Cell selection method

Abstract

Described herein are methods for identifying, selecting, or culturing producer cells and producer cells that contain a tyrosine auxotrophic selectable marker system based on a combination of a sequence encoding phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain and a sequence encoding GTP cyclohydrolase 1 (GCH1). Methods for generating producer cells and producing products in the producer cells are also described.

Description

[Technical field]

[0001] The present disclosure relates to methods and compositions for identifying, selecting, or culturing cells that contain the subject nucleic acid sequences. [Background technology]

[0002] Cellular expression systems are commonly used for the production of recombinant biological products, such as therapeutic biologics. The development of production line cells involves introducing nucleic acid constructs encoding the recombinant product of interest into host cells and selecting for cells containing these nucleic acid constructs. This generally involves exposing the cells to selective pressure so that cells that have taken up the foreign nucleic acid are favored. Early selectable marker systems used antibiotic resistance markers, but there has been a trend away from the use of such systems. Some alternative systems are based on complementation of metabolic deficiencies, for example, dihydrofolate reductase (DHFR) and glutamine synthetase (GS). However, there remains a need for new selection systems that can be used to select for cells used to produce recombinant biological products. Furthermore, as production host cell engineering strategies are increasingly used, those strategies that involve the introduction of new sequences that modify the characteristics of the host cell would benefit from a selection system separate from existing or upcoming selection systems used to introduce sequences encoding the biological product. Summary of the Invention

[0003] The present invention relates to a selection system based on tyrosine auxotrophy and the production of recombinant products that do not require the inclusion of tyrosine in the medium. It was found that a system based solely on phenylalanine hydroxylase (PAH - catalyzing the conversion of phenylalanine to tyrosine) is not effective and requires the inclusion of a second enzyme involved in tyrosine biosynthesis, namely GTP cyclohydrolase 1 (GCH1). Furthermore, it was found that using PAH with a truncation that removes the N-terminal regulatory domain offers a significant advantage compared to the full-length enzyme. Using full-length CHO PAH, cells either did not recover in tyrosine-free medium after transfection or had a much slower recovery time, whereas the truncated (tPAH) form of the molecule allowed for good recovery. The combination of PAH and GCH1 allows cells to grow at lower levels of tyrosine (e.g., in the absence of tyrosine) than similar cells that do not express these enzymes.

[0004] Thus, in a first aspect, the present invention provides a vector system comprising one or more nucleic acid vectors, the nucleic acid vector system comprising: a) a first nucleic acid sequence comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of the PAH in a host cell; b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence enabling expression of GCH1 in a host cell; c) a multiple cloning site for inserting one or more product-encoding sequences operably linked to a third control sequence enabling expression of the product of interest in the host cell.

[0005] In a related aspect, the present invention also provides a vector system comprising one or more nucleic acid vectors, the nucleic acid vector system comprising: a) a first nucleic acid sequence comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of the PAH in a host cell; b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence enabling expression of GCH1 in a host cell; c) a third nucleic acid sequence comprising a product-encoding sequence operably linked to a third control sequence enabling expression of the product of interest in the host cell.

[0006] Such vectors may be introduced into host cells, which contain the vector selected under tyrosine-limiting conditions that do not allow efficient growth of non-transformed cells. Thus, in a second aspect, the present invention provides a host cell, the host cell comprising: a) a first exogenous nucleic acid comprising a sequence encoding phenylalanine hydroxylase (PAH) operably linked to a first control sequence enabling expression of the PAH in a host cell; b) a second exogenous nucleic acid encoding GTP cyclohydrolase 1 (GCH1), operably linked to a second control sequence that allows expression of GCH1 in the host cell; and c) a third exogenous nucleic acid encoding a product operably linked to a third control sequence enabling expression of the product of interest in the host cell.

[0007] In one embodiment, the first, second, and third nucleic acid molecules are integrated into the genome of the host cell.

[0008] In one embodiment, the host cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell.

[0009] In various embodiments of the invention, the lack of a functional N-terminal regulatory domain in a PAH can result from, for example, a deletion to form a truncated PAH. Based on the human and CHO PAH amino acid sequences, this is typically a deletion of about the first 116 amino acids.

[0010] In one embodiment, the PAH is a CHO PAH or a human PAH.

[0011] In one embodiment, the first and / or second regulatory sequence comprises an SV40 promoter.

[0012] The vector system of the invention is typically used to select cells that have been successfully transformed with a nucleic acid encoding a product of interest, such as a recombinant polypeptide. Thus, in a third aspect, the invention provides a method for selecting cells that contain a nucleic acid sequence encoding a product, the method comprising: a) contacting a population of cells that are unable to survive or grow in the absence of tyrosine with a vector system of the present invention under conditions that allow for uptake of the vector system by the cells; b) culturing the cells under conditions in which the level of tyrosine is lower than that required for survival or growth of cells that do not express the PAH and GCH1 enzymes encoded by the vector system; c) selecting one or more cells capable of growing under such conditions to obtain one or more cells containing the nucleic acid sequence encoding the product.

[0013] The level of tyrosine is chosen to ensure stringent selection and is optionally supplemented with phenylalanine, hi one embodiment, the culture medium does not contain added tyrosine.

[0014] In a related aspect, the invention provides the use of the vector system of the invention to select, from a population of cells, one or more cells which contain a nucleic acid sequence which has been introduced into said cells.

[0015] The selected host cell obtained by the selection method of the invention forms another aspect of the invention. Thus, in a fourth aspect, the invention provides a host cell, the host cell comprising: a) a first exogenous nucleic acid comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of the PAH in a host cell; b) a second exogenous nucleic acid encoding GTP cyclohydrolase 1 (GCH1), operably linked to a second control sequence that allows expression of GCH1 in the host cell; and c) a third exogenous nucleic acid encoding a product operably linked to a third control sequence enabling expression of the product of interest in the host cell.

[0016] The host cells of the present invention may be genetically modified to inhibit or eliminate any endogenous PAH and / or GCH1 activity. In one embodiment, this may be accomplished by mutations (insertion, deletion, and / or substitution) in the genomic sequences that code for and / or regulate the expression of endogenous PAH and / or GCH1.

[0017] The selected host cell of the present invention, which contains a nucleic acid sequence encoding a product of interest, is typically used in producing the product. Thus, in a fifth aspect, the present invention provides a method for making a product, which comprises culturing a host cell of the present invention, which contains a nucleic acid sequence encoding the product, under conditions suitable for expressing the product, recovering the product, and optionally subjecting the recovered product to one or more processing or purification steps.

[0018] When the cell line developed using the host cell and selection process of the present invention is used for large-scale production, it may no longer be necessary to apply selection pressure by omitting tyrosine during the cultivation step.However, tyrosine, which is considered an essential amino acid, has the second lowest water solubility of any amino acid after cysteine.The low solubility of tyrosine can be a challenge to generate a feed solution of sufficient concentration to support cell cultivation under biomanufacturing conditions, such as during fed-batch bioprocessing, for example in a bioreactor.

[0019] The host cells of the invention can be grown efficiently on low levels of tyrosine (including in the absence of tyrosine), reducing the need for a high concentration tyrosine feed. In one embodiment, the medium is supplemented with phenylalanine, since phenylalanine is consumed by the cells to produce tyrosine.

[0020] The present invention also provides a culture medium, such as a feed, that includes a plurality of amino acids, such as at least three or four amino acids, and in an aqueous solution, there is less than 0.01 g / L of tyrosine, such as less than 50, 20, or 10 μM of tyrosine (e.g., no tyrosine), and at least 2, preferably at least 3, 4, 5, 6, 7, 8, or 9 mM of phenylalanine. Typically, the culture medium includes less than 10 mM of phenylalanine. The present invention also provides a culture medium mixture in a substantially dry form (e.g., includes less than 5, 4, 3, 2, or 1% water, e.g., significantly does not include water), that includes a plurality of amino acids, such as at least three or four amino acids, and has a level of tyrosine and phenylalanine such that the culture medium is prepared by adding an appropriate volume of water.

[0021] The present invention further provides the use of culture media and culture medium mixtures for selecting and / or growing cells transformed with a vector system of the present invention, e.g., in the expression of a product of interest encoded by the vector system.

[0022] The invention also provides a mixture comprising a host cell of the invention and a culture medium of the invention.

[0023] In another aspect, the invention features a bioreactor containing a population of host cells of the invention.In another aspect, the invention features a bioreactor containing a medium and a population of production cells of the invention. [Brief description of the drawings]

[0024] [Figure 1] The domain structure of PAH enzymes is shown. [Figure 2-1] (A) Histograms of mean fluorescence from cell populations following transfection and 3 weeks recovery of the same CHO cell pool obtained using flow cytometry, and (B) a table of the fluorescence data. [Figure 2-2] Continued from Figure 2-1. [Diagram 3] Graph of PAH mRNA abundance relative to control as measured by qRT-PCR is shown. [Figure 4] (A) Graph of cell growth by viable cell concentration of various cell pools, some of which overexpress truncated PAHs, in the absence of tyrosine or glutamine, optionally supplemented with phenylalanine, over 18 days, and (B) graph of culture viability of the same cell pools under the same conditions. [Diagram 5] 1 shows the growth characteristics of the tyrosine prototrophic cell pool with various phenylalanine supplements. [Figure 6] Graphs showing the growth characteristics of tyrosine prototrophic cell pools without CD CHO tyrosine with 6 mM phenylalanine (A) and (B) the viable cell concentration of various cell pools without tyrosine and optionally supplemented with phenylalanine, and the culture viability of the same pools. [Figure 7] Graphs of growth characteristics of pre-adapted tyrosine prototrophic cell pools where phenylalanine supplementation occurred prior to cell growth assessment are shown. (A) Graphs of viable cell concentration of the cell pools and (B) culture viability of the same cell pools under the same conditions are shown. [Figure 8] Shown is a graph of PAH mRNA abundance in various cell pools relative to control cells (top), and a graph of GCH1 mRNA abundance in various cell pools relative to control cells (bottom). [Figure 9-1] Graphs showing growth characteristics of co-expressing tyrosine and glutamine auxotrophic cell pools: (A) viable cell concentration, (B) viability, and (C) cell diameter. [Figure 9-2] Continued from Figure 9-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting. Headings, subtitles, or numbered or lettered elements, such as (a), (b), (i), etc., are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require that steps or elements be performed in alphabetical order or that steps or elements are necessarily separate from one another. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0026] "About" or "approximately," as the term is used herein, applies to one or more values ​​of interest and refers to a value similar to a stated reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values ​​that falls within 5%, 4%, 3%, 2%, 1% in either direction (greater or less) of the stated reference value (except where such number exceeds 100% of possible values), unless otherwise stated or otherwise clear from the context.

[0027] As used herein, the term "control element" refers to a nucleic acid suitable for regulating (e.g., increasing or decreasing) expression of a coding sequence, e.g., a gene or sequence encoding a product or enzymatic molecule. A control element may include a promoter sequence, an enhancer sequence, or both a promoter sequence and an enhancer sequence. A control element may include contiguous nucleic acid sequences, discontinuous nucleic acid sequences (sequences interrupted by other coding or non-coding nucleic acid sequences), or both. A single control element may be included on a single nucleic acid or on more than one nucleic acid. In embodiments, a control element may include sequences 5' or 3' of a coding sequence, e.g., a coding sequence of a recombinant, therapeutic, or repressor polypeptide. In embodiments, a control element may include sequences within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In embodiments, a control element may be included in part or in its entirety within sequences 5' or 3' of a coding sequence, e.g., a coding sequence of a recombinant, therapeutic, or repressor polypeptide. In embodiments, a control element may be contained in part or in its entirety within a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In embodiments, a control element may be contained in part or in its entirety within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In embodiments, a single control element may comprise a nucleic acid sequence i) proximal to (e.g., adjacent to or contained within) a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide, or ii) distal to (e.g., separated by 10 or more, 100 or more, 1000 or more, or 10,000 or more bases, or located on different, separate nucleic acids, a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide.

