Ran, Cox, Lin, and Barretto, 2013; Ran et al., 2013), the promise shown by the knockdown or knockout of individual genes provides an opportunity to more extensively screen for redundant elements within the CHO genetic code.

[0252] Combined with a set of bioinformatics tools and literature analysis, data at various omics levels are used in the form of quantitative transcriptomics and proteomics to rationally identify highly abundant non-essential proteins that are predicted to compete with the target mAb for endoplasmic reticulum (ER) space as well as post-translational modification (PTM) and secretion machinery. 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 on days 4, 7, and 9, and RNA-seq analysis was performed against the CHOK1GS (Ensembl accession number: CHOKlGS_HDvl) genomic sequence. For each gene, the transcripts per million (TPM) value provides a measure of the amount by which each gene is expressed as part of the total cellular transcript pool. Automatic gene ID assignment yielded IDs for 77.5%, 77.7%, and 77.6% of the active genes in the total transcript pool on days 4, 7, and 9, respectively. Manual assignment added gene IDs to 18.4%, 17.8%, and 18.3% of the transcript pool. A total of 13,773, 14,433, and 14,660 gene IDs were generated on days 4, 7, and 9, respectively; these assigned values represent 95.7%, 95.4%, and 95.8% of the IDs in the transcript pool on days 4, 7, and 9, respectively. Then, defined thresholds were introduced to help classify gene transcripts into different abundance levels (based on Ramskold et al. (2009), PLoS Computational Biology, December 2009; 5(12):e1000598, doi:10.1371 / journal.pcbi.1000598):

[0253] ● Genes not expressed (fragments per kilobase of exon model per million mapped reads (FPKM) < 0.3) (TPM < 0.4)

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

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

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

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

[0258] Only genes (879 genes) that consistently met the criteria for extremely highly abundant genes on all three test days (days 4, 7, and 9) were presented. Then, these genes were passed through the PANTHER classification system (http: / / www.pantherdb.org / ), using the Mus musculus database as the organism for analysis. Initially, only genes containing keywords considered as targets due to functional redundancy were highlighted for further analysis (Table E1). Thereafter, a list of functionally crucial or highly essential keywords was generated (Table E2). Any genes containing these keywords were subsequently excluded from further analysis, and in the case of genes containing keywords from both Table E1 and E2, the genes were analyzed in depth to consider evidence of how important they were for the function of CHO cells. After screening the genes by the defined criteria, 76 targets remained (112 when considering all isomers).

[0259] Then, four different tools were used for bioinformatics analysis to refine genes that further met our criteria as 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] Quantitative intracellular proteomics analysis of exponential - and stationary - phase cells of the CHO cell line generated additional data at various omics levels. When the target proteins were present, data detailing the total protein biomass (considering the average copy number of the protein and its molecular weight) were recorded.

[0265] Data from previously published quantitative proteomic analyses of extracellular proteins present in the media of CHO cell cultures (Kumar et al., 2015, Journal of Proteome Research (J Proteome Res), November 6, 2015; 14(11):4687 - 703, doi:10.1021 / acs.jproteome.5b00588; Park et al., 2017, Scientific Reports (Sci Rep), March 10, 2017; 7:44246, doi:10.1038 / srep44246) were used to attempt to identify common redundant extracellular host cell (HC) proteins. Details of the presence or absence of target proteins in a previously defined list were recorded, and if present, whether they were present at high, medium, or low levels (criteria defined in Table E3) (NSAF - Normalized Spectral Abundance Factor, NSC - Normalized Spectral Count).

[0266] The pipeline summarized is shown below:

[0267] 1. All have extremely high transcripts throughout the culture process in the bioreactor

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

[0269] 3. All have essential keywords removed (unless in conflict with point 2, where a separate assessment is made) (Table E2)

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

[0271] 5. Attempted to match against Lonza IC proteomics

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

[0273] 7. Attempted to identify glycosylation sites and glycosylation site / AA using Mus musculus Uniprot data

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

[0275] Another set of prior targets (fibronectin - Fn1 and heparan sulfate proteoglycan - HSPG2) were selected (because they have perceived redundancy), as well as two negative targets (although matching most of the criteria defined above, it seems very important for a normally operating CHO cell expression system (clusterin - Clu and 14 - 3 - 3 protein epsilon - YWHAE)). The final list of 20 targets used for testing is shown in Table E5.