[0028] The term "about" when referring to a measurable value, such as an amount, temporal duration, and the like, is meant to encompass variations of ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0029] The term "bioreactor" as used herein refers to a device in which a biological reaction or process takes place. The process can be carried out at industrial, pilot, and laboratory scales, including micro- and nano-scales.

[0030] As used herein, the term "endogenous" refers to any material that is derived from or naturally produced within an organism, cell, tissue, or system.

[0031] As used herein, the term "exogenous" refers to any material that is introduced or produced outside of an organism, cell, tissue, or system. Thus, an "exogenous nucleic acid" refers to a nucleic acid that is introduced or produced outside of an organism, cell, tissue, or system. In some embodiments, the sequence of the exogenous nucleic acid is not naturally produced or cannot be found naturally in the organism, cell, tissue, or system into which the exogenous nucleic acid is introduced. In some embodiments, the sequence of the exogenous nucleic acid is a non-naturally occurring sequence or encodes a non-naturally occurring product. In some embodiments, the sequence of the exogenous nucleic acid can also be found in the organism, cell, tissue, or system into which the exogenous nucleic acid is introduced. For example, the exogenous nucleic acid may encode an enzyme under the control of a constitutively active promoter, and the cell into which the exogenous nucleic acid is introduced contains an endogenous nucleic acid sequence (e.g., under the control of an endogenous promoter) that encodes the enzyme.

[0032] As used herein, the term "enzyme molecule" refers to a polypeptide having an enzymatic activity of interest. An enzyme molecule may share structural similarity (e.g., sequence homology) with a naturally occurring enzyme having an enzymatic activity of interest. In some cases, an enzyme molecule has at least 80% amino acid sequence identity (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with a naturally occurring enzyme having an enzymatic activity of interest. In some embodiments, an enzyme molecule is a variant of a naturally occurring enzyme (e.g., a variant that includes one or more amino acid sequence modifications (e.g., substitutions, deletions, or insertions) relative to the amino acid sequence of the naturally occurring enzyme). In some cases, the term "molecule" when used with an identifier of an enzyme (e.g., PAH or GCH1) refers to a polypeptide having the enzymatic activity of the identified enzyme. By way of example, the term "PAH molecule" or "PAH enzyme molecule" as used herein refers to a polypeptide having the enzyme activity of PAH. As a further example, the term "GCH1 molecule" or "GCH1 enzyme molecule" as used herein refers to a polypeptide having the enzyme activity of GCH1. In some embodiments, the enzyme molecule is or comprises a single polypeptide chain. In some embodiments, the enzyme molecule is or comprises a multi-polypeptide complex, e.g., an oligomer (e.g., a dimer, trimer, tetramer, pentamer, hexamer, octamer, decamer, or dodecamer).

[0033] As used herein, the term "enzymatically active fragment" refers to a portion of an enzyme or enzyme molecule that has the desired enzymatic activity of the enzyme or enzyme molecule. In some embodiments, the enzymatically active fragment is a variant of the enzyme or enzyme molecule that contains a deletion (e.g., truncation) to the enzyme or enzyme molecule. In some embodiments, the desired enzymatic activity of the enzymatically active fragment is reduced by 50, 40, 30, 20, or 10% or less relative to the enzyme or enzyme molecule from which the enzymatically active fragment is derived.

[0034] As used herein, the terms "nucleic acid", "polynucleotide", or "nucleic acid molecule" are used interchangeably and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations of DNA or RNA and polymers thereof, in either single-stranded or double-stranded form. The term "nucleic acid" includes, but is not limited to, genes, cDNA, or RNA sequences (e.g., mRNA). In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless otherwise limited, the term encompasses molecules that contain analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring or non-naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, a "subject nucleic acid" refers to any nucleic acid of interest, including, for example, a sequence encoding a product described herein, or a sequence encoding a production factor described herein (e.g., a lipid metabolism modifier (LMM) such as SCD1 and / or SREBF-1), that may desirably be introduced into or present in a cell described herein.

[0035] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of 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, with no limit on the maximum number of amino acids that a protein or peptide sequence can contain. In one embodiment, a protein can be composed of more than one polypeptide, e.g., 2, 3, 4, 5 or more, each polypeptide associated with each other by either covalent or non-covalent bonds / interactions. A polypeptide includes any peptide or protein that includes two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, e.g., also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, e.g., biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.

[0036] As used herein, the term "plurality" refers to two or more (e.g., two or more) of the grammatical object of the article. By way of example, a "plurality of cells" can mean two cells or more than two cells.

[0037] "Product," as the term is used herein, refers to an entity, e.g., a compound (e.g., a polypeptide (e.g., a glycoprotein), a nucleic acid, a lipid, a sugar, a polysaccharide, or any hybrid thereof), a vesicle, an exosome, or a virus, that is produced, e.g., expressed, by a cell, e.g., a cell that has been modified or engineered to produce a product, e.g., a production cell. In some embodiments, the product is a protein or polypeptide product. In some embodiments, the product includes a naturally occurring product. In some embodiments, the product includes a non-naturally occurring product. In some embodiments, a portion of the product is naturally occurring, while another portion of the product is non-naturally occurring. In some embodiments, the product is a polypeptide, e.g., a recombinant polypeptide. In some embodiments, the product is suitable for diagnostic or preclinical uses. In some embodiments, the product is suitable for therapeutic uses, e.g., treatment of a disease. In some embodiments, the product is a recombinant or therapeutic protein as described herein, e.g., in the section below entitled "Polypeptides." In some embodiments, the virus comprises a naturally occurring virus, a recombinant virus, a recombinant viral particle, a virus-like particle (VLP), a viral vector, an inactivated (e.g., dead or non-infectious) virus, multiple viral proteins, a viral capsid, or any fragment, subset of components, or variant thereof.

[0038] As used herein, a "production cell" refers to a cell capable of producing a product, e.g., a recombinant polypeptide. In some embodiments, a production cell comprises an exogenous nucleic acid encoding a product (e.g., a recombinant polypeptide), e.g., operably linked to a control element that regulates expression of the product in the production cell. When cultured under appropriate conditions, e.g., conditions disclosed herein, e.g., in a bioreactor and in an appropriate medium, the production cell produces, e.g., secretes, the product.

[0039] As used herein, a "production factor" refers to a polypeptide or nucleic acid that affects the properties of a production cell with respect to the expression of a recombinant product. For example, a production factor may improve the amount (e.g., specific productivity per cell or product titer) or quality (e.g., correct folding and assembly, solubility, etc.) of a product. A production factor may be, for example, a protein involved in lipid metabolism (e.g., lipid metabolism modifiers such as SCD1 and / or SREBF-1), protein synthesis, protein folding, post-translational modification, protein trafficking, and / or protein secretion. It may also be a polypeptide or nucleic acid that inhibits the expression or activity of an endogenous protein. For example, a production factor may inhibit the expression of a non-essential endogenous protein that is highly expressed and secreted to improve the production capacity of a cell.

[0040] As used herein, the term "promoter" refers to a sequence having sufficient sequence, e.g., from a naturally occurring or engineered promoter, such that operably linking a coding sequence to the promoter results in expression of the coding sequence. For example, a cytomegalovirus (CMV) promoter includes all or an active fragment of a CMV promoter, e.g., all or an active fragment of a CMV promoter, optionally including intron A and / or UTR sequences. In embodiments, the CMV promoter differs from a naturally occurring or engineered variant CMV promoter by no more than 5, 10, 20, 30, 50, or 100 nucleotides. In embodiments, the CMV promoter differs from a naturally occurring or engineered variant CMV promoter by no more than 1, 5, 10, or 50% of its nucleotides. As used herein, a promoter can be constitutive, regulated, repressible, inducible, strong, weak, or other properties of the promoter sequence it contains. In embodiments, a promoter can include sequences 5' or 3' of a coding sequence, e.g., a coding sequence of a recombinant, therapeutic, or repressor polypeptide. In embodiments, the promoter may comprise a sequence within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In embodiments, the promoter may comprise, in part or in whole, within a sequence 5' or 3' of a coding sequence, e.g., a coding sequence of a recombinant, therapeutic, or repressor polypeptide. In embodiments, the promoter may comprise, in part or in whole, within a coding sequence, e.g., a coding sequence of a recombinant, therapeutic, or repressor polypeptide. In embodiments, the promoter may comprise, in part or in whole, within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide.

[0041] As used herein, the term "operably linked" refers to the relationship between a nucleic acid sequence encoding a product (e.g., a polypeptide) or an enzyme molecule and a control element, where the product or enzyme molecule-encoding sequence and the control element are operably linked when they are positioned in a manner suitable for the control element to regulate the expression of the product or enzyme molecule-encoding sequence. Thus, with respect to different control elements, operably linked constitutes different arrangements of the product or enzyme molecule-encoding sequence relative to the control element. For example, a product (e.g., a polypeptide)-encoding sequence can be operably linked to a control element that includes a promoter element when the promoter element and the product (e.g., a polypeptide)-encoding sequence are positioned proximal to each other and on the same nucleic acid. In another example, a product (e.g., a polypeptide)-encoding sequence can be operably linked to a control element that includes an enhancer sequence that acts distally when the enhancer sequence and the product (e.g., a polypeptide)-encoding sequence are positioned on the same nucleic acid or even on different separate nucleic acids, a suitable number of bases apart.

[0042] As used herein, a selection marker refers to one or more nucleic acid sequences that confer a phenotype that can be used to select cells that contain the one or more nucleic acid sequences. In some embodiments, the one or more nucleic acid sequences include a sequence that encodes a polypeptide (e.g., and suitable control elements for the expression of the polypeptide). For example, the selection marker may include a gene that encodes a protein that confers an antibiotic resistance phenotype. Such a selection marker may be referred to as an antibiotic selection marker. In some embodiments, the selection marker includes one or more nucleic acid sequences that convey the ability to survive (e.g., grow and divide during) conditions that include reduced (e.g., absent) levels of an essential nutrient, e.g., a level that is insufficient for the cell to survive without the selection marker. For example, the selection marker may include a first nucleic acid that encodes a PAH enzyme molecule and a second nucleic acid that encodes a GCH1 enzyme molecule, where the selection marker conveys the ability to survive reduced (e.g., absent) levels of tyrosine in the culture medium. Such a selection marker may be referred to as an auxotrophic marker or an auxotrophic selection marker. By appending the compound name, e.g., amino acid name, to the auxotrophic or auxotrophic selectable marker, the selectable marker identifies the nutrient whose reduced levels or absence convey the ability to survive.

[0043] Vectors and Vector Systems The present invention employs vectors encoding components that enable transformed host cells to express a product of interest, such as a recombinant polypeptide, and to grow at low levels and in the absence of tyrosine, an essential amino acid for the cell, the absence of which would result in cell death and / or poor growth.