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

[0277] 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)) was performed using Cas9 nuclease, and the effect on recombinant protein production was evaluated. Using specifically enhanced Cas9 (eCas9 1.1) (Slaymaker et al., 2016), the 2A peptide fused to GFP can allow for high-throughput isolation of cells with Cas9 nuclease activity, thus enabling multiplex genome editing (Lonowski et al., 2017, Nat Protoc, March 2017; 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 lacking competing redundant proteins (e.g., proteins (i.e., mRNA-encoded proteins) that compete for translation and ER / Golgi resources with the product (i.e., the mRNA-encoded product)) can be created. This will contribute to generating a more streamlined genome and proteome with improved growth and production properties, better suited to the high titers and growth rates required for the competitive production of biopharmaceuticals.

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

[0280] To preliminarily screen gene knockout targets identified in Table E5, each gene target was individually knocked out from the IgG4 Mab-producing 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 the constitutive U6 promoter. Forty-eight hours after transfection, the heterogeneous pool of transfected cells was sorted by FACS for high GFP expression (representing high Cas9 nuclease activity), where ≥0.5x 10^6 live cells were selected and pooled for further culture, and subsequently growth, Mab productivity, and gene knockout success were evaluated.

[0281] Specifically, the GS-CHOK1V cell line that produces IgG4 Mab was transiently transfected with plasmid (pX458) containing: 1) a Cas9-GFP nuclease fusion protein expressed from the CMV promoter and 2) one or more guide RNAs specific for a target gene in its genome expressed from the constitutive U6 promoter. 48 hours after transfection, the transfected pool was sorted for high GFP expression by FACS, where ≥0.5x 10^6 cells were collected and pooled into a new flask. 72 hours later, in the 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 automated cell counting (ViCell - Beckman Coulter, High Wycombe, UK). The percentage change in qP in the FACS sorted pool of GS-CHOK1SV cells transfected with the pX458 vector containing the gRNA specific for the indicated target gene was calculated. All data points were expressed relative to control GS-CHOK1SV cells transfected with pX458 expressing the Cas9-GFP nuclease fusion protein but lacking the gRNA.

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

[0283] To determine the success of Cas9-GFP gene editing targeting specific genes, genomic DNA was extracted from the FACS sorted cells and evaluated by TIDE analysis (Brinkman et al., Nucleic Acids Res, December 16, 2014; 42(22):e168, doi:10.1093 / nar / gku936). The results showed a high targeted gene indel rate (ranging from ≥60% to 90%) obtained by this method ( Figure 1B ), strongly indicating that the phenotypic readout (i.e., the observed increase in qP) varied with gene editing in the pool.

[0284] Example 3: Multigene knockout of endogenous targets and increase in product yield.

[0285] The data provided in Example 2 partially shows that several endogenous genes encoding non-essential, redundant, and / or secreted endogenous proteins in cells can be individually knocked out to increase the production of Mab from the CHO cell line. Since the number of endogenous proteins passing through the cellular secretory apparatus is very large, 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 individually knocked out (Figure 1) were knocked out in random triplicates 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. Compared to control GS-CHOK1SV cells transfected with the pX458 vector lacking gRNA (Table E7), some combinations of gene targets (e.g., C1S, Dcn, and Lambl) associated with a significant increase in average titer and qP showed an average 25% increase in titer and an average 28% increase in qP when combined in the triple knockout pool in triplicates.

[0286] Example 4: Table

[0287] Table E1 - PANTHER-Assigned Keywords - Genes to be Further Analyzed Highlighted

[0288] Coagulation Vesicle-Associated Vesicle Coat Protein Membrane Component Complement Component Amyloid Huntington's (Disease) Placenta Chemokine CD Communication Bone Marrow Estrogen Cell Adhesion Immunity Galectin Epithelium Alzheimer's Neuron Parkinson's Cytokine Embryo Apoptosis Transmembrane Receptor Secretion Exosome Endoplasmic Reticulum Extracellular Space Extracellular Matrix Extracellular Region Histocompatibility MHC Immunity

[0289] Table E2 - PANTHER-Assigned Keywords - Genes Removed from Further Analysis

[0290] Ribosome Eukaryotic Initiation ATP ADP Actin Myosin RNP NADH Ubiquitin Peptidyl Mitochondria Transporter Polymerase Heat Shock Splicing Nucleosome RNA / DNA Binding Kinase Cell Division Cytochrome C Histone Transcription Translation cAMP Chaperone Folding Glycosylation Cell Cycle Tubulin Kinesin Dynein Metabolism Cytoskeleton Transport Calmodulin Ras Collagen Endoplasm Golgi Apparatus