[0044] The vector comprises three components: a first nucleic acid sequence encoding a phenylalanine hydroxylase (PAH) enzyme molecule, typically lacking a functional N-terminal regulatory domain, and a second nucleic acid sequence encoding a GTP cyclohydrolase 1 (GCH1) enzyme molecule. These sequences are operably linked to a control sequence that allows expression of the enzyme in a suitable host cell. In one embodiment, the control sequence comprises a CMV promoter or an SV40 promoter, for example, a sequence encoding a human PAH sequence can be operably linked to a control sequence comprising an SV40 promoter, and / or a sequence encoding GCH1 can be operably linked to a control sequence comprising an SV40 promoter.

[0045] The third sequence comprises an insertion site, e.g., multiple cloning sites, into which the nucleic acid sequence encoding the product of interest can be cloned. This site is positioned and operably arranged to control the sequence so that the desired sequence can be expressed in a suitable host cell when introduced. In one embodiment, the three sequences that can be considered as expression cassettes are present in the same vector. In another embodiment, the first and second nucleic acid sequences can be on separate vectors, provided that the third nucleic acid sequence is on the same vector as one of them, to ensure that the selection of the sequence of interest is linked to the presence of a selectable marker.

[0046] The vector may contain additional expression cassettes for the product of interest, i.e., the vector system may contain an insertion site, e.g., a fourth, and optionally a fifth, and optionally a sixth nucleic acid sequence, each of which may contain a multiple cloning site, into which a nucleic acid sequence encoding the product of interest may be cloned. With respect to the third nucleic acid sequence, these sites are positioned and operably arranged to control the sequence so that the desired sequence, when introduced, may be expressed in a suitable host cell. For example, bispecific antibodies have at least three different, usually at least four different chains. These expression cassettes are ready for insertion of the sequence of interest and may be configured in a variety of ways. When the PAH and GCH1 sequences are on different vectors, each vector may contain one or more expression cassettes with multiple cloning sites, e.g., each may contain two such expression cassettes. In some embodiments, the expression cassettes, each of which has multiple cloning sites, may be in a single vector with only one of the selection markers. Thus, one vector can carry three or four expression cassettes, each with multiple cloning sites for introduction of sequences of interest, such as heavy or light chains for bispecific antibody production.

[0047] In one embodiment, and to take full advantage of the ability to introduce multiple sequences in the same step, all components of the vector system can be introduced into the host cell simultaneously.

[0048] In another embodiment, a suitable host cell may already be engineered to contain one of the first or second nucleic acid sequences. Thus, the invention further provides a selection system, the selection system comprising: a) a first nucleic acid comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of the PAH in a host cell; b) a second nucleic acid encoding GTP cyclohydrolase 1 (GCH1), operably linked to a second control sequence that allows expression of GCH1 in the host cell; c) (i) a multiple cloning site for inserting a sequence encoding a product of interest operably linked to a third control sequence that allows for expression of the product in the host cell, or (ii) a third nucleic acid encoding a product of interest operably linked to a third control sequence that allows for expression of the product in the host cell; d) a host cell, (a) and (c) are present in a vector and (b) is present in a host cell (typically integrated into the host cell genome), or (b) and (c) are present in a vector and (a) is present in a host cell (typically integrated into the host cell genome).

[0049] The nucleic acid sequence encoding the recombinant product and PAH, GCH1 enzyme can be cloned into several types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors and replication vectors. In embodiments, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and other virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain a functional replication origin in at least one organism (so that vectors can be self-replicating), control elements including promoter elements and optionally enhancer elements, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).Virus-derived vectors are suitable tools for achieving long-term gene transfer, because they allow the long-term stable integration of transgene and its propagation in daughter cells.

[0050] Vectors may also include, for example, signal sequences facilitating secretion, polyadenylation signals, and transcription terminators (e.g., from the bovine growth hormone (BGH) gene), elements allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColE1 or others known in the art), and / or elements allowing selection, such as a selectable marker or reporter gene.

[0051] Contemplated vectors may include insertion sites suitable for inserting sequences encoding polypeptides, such as exogenous therapeutic polypeptides. The insertion sites may include restriction endonuclease sites.

[0052] A sequence encoding a product of interest (described in the section below entitled Recombinant Products) can be introduced into the vector system described herein using cloning techniques well known in the art. The resulting vector system then contains, in addition to the first and second nucleic acid sequences, at least a third nucleic acid sequence comprising a sequence encoding the product of interest operably linked to a third control sequence that allows for expression of the product in a host cell, where this third sequence is present in the same vector as the first and / or second nucleic acid sequence (ensuring that a selectable marker functions to select for cells containing the third nucleic acid sequence).

[0053] As mentioned above, the vector system of the present invention can be used to express multiple sequences of interest, for example for proteins with multiple subunits, including antibodies (standard and bispecific antibodies). Thus, the vector can include additional expression cassettes of products of interest, and the multiple sequences of interest can be introduced into the multiple cloning site to produce a vector ready to be introduced into a host cell capable of expressing multiple products of interest. Thus, after introduction of the sequences of interest, in addition to the third nucleic acid sequence comprising a sequence encoding the product of interest operably linked to a third control sequence that allows expression of the product in the host cell, the vector system may include a fourth, and optionally a fifth, and optionally a sixth nucleic acid sequence, etc., each comprising a sequence encoding the product of interest operably linked to a control sequence that allows expression of the product in the host cell. These sequences are present in the same vector as the first and / or second nucleic acid sequence (to ensure that they can be selected as a result of being associated with a selectable marker).

[0054] Again, as mentioned above, these expression cassettes can be configured in various ways. When the PAH and GCH1 sequences are on different vectors, each vector can contain one or more expression cassettes, each encoding a product of interest, for example, each can contain two such expression cassettes. In some embodiments, the expression cassettes can be in a single vector with only one of the selection markers. Thus, one vector can have three or four expression cassettes, each with a sequence encoding a product of interest, such as a heavy or light chain for bispecific antibody production.

[0055] The vector may also contain sequences that aid in integration into the host cell genome either randomly or in a site-specific manner, such as the PiggyBac™ system, which uses inverted terminal repeats (ITRs) located at both ends of the vector. Sequence-specific transposases, site-specific integration methods, and sequences involved in the transfection process are also described in WO2013 / 190032 and WO2018 / 150269.

[0056] In some embodiments, the vector containing the nucleic acid sequence encoding the product contains an additional selection marker, such as glutamine synthetase, as described below. Typically, the vector system contains a separate vector containing the additional selection marker, as described below, and a multiple cloning site for inserting one or more sequences encoding the product(s) of interest operably linked to a control sequence that allows expression of the product in the host cell. When the sequence of interest is cloned into the multiple cloning site, the vector contains an additional selection marker and a nucleic acid sequence containing a sequence encoding the product of interest operably linked to a control sequence that allows expression of the product in the host cell, as described below. Such vectors generally do not contain PAH or GCH1 sequences.

[0057] The vector(s) may be provided in a kit, including instructions for use, and optionally, transfection reagents and the like.

[0058] Also provided herein are products described herein, such as nucleic acids encoding recombinant polypeptides, such as the subject nucleic acids. The nucleic acid sequence encoding the desired recombinant polypeptide can be obtained using recombinant methods known in the art, such as by screening a library from cells expressing the desired nucleic acid sequence, such as a gene, by deriving the nucleic acid sequence from a vector known to contain it, or by directly isolating it from cells and tissues containing it, using standard techniques. Alternatively, the nucleic acid encoding the recombinant polypeptide can be produced synthetically, rather than cloned. Recombinant DNA techniques and technologies are highly advanced and well established in the art. Thus, a person skilled in the art with knowledge of the amino acid sequence of the recombinant polypeptide described herein can easily envision or generate the nucleic acid sequence encoding the recombinant polypeptide.

[0059] GCH1 enzyme molecule Naturally occurring GCH1 enzyme catalyzes the conversion of GTP to 7,8-dihydroneopterin 3'-triphosphate (consuming two water molecules and also producing acetate), which is the first step in the production of BH4. In some embodiments, the GCH1 enzyme molecule has the same or similar activity as the naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule has increased or decreased activity relative to the naturally occurring GCH1 enzyme.

[0060] In some embodiments, the GCH1 enzyme molecule is a naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule comprises a full-length (e.g., uncleaved) GCH1 enzyme. In some embodiments, the GCH1 molecule has at least 50% amino acid sequence identity (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a mammalian GCH1 enzyme.

[0061] In some embodiments, the GCH1 enzyme molecule is a variant of a naturally occurring or non-naturally occurring (e.g., synthetic) GCH1 enzyme (e.g., a variant that includes one or more amino acid sequence modifications (e.g., substitutions, deletions, or insertions) relative to the amino acid sequence of the naturally occurring or non-naturally occurring enzyme). In some embodiments, the GCH1 enzyme molecule is or includes a deletion mutation, e.g., a truncation, e.g., a truncation of the N-terminal region, relative to the naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule is or includes at least 75, 80, 85, 90, 95, or 99% of the amino acid sequence (and optionally up to 100, 99, 95, 90, 85, 80, 79, 78, 77, 76, or 75% of the amino acid sequence) of a naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule comprises no more than 99, 95, 90, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, or 75% of the amino acid sequence of a naturally occurring GCH1 enzyme.

[0062] In some embodiments, the GCH1 enzyme molecule is a monomer, e.g., an active enzyme as a monomer. In some embodiments, the GCH1 enzyme molecule forms a multimer (e.g., under suitable conditions for enzyme activity, e.g., under cellular or physiological conditions, e.g., during a biomanufacturing process), e.g., an active enzyme as a multimer. In some embodiments, the GCH1 enzyme molecule multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer, e.g., decamer.

[0063] The sequence for use in the GCH1 enzyme molecule of the present disclosure can be obtained from any known GCH1 enzyme sequence.In some embodiments, the GCH1 enzyme molecule comprises human GCH1 enzyme, its variant, or its enzymatically active fragment.In some embodiments, the GCH1 enzyme molecule comprises CHO GCH1 enzyme, its variant, or its enzymatically active fragment.

[0064] In some embodiments, the GCH1 enzyme molecule comprises an amino acid sequence encoded by SEQ ID NO: 1, e.g., the amino acid sequence of SEQ ID NO: 2. In some embodiments, the GCH1 enzyme molecule comprises an amino acid sequence encoded by NCBI Reference Sequence: NM_001024024 (e.g., as of October 6, 2019). In some embodiments, the GCH1 enzyme molecule comprises an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 1, e.g., the amino acid sequence of SEQ ID NO: 2. In some embodiments, the exogenous nucleic acid encoding a GCH1 enzyme molecule comprises a nucleic acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the nucleic acid sequence of SEQ ID NO:1. NCBI reference sequence: NM_001024024 (query number 1) MEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS (SEQ ID NO: 2)

[0065] PAH enzyme molecule Naturally occurring PAH enzymes catalyze the conversion of phenylalanine to tyrosine using molecular oxygen and tetrahydrobiopterin (BH4). In some embodiments, the PAH enzyme molecule has the same or similar activity as the naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule has increased or decreased activity relative to the naturally occurring PAH enzyme.

[0066] In some embodiments, the PAH enzyme molecule is a naturally occurring PAH enzyme, hi some embodiments, the PAH enzyme molecule comprises a full-length (e.g., uncleaved) PAH enzyme.

[0067] In some embodiments, the PAH enzyme molecule is a variant of a naturally occurring or non-naturally occurring (e.g., synthetic) PAH enzyme (e.g., a variant that includes one or more amino acid sequence modifications (e.g., substitutions, deletions, or insertions) relative to the amino acid sequence of the naturally occurring or non-naturally occurring enzyme). In some embodiments, the PAH enzyme molecule is or includes a deletion mutation, e.g., a truncation, e.g., a truncation of the N-terminal region, relative to a naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule is or includes at least 75%, 80%, 85%, 90%, 95%, or 99% of the amino acid sequence (and optionally up to 100%, 99%, 95%, 90%, 85%, 80%, 79%, 78%, 77%, 76%, or 75% of the amino acid sequence) of a naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule comprises no more than 99, 95, 90, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, or 75% of the amino acid sequence of a naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule is or comprises at least 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 335, or 336 amino acids (and optionally no more than 450, 400, 390, 380, 370, 360, 350, 340, or 336 amino acids) of a naturally occurring PAH enzyme molecule. In some embodiments, the PAH enzyme molecule is or comprises no more than 450, 400, 390, 380, 370, 360, 350, 340, or 336 amino acids (and optionally at least 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 335, or 336 amino acids) of a naturally occurring PAH enzyme molecule. For example, the PAH enzyme molecule can comprise the first amino acids 1-14 and 37 onwards, including a deletion of amino acids 15-37. As a further example, the PAH enzyme molecule can comprise a deletion of amino acids 1-116. As a further example, the PAH enzyme molecule can comprise a deletion of amino acids 1-10 and 30-40.As a further example, a PAH enzyme molecule can include 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, or 350 C-terminal amino acids, e.g., 343 C-terminal amino acids, of a naturally occurring PAH enzyme.

[0068] In a preferred embodiment, the PAH enzyme molecule lacks part or all of the regulatory domain of a naturally occurring PAH enzyme, such that, for example, the PAH enzyme molecule is constitutively active relative to the naturally occurring PAH enzyme. Without wishing to be bound by theory, it is understood that the PAH enzyme comprises an N-terminal region that includes one or more regulatory domains that regulate the enzymatic activity of the PAH, for example, by regulating access to the enzyme active site. The regulatory region may include an ACT domain, which is known to allow allosteric regulation of metabolic enzymes, and / or an active site lid, which can conditionally block access to the enzyme active site. We believe that a PAH enzyme molecule lacking part or all of the regulatory domain is useful for a production cell, for example, a selection marker described herein, because such a PAH enzyme molecule may be more active (e.g., constitutively active) than a full-length PAH enzyme, for example, a PAH enzyme molecule that includes a PAH enzyme molecule that is subject to allosteric regulation of a regulatory domain. In some embodiments, the PAH enzyme molecule lacks an active site lid. In some embodiments, the PAH enzyme molecule lacks an ACT domain. In some embodiments, the PAH enzyme molecule includes a modification (e.g., substitution, deletion, or insertion) that eliminates the regulatory (e.g., inhibitory) function of the N-terminal regulatory region (e.g., active site lid and / or ACT domain). In some embodiments, the PAH enzyme molecule is not significantly inhibited (e.g., is not inhibited) by the presence of phenylalanine. In some embodiments, the PAH enzyme molecule includes a deletion of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, or 1-116 (e.g., 1-116) of human PAH, or a deletion of residues corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, or 1-116 (e.g., 1-116) of human PAH. In some embodiments, the PAH enzyme molecule lacks the N-terminal 116 amino acids of a naturally occurring PAH enzyme (eg, a naturally occurring human PAH enzyme), or the corresponding amino acids of a different naturally occurring PAH enzyme.See Daubner et al., 1997, Arch. Biochem. Biophys 348(2):295 (describing a truncated PAH lacking the regulatory domain (first 116 amino acids). This truncated PAH expressed in E. coli was more stable, more soluble, did not require preincubation with phenylalanine to be active, and had a higher affinity for substrates). In some embodiments, the PAH enzyme molecule comprises the C-terminal region of a naturally occurring PAH enzyme, e.g., the catalytic and multimerization portions of the PAH enzyme.

[0069] In some embodiments, the PAH enzyme molecule is a monomer, e.g., an active enzyme as a monomer. In some embodiments, the PAH enzyme molecule forms a multimer (e.g., under appropriate conditions for enzyme activity, e.g., under cellular or physiological conditions, e.g., during a biomanufacturing process), e.g., an active enzyme as a multimer. In some embodiments, the PAH enzyme molecule multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer, e.g., a tetramer.

[0070] The sequences for use in the PAH enzyme molecules of the present disclosure can be obtained from any known PAH enzyme sequence. In some embodiments, the PAH enzyme molecule comprises a human PAH enzyme, a variant thereof, or an enzymatically active fragment thereof. In some embodiments, the PAH molecule has at least 50% amino acid sequence identity (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a human PAH enzyme. In some embodiments, the PAH enzyme molecule comprises a CHO PAH enzyme, a variant thereof, or an enzymatically active fragment thereof. In some cases, the PAH molecule has at least 50% amino acid sequence identity (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a CHO PAH enzyme.

[0071] In some embodiments, the PAH enzyme molecule comprises an amino acid sequence encoded by any of SEQ ID NOs:3 or 4, e.g., any of SEQ ID NOs:5 or 6. In some embodiments, the PAH enzyme molecule comprises an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to an amino acid sequence encoded by any of SEQ ID NOs:3 or 4, e.g., any of SEQ ID NOs:5 or 6. In some embodiments, the exogenous nucleic acid encoding the PAH enzyme molecule comprises a nucleic acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence of any of SEQ ID NOs:3 or 4. Exemplary CHO PAH Nucleic Acid Sequences (NCBI Reference Sequence: XM_027434726.1) Exemplary CHO PAH Amino Acid Sequences MVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKKTWGTVFKTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCLSDKPKLLPLDLEKTASQEYNVTEFQPLYYVAESFNDAKEKVRAFAATIPRPFSVRYDPYTQRVEVLDNTQQLKILADSINSEVGILCSALHKIKS (SEQ ID NO: 5) Exemplary Human PAH Nucleic Acid Sequences (GenBank: K03020.1) Exemplary Human PAH Amino Acid Sequences MVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK (SEQ ID NO: 6)

[0072] host cell The present disclosure is directed, in part, to a host cell comprising a tyrosine auxotrophic selection marker, e.g., a first nucleic acid encoding a phenylalanine hydroxylase (PAH) enzyme molecule, and a second nucleic acid encoding a GTP cyclohydrolase 1 (GCH1) enzyme molecule. At least one of these sequences is exogenous to the host cell, i.e., not naturally occurring. Both sequences may be exogenous to the host cell.

[0073] As described above in the section on vectors, the nucleic acid sequences can be present in the same or different vectors, so they can be present in the host cell in the same or different nucleic acid molecules / vectors.These vectors can be self-replicating vectors, especially when maintained extrachromosomally.In some embodiments, the first and / or second nucleic acid is integrated into the genome of the production cell.

[0074] The host cell after introduction of the vector system typically also contains a third exogenous nucleic acid sequence encoding a product of interest, and these cells are also referred to herein as "production cells". The product typically does not naturally occur in the unmodified host cell, e.g., a biotherapeutic protein. The third nucleic acid sequence is present in the same nucleic acid as the first nucleic acid sequence and / or the second nucleic acid sequence, depending on the number of vectors used to produce the cell. In some embodiments, the third exogenous nucleic acid is integrated into the genome of the host cell. There may also be additional exogenous nucleic acids introduced using the vector system of the present invention.

[0075] The first, second, and / or third exogenous nucleic acid may include one or more control elements. The control elements, e.g., promoters and / or enhancers, may be operably linked to the sequence encoding the PAH enzyme molecule, the sequence encoding the GCH1 molecule, or the sequence encoding the product. In some embodiments, the first and second exogenous nucleic acids include one or more control elements sufficient to express the PAH enzyme molecule and the GCH1 enzyme molecule in the production cell. In some embodiments, the third exogenous nucleic acid includes one or more control elements sufficient to express the product, e.g., a polypeptide product, in the production cell. Control elements suitable for use in the present invention are known to those of skill in the art, and examples are also described herein.

[0076] host cell type In one aspect, the host cell of the present disclosure may be, be made from, or be derived from any cell type, strain, or cell line described herein.Generally, the methods herein can be used to produce a host cell, for example, a cell or cell line that comprises a nucleic acid construct (e.g., a vector or a heterologous nucleic acid integrated into a genome) that comprises (i) a subject nucleic acid sequence that encodes a product of interest, and (ii) one or more exogenous nucleic acid sequences that encode one or more enzyme molecules involved in the biosynthetic pathway of an amino acid, and the cell or cell line does not endogenously express the enzyme molecule.

[0077] The host cell can be any suitable cell that can be genetically engineered and grown. Typically, the cell is one that is suitable for large-scale culture to produce the product of interest.

[0078] The host cell before the introduction of the vector system of the present invention cannot produce a sufficient level of tyrosine to support cell growth in the absence of tyrosine.This may be because it does not express one or more of the enzymes required for tyrosine biosynthesis to a sufficient level or is engineered to knock out the relevant gene.Thus, in one embodiment, the host cell of the present invention is genetically modified to inhibit or eliminate any endogenous PAH and / or GCH1 activity.This can be achieved, for example, by mutation (insertion, deletion, and / or substitution) in the genomic sequence that codes and / or regulates the expression of endogenous PAH and / or GCH1.

[0079] In some embodiments, the host cell is a eukaryotic cell, such as a mammalian, yeast, or insect cell.

[0080] In one embodiment, the host cell is a mammalian cell. Examples of species from which the host cell may be derived include human, mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, and cat.

[0081] In an embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell. In one embodiment, the host cell is a CHO-K1 cell, a CHOK1SV® cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHO-S, a CHO GS knockout cell (a CHO cell in which all endogenous copies of the glutathione synthetase (GS) gene have been inactivated), a CHOK1SV® FUT 8 knockout cell, a CHOZN, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GS-KO cell) is, for example, a CHOK1SV® GS knockout cell (GS Xceed® cell-CHOK1SV GS-KO®, Lonza Biologics, Inc.). A CHO FUT8 knockout cell is, for example, a Potelligent® CHOK1SV® FUT8 knockout (Lonza Biologics, Inc.).

[0082] In embodiments, the host cell is a HeLa, MDCK, Sf9, Sf21, Tn5, HT1080, NB324K, FLYRD18, HEK293, HEK293T, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney), VERO, SP2 / 0, NS0, YB2 / 0, Y0, EB66, C127, L cell, COS (e.g., COS1 and COS7), QC1-3, CHOK1, CHOK1SV, Potelligent® (CHOK1SV FUT8-KO), CHO GS knockout, GS Xceed™ (CHOK1SV GS-KO), CHOS, CHO DG44, CHO DXB11, or CHOZN cell, or any cell derived therefrom.

[0083] In other embodiments, the host cell is a non-mammalian cell, such as an avian, fish, insect, plant, fungal, or yeast cell.

[0084] In some embodiments, the host cell or cell line of the host cell is formed by a process involving the fusion of multiple cells (e.g., the fusion of two cells of the same type (e.g., two CHO cells) or two cells of different types (e.g., different species). Examples of host cells or cell lines formed by a process involving the fusion of multiple cells include, but are not limited to, hybridomas, triomas, and quadromas.