[0291] Table E3 - Abundance Level Criteria of Kumar et al. (2015) and Park et al. (2017)

[0292]

[0293] Table E4 - Final Criteria Considered for Target Ranking

[0294] Protein Abundance Classification Protein Level Kumar - High NSAF ≥ -8.8939 Kumar - Medium -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]

[0299] Key to Table E5

[0300]

[0301] Table E6 Random Triplet Combinations of Knockout Genes

[0302]

[0303] Table E7 Changes in Mab Titer, qP, and Growth (Integrated Viable Cell Density, IVCD) of FACS-Sorted Pools Transfected with pX458 Containing gRNAs Specific to Random Triplet Combinations of Target Genes All values are expressed relative to control cells transfected with pX458 lacking gRNA.

[0304] Multiplex

[0305] IVCD% Titer% qP% Random 1 Random 2 2.77 24.14 21.70 Random 3 -3.75 -12.42 -8.78 Random 4 -4.35 5.54 10.83 Random 5 -8.60 -1.81 6.55 Random 6 -1.42 24.60 27.73 Random 7 -4.17 2.16 7.26 Random 8 -2.84 2.99 4.61 Random 9 -1.45 14.23 17.19 Random 10 1.18 13.93 12.17 Example 5: References -15.71 -16.00 -0.33

[0306] ​

[0307] 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.

[0308] Brown, A.J., Gibson, S., Hatton, D. and James, D.C. (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

[0309] Cockett, I.M., Bebbington, R.C., and Yarranton, T.G. (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

[0310] 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

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[0312] 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

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[0314] Lewis, N. E., Liu, X., Li, Y., Nagarajan, H., Yerganian, G., O'Brien, E., …… Palsson, B. O. (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

[0315] Park, J.H., Jin, J.H., Lim, M.S., An, H.J., Kim, J.W., & Lee, G.M. (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

[0316] Pieper, L.A., Strotbek, M., Wenger, T., Gamer, M., Olayioye, M.A., & 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

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[0318] Slaymaker, I.M., Gao, L., Zetsche, B., Scott, D.A., Yan, W.X., and Zhang, F. (2016), "Rationally engineered Cas9 nucleases with improved specificity," Science, 351(6268), 84 - 88. https: / / doi.org / 10.1126 / science.aad5227

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[0321] Equivalent

[0322] Through routine experimentation alone, one of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein. These equivalents are intended to be covered by the following claims.

[0323] The present invention encompasses all variations, combinations, and permutations of introducing one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims into another claim. In the case where elements are presented as a list (e.g., in Markush group format) or as an alternative representation, each subgroup of the elements is also disclosed, and any one or more elements may be removed from the group.

[0324] It should be understood that, generally, 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 consist of or consist essentially of these elements and / or features. 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, the endpoints are also included. Further, unless otherwise indicated or obvious from the context and the understanding of those of ordinary skill in the art, in different embodiments of the present invention, 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 dictates otherwise.

Claims

1. A method for producing a product, such as a recombinant polypeptide, in a cell, such as a production cell, the method comprising: Reduce the level of an endogenous protein in said cell, e.g., a production cell, thereby producing said product.

2. The method according to claim 1, wherein the cell is a production cell.

3. The method according to claim 1 or 2, further comprising culturing the cell under conditions suitable for producing the product, such as a recombinant polypeptide.

4. The method according to any one of the preceding claims, further comprising providing a cell, such as a production cell.

5. A method for producing a product, such as a recombinant polypeptide, in a cell, such as a production cell, the method comprising: Provide a cell, e.g., a production cell, reduce the level of an endogenous protein in said cell, e.g., a production cell, and culture said cell under conditions suitable for producing said product, e.g., a recombinant polypeptide, thereby producing said product.

6. The method according to any one of the preceding claims, further comprising harvesting the product, such as separating the product from the production mixture and / or providing a purified product formulation, for example by methods described herein or known in the art.

7. A method for producing a cell, such as a production cell, the method comprising: Reduce the level of an endogenous protein in said cell, e.g., a production cell, wherein said cell, e.g., a production cell is capable of expressing a product, e.g., a recombinant polypeptide.

8. The method according to claim 7, further comprising providing a cell, such as a production cell.

9. The method according to any one of the preceding claims, wherein the endogenous protein is a non-essential protein (e.g., wherein a decrease or complete loss of the level of the protein does not significantly and detrimentally affect cell viability or product production).

10. The method according to any one of the preceding claims, wherein the endogenous protein is an extracellular protein.

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