[0085] In some embodiments, the derivatives include, but are not limited to, cells described herein that further include modifications such as mutations (e.g., substitutions, deletions, or insertions) or addition of nucleic acids (e.g., vectors) (e.g., gene knock-ins, gene knock-outs, or gene multiplicities). In some embodiments, the derivatives include cells described herein that have undergone directed evolution. In some embodiments, the derivatives include combinations of these exemplary modifications described herein.

[0086] Eukaryotic cells include stem cells. Stem cells may be, for example, pluripotent stem cells, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs).

[0087] In embodiments, the host cell is a differentiated form of any of the cells described herein. In one embodiment, the host cell is a cell derived from any primary cell in culture.

[0088] In embodiments, the host cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte, or a non-parenchymal cell.For example, the host cell can be a plateable metabolically limited human hepatocyte, a plateable induction limited human hepatocyte, a plateable Qualyst Transporter Certified™ human hepatocyte, a suspension limited human hepatocyte (including 10 donor and 20 donor pooled hepatocytes), a human hepatic Kupffer cell, a human hepatic stellate cell, a dog hepatocyte (including single and pooled beagle hepatocytes), a mouse hepatocyte (including CD-1 and C57BI / 6 hepatocytes), a rat hepatocyte (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), a monkey hepatocyte (including Cynomolgus or Rhesus hepatocytes), a cat hepatocyte (including Domestic Shorthair hepatocytes), and a rabbit hepatocyte (including New Zealand White hepatocytes).Exemplary hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive Research Triangle Park, North Carolina, USA 27709.

[0089] In some embodiments, the host cell comprises a knockout of glutamine synthetase (GS). In embodiments, the host cell does not comprise a functional GS gene. In embodiments, the host cell does not comprise a GS gene. In embodiments, the GS gene in the host cell comprises a mutation that renders the gene unable to encode a functional GS protein.

[0090] In embodiments, the eukaryotic cell is, for example, a yeast cell (e.g., a species of the genus Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), the genus Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), the genus Saccharomyces (e.g., Saccharomyces cerevisae, cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum), the genus Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), the genus Candida (e.g., Candida utilis, Candida cacaoi, Candida boidinii), the genus Geotrichum (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia In some embodiments, the eukaryotic cell is a lower eukaryotic cell, such as Pichia lipolytica, or Schizosaccharomyces pombe. In some embodiments, the eukaryotic cell is of the species Pichia pastoris. Examples of Pichia pastoris strains include, but are not limited to, X33, GS115, KM71, KM71H, and CBS7435.

[0091] In embodiments, the eukaryotic cell is a fungal cell (e.g., Aspergillus sp. (such as A. niger, A. fumigatus, A. orzyae, A. nidula), Acremonium sp. (such as A. thermophilum), Chaetomium sp. (such as C. thermophilum), Chrysosporium sp. (such as C. thermophile), Cordyceps sp. (such as C. militaris), Corynascus sp., Ctenomyces sp., Fusarium sp. (such as F. oxysporum), Glomerella sp. (such as G. graminicola), Hypocrea sp. (such as H. jecorina), Magnaporthe sp. (such as M. orzyae), Myceliophthora sp. (such as M. thermophile), Nectria sp. (such as N. heamatococca), Neurospora sp. (e.g. N. crassa), Penicillium sp., Sporotrichum sp. (e.g. S. thermophile), Thielavia sp. (e.g. T. terrestris, T. heterothallica), Trichoderma sp. (e.g. T. reesei), or Verticillium sp. (e.g. V. dahlia).

[0092] In embodiments, the eukaryotic cell is an insect cell (e.g., an Sf9, Mimic™ Sf9, Sf21, High Five™ (BT1-TN-5B1-4), or BT1-Ea88 cell), an algae cell (e.g., of the genus Amphora sp., Bacillariophyceae sp., Dunaliella sp., Chlorella sp., Chlamydomonas sp., Cyanophyta sp. (cyanobacteria), Nannochloropsis sp., Spirulina sp., or Ochromonas sp.), or a plant cell (e.g., a cell from a monocotyledonous plant (e.g., corn, rice, wheat, or Setaria sp.), or a dicotyledonous plant (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens, or Arabidopsis sp.)).

[0093] In embodiments, the host cell is a prokaryotic cell, such as a bacterial cell.

[0094] In an embodiment, the prokaryotic cell is a gram-positive cell, such as Bacillus sp., Streptomyces sp., Streptococcus sp., Staphylococcus sp., or Lactobacillus sp. Bacillus sp. that can be used are, for example, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. natto, or B. megaterium. In an embodiment, the cell is B. subtilis, such as B. subtilis 3NA and B. subtilis 168. Bacillus sp. can be, for example, Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12 th Avenue, Columbus OH 43210-1214.

[0095] In embodiments, the prokaryotic cell is a gram-negative cell, such as a Salmonella sp. or Escherichia coli, such as TG1, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue, and Origami, and from an E. coli B strain, such as BL-21 or BL21(DE3), or BL21(DE3)pLysS, all of which are commercially available.

[0096] In some embodiments, the prokaryotic cell is a cyanobacteria cell. In some embodiments, the cyanobacteria cell is a blue-green alga, such as a Synechocystis cell.

[0097] Suitable host cells are commercially available from culture collections such as, for example, DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0098] Additional selection markers In some embodiments, the host cell comprises one or more selection markers in addition to the tyrosine auxotrophic selection marker. In some embodiments, the second selection marker is a different auxotrophic selection marker, such as a different amino acid auxotrophic selection marker. In one embodiment, the amino acid is proline or glutamine. Examples of nucleic acid sequences required for such selection markers are sequences encoding glutamine synthetase (for glutamine) and pyrroline-5-carboxylate synthetase (P5CS) (for proline).

[0099] Another selection marker is an exogenous nucleic acid encoding a dihydrofolate reductase (DHFR), e.g., a DHFR enzyme molecule that confers resistance to, e.g., methotrexate (MTX). In some embodiments, the DHFR selection marker is also a thymidine auxotrophic selection marker and / or a hypoxanthine auxotrophic selection marker. In some embodiments, the host cell does not contain an endogenous functional DHFR gene, e.g., does not contain a mutation that renders the endogenous DHFR gene unable to encode a functional DHFR enzyme.

[0100] Additional selectable markers include hypoxanthine-guanine phosphoribosyltransferase (HPRT) selectable markers, e.g., exogenous nucleic acid encoding an HPRT enzyme molecule. In some embodiments, the production cells are unable to grow and / or divide in the presence of aminopterin without HPRT (e.g., supplemental HPRT encoded by an exogenous nucleic acid) and supplemental purine, e.g., hypoxanthine. In some embodiments, the HPRT selectable marker is also a purine (e.g., hypoxanthine or guanine) auxotrophic selectable marker. In some embodiments, the production cells do not contain an endogenous functional HPRT gene, e.g., do not contain a mutation that renders the endogenous HPRT gene incapable of encoding a functional HPRT enzyme.

[0101] In one embodiment, the selectable marker is compatible with the Selexis selection system (eg, SUREtechnology Platform™ and Selexis Genetic Elements™, commercially available from Selexis SA) or the Catalent GPEx® selection system.

[0102] A selection marker for use in a production cell may be associated with a subject nucleic acid. As used herein with respect to the relationship between a selection marker and a subject nucleic acid, associated with refers to a relationship in which the presence of the selection marker in a production cell correlates with the presence of the subject nucleic acid. The selection marker is associated with a subject nucleic acid such that selecting for (e.g., requiring) the presence of the selection marker in a production cell selects for the presence of the subject nucleic acid. In some embodiments, the selection marker, e.g., at least one component of the selection marker, is located on the same nucleic acid molecule as the subject nucleic acid, e.g., on the same vector as the subject nucleic acid. For example, a production cell comprising a tyrosine auxotrophic selection marker comprising a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule may comprise a subject nucleic acid located on the same vector as either the first exogenous nucleic acid or the second exogenous nucleic acid. In a production cell comprising two or more selection markers, each selection marker may be associated with a different subject nucleic acid. In some embodiments, the production cell comprises a first selection marker associated with the first subject nucleic acid (e.g., encoding a product) and a second selection marker associated with the second subject nucleic acid (e.g., encoding a production factor, e.g., lipid metabolism regulator (LMM), such as SCD1 and / or SREBF-1, as described in WO2017 / 191165 and WO2019 / 152876, which are incorporated by reference herein). Thus, the additional selection marker is used to maintain the exogenous production factor introduced into the host cell (including in the previous case for producing a stable cell line). In some embodiments, the production cell comprises a first selection marker associated with the first subject nucleic acid (e.g., encoding a first product) and a second selection marker associated with the second subject nucleic acid (e.g., encoding a second product). In some embodiments, the production cell comprises a first selection marker associated with a first subject nucleic acid (e.g., encoding a first product of a multi-polypeptide product) and a second selection marker associated with a second subject nucleic acid (e.g., encoding a second product of a multi-polypeptide product).It is understood that additional subject nucleic acids may be included that may be associated with different markers or the same marker.

[0103] Inhibitors The host cells and / or cultures comprising the host cells may include one or more enzyme molecule inhibitors (also referred to herein as inhibitors). Enzyme molecule inhibitors may be used to increase the stringency of the selection process described herein, for example, by reducing or preventing endogenous enzyme molecule activity, such that cells that do not incorporate an exogenous nucleic acid encoding the enzyme molecule (e.g., and including the subject nucleic acid sequence) exhibit reduced or undetectable levels of endogenous enzyme molecule activity. Cells that exhibit reduced or undetectable levels of endogenous enzyme molecule activity may not be able to grow and / or survive in the absence of an external supply of an amino acid (e.g., proline, tyrosine, or glutamine) whose synthesis requires the activity of the enzyme molecule. In some embodiments, the inhibitor binds to the enzyme molecule, e.g., binds to and inhibits the enzyme molecule. In embodiments, the inhibitor is an allosteric inhibitor of the enzyme molecule. In embodiments, the inhibitor is a competitive inhibitor of the enzyme molecule.

[0104] The production cells described herein may, in some embodiments, further comprise an inhibitor of the enzyme molecule (e.g., PAH or GCH1) expressed by the exogenous nucleic acid introduced into the cell. In some embodiments, the level of the inhibitor in the cell is sufficient to reduce the endogenous enzyme molecule activity to less than about 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, or 10% of the activity observed in a cell lacking the inhibitor. In some embodiments, less than about 0.001%, 0.01%, 0.1%, 1%, 5%, or 10% of the cells selected based on growth in a medium lacking an amino acid do not comprise a subject nucleic acid. In some embodiments, the ratio of enzyme molecules and inhibitor molecules in a cell is about: 1:1000, 1:500, 1:250, 1:200, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 250:1, 500:1, or 1000:1.

[0105] The inhibitor can be, for example, an amino acid or analog thereof, a polypeptide, a nucleic acid, or a small molecule. In some embodiments, the inhibitor is an analog of an amino acid produced by a biosynthetic pathway involving an enzyme molecule. In some embodiments, the inhibitor is an analog of a substrate of the enzyme molecule. In some embodiments, the inhibitor is an antibody molecule (e.g., an antibody or antibody fragment, such as those described herein), a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, an aptamer, or a structural and / or functional fragment or hybrid of any of these. In some embodiments, the inhibitor is an antisense RNA, siRNA, tRNA, ribosomal RNA, microRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, RNA aptamer, or a long non-coding RNA.

[0106] In some embodiments, the inhibitor inhibits an enzyme molecule in the biosynthetic pathway of proline, tyrosine, or glutamine. In one embodiment, the inhibitor inhibits the activity of PAH (e.g., a phenylalanine analog) or GCH1. In an embodiment, the inhibitor is a tetrahydrobiopterin (BH4) analog. In some embodiments, the inhibitor is a GTP analog. In one embodiment, the inhibitor is selected from α-methyltyrosine (e.g., at 50-100 μM), α-methylphenylalanine, and 2,4-amino-6-hydroxypyrimidine.

[0107] In embodiments, the inhibitor inhibits the activity of an enzyme that, when used, forms the basis of one of the additional selection markers, such as a pyrroline-5-carboxylate synthase (P5CS) molecule. In embodiments, the inhibitor inhibits the activity of P5CS. In embodiments, the inhibitor is a proline analog. In embodiments, the inhibitor is L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline, or L-4-thiazolidine carboxylic acid. In some embodiments, the inhibitor inhibits the activity of DHFR, for example, methotrexate. In some embodiments, the inhibitor inhibits glutamine synthetase (e.g., a glutamine analog, methionine sulfoximine (MSX) or an analog thereof (e.g., alpha-methyl or alpha-ethyl MSX)). In some embodiments, the production cell comprises more than one selection marker and comprises an enzyme molecule inhibitor of each selection marker.

[0108] Introduction of Nucleic Acid into Host Cells and Selection Steps Many suitable methods for introducing exogenous nucleic acid into a host cell are known in the art, including, for example, transfection, transduction (e.g., viral transduction), or electroporation of nucleic acid, e.g., vector, into the cell. Examples of physical methods for introducing nucleic acid, e.g., heterologous nucleic acid or vector described herein, into a host cell include, but are not limited to, calcium phosphate precipitation, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). Examples of chemical means for introducing nucleic acid, such as heterologous nucleic acid or vector described herein, into host cells include, but are not limited to, lipofection, colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.Exemplary colloidal systems for use as in vitro and in vivo delivery vehicles are liposomes (e.g., artificial membrane vesicles).Other state-of-the-art methods of targeted delivery of nucleic acid are available, such as delivery of polynucleotides or other suitable submicron-sized delivery systems in targeted nanoparticles.

[0109] Host cells can be transiently or stably transfected with the nucleic acid.

[0110] Selection of host cells containing the introduced nucleic acid can be achieved by culturing the cells under stringent selective conditions that allow cells containing the introduced nucleic acid to grow, while limiting the ability of non-transformed cells to grow, based on the tyrosine auxotrophic selection system of the present invention (and any other additional selection markers that may have been included).

[0111] The transfected / transformed cell population is cultured under conditions where the level of tyrosine allows easy selection of cells containing the introduced nucleic acid. Thus, cells are cultured in the presence of a lower level of tyrosine than that required for the survival or growth of the cell condition. Typically, this involves the use of a medium lacking tyrosine, so that cells are cultured in the absence of tyrosine. Nevertheless, low levels of tyrosine can be tolerated as long as the selection conditions are sufficiently stringent, such as a culture medium containing less than 0.01 g / L tyrosine, or less than 50, 20, or 10 μM tyrosine. Those skilled in the art will be able to easily determine the desired tyrosine level to obtain sufficient selection stringency.

[0112] Since the enzyme molecule provided by one or more exogenous nucleic acids provides the activity to convert phenylalanine to tyrosine, it may be desirable to supplement the culture medium with additional phenylalanine to meet the normal requirement of the host cells for phenylalanine and provide a precursor for tyrosine production. Thus, the population of cells may be cultured in the presence of a level of phenylalanine that is higher than the level required for survival or growth of the production cells cultured in the presence of a level of tyrosine required for survival or growth. Thus, in some embodiments, phenylalanine is provided (e.g., as part of the culture and / or as a component of the culture medium) at a level of at least 0.035 g / L. Thus, the cells may be cultured in the presence of a level of phenylalanine of at least 2, 3, or 4 mM. Typically, the level of phenylalanine is less than 10 mM, e.g., less than 9, 8, 7, or 6 mM, since high levels of phenylalanine can inhibit cell growth.

[0113] For CHO PAH enzymes, in one embodiment, the phenylalanine level in the culture medium is preferably 2-9 mM, e.g., 2 or 3 mM to 6 or 7 mM phenylalanine, while for human PAH enzymes, in one embodiment, the preferred range is 4-9 mM phenylalanine.

[0114] The cells may be subjected to an adaptation step so that they can adjust to higher levels of phenylalanine. This step may involve passaging the cells at one or more progressively higher concentrations of phenylalanine in the cell culture medium, for example, 3 mM for one or two passages, and then at a final desired concentration, for example, 6 mM. This may be done before or after transfection, for example, so that the cells are recovering before the growth phase.

[0115] In some embodiments, the level of phenylalanine is established and / or maintained using an autoregulatory system that detects and / or monitors the level of phenylalanine in the culture and provides phenylalanine in response to the detected level being below a threshold (e.g., until the detected level is equal to or greater than the threshold). In some embodiments, such an autoregulatory system utilizes spectroscopy (e.g., Raman spectroscopy) to detect and / or monitor the level of phenylalanine. Similar considerations apply when additional selectable markers are used.

[0116] When two selection markers (e.g., the selection system of the present invention and the GS selection system) are used, the relevant vectors may be introduced simultaneously and the cell culture medium may be formulated to provide stringent selection for both types of markers, i.e., medium lacking tyrosine and glutamine, optionally supplemented with phenylalanine, for example, as described above. Alternatively, the selection may be a two-step process in which one vector system is introduced and selected under stringent conditions for the first marker, and then the resulting selected cells are transfected / transformed with the second marker and, optionally, under stringent conditions for the first marker, e.g., medium lacking tyrosine and glutamine, optionally supplemented with phenylalanine. Alternatively, less stringent conditions for the first marker may be used when subsequently selecting for the second marker. The culture conditions described above apply mutatis mutandis to this two selection marker procedure (and when using additional markers).

[0117] Functional characteristics of producer cells containing introduced nucleic acid sequences In some embodiments, the host cell comprises a tyrosine auxotrophic selection marker (e.g., a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule) and is capable of growing and / or dividing in a culture medium containing reduced levels of tyrosine (e.g., in the absence of tyrosine). Such cells are also referred to herein as production cells. The ability to grow and / or divide is known to those of skill in the art and can be assessed by methods described herein. In some embodiments, the host cell is capable of growing and / or dividing in a culture medium containing less than 0.01 g / L, or less than 50, 20, or 10 μM tyrosine, e.g., in the absence of tyrosine. In some embodiments, the host cell is capable of growing and / or dividing in a culture medium lacking tyrosine.

[0118] In some embodiments, the host cells comprise a subject nucleic acid associated with a selectable marker (e.g., a tyrosine auxotrophic selectable marker, comprising a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule). In some embodiments, the host cells comprise at least a threshold copy number of the subject nucleic acid (e.g., a copy number sufficient to efficiently produce a product), e.g., at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 copies of the subject nucleic acid.

[0119] In some embodiments, the host cell comprises a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule. In some embodiments, the host cell comprises at least a threshold copy number of the first exogenous nucleic acid (e.g., a copy number sufficient for the host cell to grow and / or divide at reduced levels (e.g., in the absence of tyrosine), e.g., at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 copies of the first exogenous nucleic acid). In some embodiments, the host cell comprises at least a threshold copy number of a second exogenous nucleic acid (e.g., a copy number sufficient for the host cell to grow and / or divide at a reduced level (e.g., in the absence of tyrosine)), e.g., at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, or 10,000 copies of the second exogenous nucleic acid.

[0120] In some embodiments, a subject nucleic acid persists in a host cell (e.g., or its daughter cells or progeny) for a specified interval, for example, due to its association with a selectable marker (e.g., a tyrosine auxotrophic marker, including a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule). In some embodiments, a first exogenous nucleic acid persists in a host cell (e.g., or its daughter cells, progeny, generation, or population doubling) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (and optionally persists indefinitely). In some embodiments, the first exogenous nucleic acid remains in the host cell (e.g., or a daughter cell or progeny thereof) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 cell divisions (and optionally, remains indefinitely). In some embodiments, the first exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings), for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 host cycles, population doublings, or generations (and optionally persists indefinitely), e.g., of a bioreactor described herein. In some embodiments, the second exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (and optionally persists indefinitely).In some embodiments, the second exogenous nucleic acid remains in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 cell divisions (and optionally, remains indefinitely). In some embodiments, the second exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings), for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 host cycles, population doublings, or generations of, e.g., a bioreactor described herein (and optionally persists indefinitely). In some embodiments, the first exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings), for as long as the host cell is maintained in a medium that includes reduced levels of tyrosine (e.g., no tyrosine). In some embodiments, the second exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings) as long as the host cell is maintained in a medium with reduced levels of tyrosine (e.g., no tyrosine). In some embodiments, persistence of the first, second, or first and second exogenous nucleic acid in the host cell is assessed functionally, e.g., by whether the host cell grows and produces a product in a medium with reduced levels of tyrosine (e.g., no tyrosine). In some embodiments, persistence of the first, second, or first and second exogenous nucleic acid in the host cell is assessed (e.g., confirmed) by using RT-PCR.

[0121] In some embodiments, a host cell that includes a tyrosine auxotrophic selection marker (e.g., a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule) grows and / or divides faster in a culture medium that includes reduced levels of tyrosine (e.g., in the absence of tyrosine) than an otherwise similar cell that does not include a tyrosine auxotrophic selection marker. In some embodiments, the host cell grows and / or divides at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% faster, or 10-fold, 10-fold, or 10-fold faster, in a culture medium that includes reduced levels of tyrosine (e.g., in the absence of tyrosine) than an otherwise similar cell that does not include a tyrosine auxotrophic selection marker. 2 Double, 10 3 Double, 10 4 Double, 10 5 Double, or 10 6 grow and / or divide twice as fast.

[0122] In some embodiments, host cells containing a selectable marker comprising an exogenous nucleic acid encoding an enzyme molecule (e.g., a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule) (and optionally a subject nucleic acid associated with the exogenous nucleic acid) exhibit increased enzyme molecule activity compared to cells lacking the exogenous nucleic acid and / or the subject nucleic acid. In some embodiments, the level of enzyme molecule activity is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to enzyme molecule activity detectable in cells lacking the exogenous nucleic acid encoding the enzyme molecule and / or the associated subject nucleic acid. In some embodiments, cells with increased activity may grow faster than cells lacking the exogenous nucleic acid encoding the enzyme molecule and / or the associated subject nucleic acid. In some embodiments, the rate of cell growth and / or division is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to similar cells under similar culture conditions lacking exogenous nucleic acids encoding the enzyme molecules and / or related subject nucleic acids. In some embodiments, the host cells grow at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 5000, or 10,000 times faster on medium lacking amino acids than similar cells lacking the subject nucleic acid and / or exogenous nucleic acid encoding the enzymatic molecule.

[0123] Methods for Producing Recombinant Products Using Host (Producer) Cells The host cells of the present invention, also referred to as production cells, can be used to express the product encoded by the introduced nucleic acid. These production cells are typically stably transfected into the cells with the first, second, and / or third exogenous nucleic acid (and optionally further exogenous nucleic acids as described herein, which produce two or more products of interest, including multiple subunit products) to create the production cells. In alternative embodiments, the host cells may be transiently transfected into suitable cells with the first, second, and / or third exogenous nucleic acid.

[0124] The recombinant product may be expressed by culturing the production cells of the invention by any method known in the art suitable for the production of the product, taking into consideration the methods described below. In some embodiments, the culture medium lacks tyrosine or contains a level of tyrosine of 0.01 g / L or less or 50, 20, or 10 μM or less (e.g., a level of tyrosine insufficient for culturing a similar cell that does not contain one or more exogenous nucleic acids encoding one or more enzyme molecules and / or a subject nucleic acid). In some embodiments, the culturing includes culturing the production cells in the presence of a level of tyrosine lower than that required for the survival or growth of a cell that does not contain one or more exogenous nucleic acids (e.g., a cell similar to the production cell), e.g., in the absence of tyrosine. Since it may not be necessary to apply selective pressure on the production cell line during expression of the recombinant product, the culture medium may contain tyrosine at various stages during the growth and production phases. However, since tyrosine is difficult to handle in cell culture medium due to its low solubility, it may be advantageous to omit it entirely from the cell culture medium.

[0125] On the other hand, when tyrosine is absent (or at low levels), cells consume more phenylalanine, so various media used, such as feed solutions, can be supplemented with phenylalanine. For example, phenylalanine can be included at a level of at least 0.035 g / L. Thus, in some embodiments, phenylalanine is provided (e.g., as part of the culture and / or as a component of the culture medium) at a level of at least 0.035 g / L. Thus, cells can be cultured in the presence of phenylalanine at a level of at least 2, 3, 4, 5, 6, 7, 8, or 9 mM. Typically, the level of phenylalanine is less than 10 mM, for example, less than 9, 8, 7, or 6 mM, because high levels of phenylalanine can inhibit cell growth.

[0126] For CHO PAH enzymes, in one embodiment, the phenylalanine level in the culture medium is preferably 2-9 mM, e.g., 2 or 3 mM to 6 or 7 mM phenylalanine, while for human PAH enzymes, in one embodiment, the preferred range is 4-9 mM phenylalanine. Our results show that truncated human PAH enzymes provide superior cell performance when the cell culture medium is supplemented with phenylalanine.

[0127] The cells may be subjected to an adaptation step so that they can adjust to higher levels of phenylalanine. In one embodiment, the cells are already adapted during the selection phase to grow in phenylalanine-supplemented medium. Alternatively, this may be done during the production or pre-production phase, for example in an N-1 bioreactor producing inoculum for the N bioreactor. Again, adaptation may be done for another period with increased concentrations of phenylalanine, or the cells may be seeded into the cell culture medium already at the final level of supplementation.

[0128] In some embodiments, the level of phenylalanine is established and / or maintained using an autoregulatory system that detects and / or monitors the level of phenylalanine in the culture and provides phenylalanine in response to the detected level being below a threshold (e.g., until the detected level is equal to or greater than the threshold). In some embodiments, such an autoregulatory system utilizes spectroscopy (e.g., Raman spectroscopy) to detect and / or monitor the level of phenylalanine. Similar considerations apply when additional selectable markers are used.

[0129] In embodiments, the cell culture is performed as a batch culture, a fed-batch culture, an abbreviated fed-batch overgrowth (aFOG), a draw-and-fill culture, a continuous culture, or a semi-continuous culture, including a perfusion culture. In some embodiments, the bioreactor is capable of or configured to operate continuously or semi-continuously. In one embodiment, the cell culture is a suspension culture. In one embodiment, the cell or cell culture is placed in vivo for expression of a recombinant polypeptide, e.g., placed in a model organism or a human subject. In some embodiments, the cell culture utilizes solid microcarriers (e.g., growth on the surface of a solid microcarrier), porous microcarriers (e.g., growth on and / or within a microcarrier), or a support substrate (e.g., growth on and / or within a matrix). In some embodiments, the cell culture is a perfusion culture. In some embodiments, the cell culture is shaken. In some embodiments, the cell culture is a microfluidic culture.

[0130] In one embodiment, the culture medium is serum-free. Serum-free, protein-free, and chemically defined, animal component-free (CDACF) media are commercially available, for example, from Lonza Bioscience.

[0131] In some embodiments, lipid additives (including, for example, cholesterol, oleic acid, linoleic acid, or combinations thereof) can be added to the culture medium.

[0132] Suitable culture media and culture methods for mammalian cell lines are well known in the art, for example, as described in US Pat. No. 5,633,162. Examples of standard cell culture media for 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, 1 p. 173 ff, 1963), Dulbecco's Modified Eagle's Medium (DMEM), Eagle's Minimum Essential Medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc. 53, p288 ff. 1965) or Iscoves' 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 was specially designed for CHO cell culture. Other media specially adapted for CHO cell culture are described in EP 481 791. Such culture media may be supplemented with fetal bovine serum (FBS, also called fetal serum FCS), the latter being known to provide a natural source of numerous hormones and growth factors. Cell culture of mammalian cells is now a routine operation well documented in scientific textbooks and manuals, see for example R. Ian Fresney, Culture of Animal cells, a manual, 4 thedition, Wiley-Liss / NY, 2000. Any of the cell culture media described herein can be formulated to lack a particular amino acid, e.g., an amino acid whose biosynthesis can be rescued when cells take up a subject nucleic acid, such as tyrosine.

[0133] Other suitable culture methods are known to those of skill in the art and may depend on the recombinant polypeptide product and the host cell utilized. It is within the skill of the artisan to determine or optimize conditions suitable for expression and production of a recombinant or therapeutic polypeptide expressed by a cell.

[0134] In one aspect, the disclosure is directed to a method of making or producing a polypeptide product, the method comprising harvesting the polypeptide product. In some embodiments, harvesting comprises separating the polypeptide product from the producing cells and / or culture medium, e.g., by methods described herein or known in the art.

[0135] The culturing may include different culturing steps. Thus, in some embodiments, the culturing step includes culturing the production cells in a first culture medium and then in a second culture medium (i.e., using different media that may have, for example, different levels of tyrosine and / or phenylalanine).

[0136] The production cells may be cultured in any suitable vessel at various scales. For industrial production, bioreactors may be used, e.g., bioreactors having a volume of at least 10 liters, e.g., at least 50 liters, 50 to 800 liters, or 800 to 200,000 liters. The bioreactor may be a single-use bioreactor. In an embodiment, the bioreactor comprises a bioprocess vessel, a shell, at least one agitator, at least one sparger, at least one gas filter inlet port for the sparger and headspace overlay, at least one fill port, at least one harvest port, at least one sample port, and at least one probe. The bioreactor may also comprise processes and probes for monitoring and maintaining one or more parameters, e.g., pH, dissolved oxygen pressure (DOT), phenylalanine levels, and / or temperature. The bioreactor may be operably coupled to a harvest vessel. Further details and embodiments are provided in the "Applications" section below.

[0137] Once biosynthesis of the product by the production cells has progressed to a sufficient point, the product may be harvested, e.g., the culture medium is removed and the supernatant is separated from the cells and cell debris. The product may be subjected to one or more purification / processing steps to obtain a purified product, such as affinity chromatography, ion exchange chromatography, filtration, and / or viral inactivation. The product may also be combined with one or more pharma- ceutically acceptable carriers, excipients, or diluents to produce compositions, such as formulated pharmaceutical compositions that include, for example, one or more of buffers, surfactants, stabilizers (such as trehalose, sucrose, glycerol), amino acids (such as glycine, histidine, arginine), metal ions / chelators, salts, and / or preservatives.

[0138] Recombinant products Provided herein are compositions and methods for identifying, selecting, or culturing production cells or cell lines capable of producing high yield products, e.g., polypeptides, e.g., therapeutic polypeptides, as well as methods for producing the products. Products encompassed by the present disclosure include, but are not limited to, molecules, nucleic acids (e.g., non-coding nucleic acids, e.g., non-coding RNA molecules, e.g., antisense RNA, siRNA, tRNA, ribosomal RNA, microRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, or long non-coding RNA, e.g., Xist or HOTAIR), polypeptides (e.g., recombinant polypeptides and / or therapeutic polypeptides), or hybrids thereof, that can be produced, e.g., by expression in cells. In some embodiments, cells are engineered or modified to produce products. Such modifications include introducing molecules that control or result in the production of the product. For example, cells are modified by introducing an exogenous nucleic acid that encodes a polypeptide, e.g., a recombinant polypeptide, and the cells are cultured under conditions suitable for the production, e.g., expression and secretion, of the polypeptide, e.g., a recombinant polypeptide. In another example, a cell is modified by introducing an exogenous nucleic acid that controls, e.g., increases, expression of a polypeptide endogenously expressed by the cell, such that the cell produces a level or amount of the polypeptide that is higher than the level or amount endogenously produced in an unmodified cell. In embodiments, the cells or cell lines identified, selected, or generated by the methods described herein produce a product, e.g., a recombinant polypeptide, that is useful for the treatment of a medical condition, disorder, or disease.

[0139] Polypeptides In some embodiments, the product of interest comprises one or more polypeptides, e.g., recombinant polypeptides, which are typically heterologous polypeptides, i.e., products that are not naturally expressed by cells. The product may be, for example, a therapeutic or diagnostic protein useful for drug screening. The therapeutic or diagnostic protein may be an antibody molecule, e.g., an antibody or antibody fragment, a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, an aptamer, or a structural and / or functional fragment or hybrid of any of these. In one embodiment, the product comprises multiple polypeptide chains, e.g., an antibody or antibody fragment comprising a heavy chain and a light chain.

[0140] In some embodiments, the product is an antibody molecule. The products encompassed herein are diagnostic antibody molecules, e.g., monoclonal antibodies or antibody fragments thereof, useful for imaging techniques, and therapeutic antibody molecules, e.g., useful for treating a disease or disorder, suitable for administration to a subject. An antibody molecule is a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. In embodiments, the antibody molecule is a full-length antibody or an antibody fragment. The antibodies and multiformat proteins may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. The antibody may be a multimer of immunoglobulin molecules, e.g., a tetramer of immunoglobulin molecules. In embodiments, the antibody is a monoclonal antibody. The antibody may be a human or humanized antibody. In one embodiment, the antibody is an IgA, IgG, IgD, IgM, or IgE antibody. In one embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody molecule is or comprises a multispecific antibody, e.g., a bispecific, trispecific, or tetraspecific antibody, e.g., a BiTE.

[0141] "Antibody fragment" refers to at least a portion of an intact antibody, or a recombinant variant thereof, including an antigen-binding domain, e.g., an antigen-determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include Fab, Fab', F(ab') 2 , and multispecific antibodies formed from antibody fragments such as Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and bivalent fragments including two Fab fragments linked by a disulfide bridge at the hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0142] Examples of polypeptides of interest include, but are not limited to, those listed below: Hormones: Erythropoietin, epoein-α, darbepoietin-α, growth hormone (GH), somatotropin, human follicle-stimulating hormone (FSH), human chorionic gonadotropin, lutropin-α, glucagon, growth hormone-releasing hormone (GHRH), insulin. Clotting / Coagulation Factors: Factor VIIa, Factor VIII, Factor IX, Antithrombin III (AT-III), Protein C Concentrate Cytokines / Growth Factors: Type I alpha-interferon, interferon-αn3 (IFNαn3), interferon-β1a (rIFN-β), interferon-β1b (rIFN-β), interferon-γ1b (IFNγ), aldesleukin (interleukin 2 (IL2), epidermal thymocyte-activating factor; ETAF), palifermin (keratinocyte growth factor; KGF), becaplumin (platelet-derived growth factor; PDGF), anakinra (recombinant IL1 antagonist).

[0143] Antibodies: bevacizumab (VEGFA mAb), cetuximab (EGFR mAb), panitumumab (EGFR MAb), alemtuzumab (CD52 mAb), rituximab (CD20 chimeric Ab), trastuzumab, adalimumab, infliximab, tositumomab, acritumomab, ranibizumab, abciximab, omalizumab, palivizumab, natalizumab, daclizumab, basiliximab, eculizumab.

[0144] Vaccine antigens: Hepatitis B surface antigen (HBsAg), HPV antigen, HIV antigen, influenza antigen.

[0145] Other: Albumin, Anti-Rhesus (Rh) Immunoglobulin G, Enfuvirtide, Spider Silk Proteins, e.g., Fibrion, Botulinum Toxin Type A, Alglucerase, Imiglucerase, Recombinant Human Hyaluronidase, Palifermin, Anakinra, Dornase Alfa, Synthetic Porcine Secretin.

[0146] The recombinant polypeptide of interest may be a multispecific protein, for example a bispecific antibody, many formats of which are available, such as BsIgG (Triomab), BiTE, DART, TandB, etc.

[0147] In some embodiments, the polypeptide (e.g., produced by a cell and / or by the methods described herein) is an antigen expressed by a cancer cell. In some embodiments, the recombinant or therapeutic polypeptide is a tumor-associated or tumor-specific antigen. In some embodiments, the recombinant or therapeutic polypeptide is selected from the group consisting of HER2, CD20, 9-O-acetyl-GD3, βhCG, A33 antigen, CA19-9 marker, CA-125 marker, calreticulin, carboanhydrase IX (MN / CA IX), CCR5, CCR8, CD19, CD22, CD25, CD27, CD30, CD33, CD38, CD44v6, CD63, CD70, CC123, CD138, carcinoembryonic antigen (CEA; CD66e), desmoglein 4, E-cadherin neoepitope, 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, gangloside 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, MUCl, MUC2, MUC3, MUC4, MUC5 AC , MUC5 B , MUC7, MUC16, Mullerian inhibitory substance (MIS) receptor type II, plasma cell antigen, polySA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-alpha precursor, and combinations thereof.

[0148] In some embodiments, the polypeptide (e.g., produced by a cell and / or by the methods described herein) is an activating receptor, such as 2B4 (CD244), α 4 β 1 Integrin, β 2and optionally selected from integrins, CD2, CD16, CD27, CD38, CD96, CD100, CD160, CD137, CEACAMl (CD66), CRTAM, CSl (CD319), DNAM-1 (CD226), GITR (TNFRSF18), activating forms of KIR, NKG2C, NKG2D, NKG2E, one or more natural cytotoxicity receptors, NTB-A, PEN-5, and combinations thereof. 2 The integrin comprises CD11a-CD18, CD11b-CD18, or CD11c-CD18; optionally, the activating form of KIR comprises KlR2DSl, KIR2DS4, or KIR-S; and optionally, the natural cytotoxicity receptor comprises NKp30, NKp44, NKp46, or NKp80.

[0149] In some embodiments, the polypeptide (e.g., produced by a cell and / or by a method described herein) is an inhibitory receptor and is selected from KIR, ILT2 / LIR-1 / CD85j, an inhibitory form of KIR, KLRG1, LAIR-1, NKG2A, NKR-P1A, Siglec-3, Siglec-7, Siglec-9, and combinations thereof, optionally wherein the inhibitory form of KIR includes KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, or KIR-L.

[0150] In some embodiments, the polypeptide (e.g., produced by a cell and / or by the methods described herein) is an activating receptor and is selected from the group consisting of CD3, CD2 (LFA2, OX34), CD5, CD27 (TNFRSF7), CD28, CD30 (TNFRSF8), CD40L, CD84 (SLAMF5), CD137 (4-1BB), CD226, CD229 (Ly9, SLAMF3), CD244 (2B4, SLAMF4), CD319 ( CRACC, BLAME), CD352 (Lyl08, NTBA, SLAMF6), CRTAM (CD355), DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LTβR (TNFRSF3), OX40 (CD134), NKG2D, SLAM (CD150, SLAMF1), TCRα, TCRβ, TCRδγ, TIM1 (HAVCR, KIM1), and combinations thereof.

[0151] In some embodiments, the polypeptide (e.g., produced by a cell and / or by a method described herein) is an inhibitory receptor and is selected from PD-1 (CD279), 2B4 (CD244, SLAMF4), B71 (CD80), B7Hl (CD274, PD-L1), BTLA (CD272), CD160 (BY55, NK28), CD352 (Ly108, NTBA, SLAMF6), CD358 (DR6), CTLA-4 (CD152), LAG3, LAIR1, PD-1H (VISTA), TIGIT (VSIG9, VSTM3), TIM2 (TIMD2), TIM3 (HAVCR2, KIM3), and combinations thereof.

[0152] Other recombinant protein products (e.g., produced by cells and / or by the methods described herein) include non-antibody or alternative protein scaffolds, such as, but not limited to, DARPins, affibodies, and adnectins. Such non-antibody or alternative protein scaffolds can be engineered to recognize or bind one or more, e.g., 1, 2, 3, 4, or 5 or more, different targets or antigens.

[0153] Purpose The present disclosure features, among other things, production cells, methods of using production cells to make or manufacture polypeptide products, methods of identifying, selecting, and / or culturing cells (e.g., production cells), and methods of making or producing production cells. The methods of identifying, selecting, and / or culturing cells disclosed herein can be used to generate cells, e.g., production cells, evaluate various cell lines, or evaluate the production of various cell lines for use in bioreactors or processing vessels or tanks, or more generally with any source, that are useful for producing various products. The compositions and methods described herein are suitable for culturing any desired cell line, including, for example, prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the compositions and methods described herein are suitable for culturing suspension or anchorage-dependent (adherent) cells, and are suitable for production operations configured for the production of pharmaceuticals and biopharmaceuticals, such as polypeptide products, nucleic acid products (e.g., DNA or RNA), exosomes, vesicles, or cells and / or viruses (such as those used in cell and / or viral therapy or as vaccines).

[0154] In embodiments, the cells, e.g., production cells, express or produce a product, such as a therapeutic or diagnostic recombinant product. As described in more detail below, examples of products produced by the cells include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to an antigen but are not structurally related to antibodies, e.g., DARPins, affibodies, adnectins, or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cellular therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA, etc.) or DNA (e.g., plasmid DNA, etc.), antibiotics, or amino acids. In embodiments, the compositions and methods described herein can be used to produce biosimilars.

[0155] As mentioned, in embodiments, the compositions and methods described herein allow for the production of eukaryotic cells, e.g., mammalian cells or lower eukaryotic cells, e.g., yeast cells or filamentous fungal cells, or prokaryotic cells, e.g., gram-positive or gram-negative cells, and / or eukaryotic or prokaryotic products, e.g., proteins, peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA), which are synthesized in a large-scale manner by eukaryotic cells. Unless otherwise stated herein, the compositions and methods described herein can include any desired volume or production capacity, including, but not limited to, bench scale, pilot scale, and full production scale capacity.

[0156] Additionally, and unless otherwise stated herein, the compositions and methods described herein can be used with any suitable reactor, including, but not limited to, stirred tank, air-lift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors, with or without solid or porous microcarriers or supports. As used herein, a "reactor" can include a fermenter or fermentation unit, or any other reaction vessel, and the term "reactor" is used synonymously with "fermenter." For example, in some embodiments, a bioreactor unit may be configured to provide the following: supply of nutrients and / or carbon sources, injection of a suitable gas (e.g., oxygen), inflow and outflow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, oxygen and CO2. 2The bioreactor may perform one or more or all of the following: maintaining pH levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, may include multiple reactors within the unit, e.g., a 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 within each unit, and / or a facility may include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-fed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactor may have a volume of about 100 ml to about 50,000 L. Non-limiting examples include volumes of 10 ml, 50 ml, 100 ml, 250 ml, 500 ml, 750 ml, 1 liter, 2 liter, 10 liter, 50 liter, 100 liter, 500 liter, 1000 liter, 2000 liter, 5000 liter, 10,000 liter, 15,000 liter, 20,000 liter, and / or 50,000 liter, or approximately those volumes. In the context of industrial-scale manufacturing required to make enough product for clinical or commercial use, the volume is typically at least 10 liters. In some embodiments, the bioreactor is configured to grow microfluidic cultures. Furthermore, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed of any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastic, and / or glass. In some embodiments, suitable reactors can be circular, e.g., cylindrical. In some embodiments, a suitable reactor may be square, for example rectangular.Square reactors may in some cases offer advantages over circular reactors, such as ease of use (e.g., packing and setting up by one of ordinary skill in the art), better mixing and homogeneity of the reactor contents, and lower bed footprint.

[0157] In embodiments, and unless otherwise stated herein, the compositions and methods described herein can be used with any suitable unit operations and / or equipment not specifically mentioned, such as operations and / or equipment for separation, purification, and isolation of such products. Any suitable equipment and environment can be used, such as conventional stick-built equipment, modular, mobile, and temporary equipment, or any other suitable construction, equipment, and / or layout. For example, in some embodiments, modular clean rooms can be used. In addition, and unless otherwise stated herein, the compositions and methods described herein can be housed and / or performed in a single location or facility, or alternatively housed and / or performed in separate or multiple locations and / or facilities.

[0158] By way of non-limiting example, and without 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 incorporated by reference in their entireties herein, describe exemplary equipment, devices, and / or systems that may be suitable for use with the compositions and methods described herein.

[0159] The compositions and methods described herein can utilize a wide range of cells as described in the above section on host cells. In a preferred embodiment, the mammalian cell is a CHO cell line. Examples include CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN, and CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHOK1SV® GS knockout cells. CHO FUT8 knockout cells are, for example, Potelligent® CHOK1SV® (Lonza Biologics, Inc.).

[0160] In one embodiment, the eukaryotic cell is, for example, a yeast cell (e.g., a species of the genus Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), the genus Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), the genus Saccharomyces (e.g., Saccharomyces cerevisae, Saccharomyces kluyveri, Saccharomyces uvarum), the genus Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), the genus Candida (e.g., Candida utilis, Candida cacaoi, Candida boidinii), the genus Geotrichum (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces and lower eukaryotic cells such as P. pombe. The species Pichia pastoris is preferred. Examples of Pichia pastoris strains are X33, GS115, KM71, KM71H, and CBS7435.

[0161] In embodiments, the cultured cells are used to produce proteins, e.g., antibodies, e.g., monoclonal antibodies, and / or recombinant proteins, for therapeutic use. In embodiments, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in embodiments, molecules having molecular weights of about 4000 Daltons to greater than about 140,000 Daltons can be produced. In embodiments, these molecules can have a range of complexity and can include post-translational modifications, including glycosylation.

[0162] The invention is further illustrated by reference to the following non-limiting examples. EXAMPLES

[0163] Example 1: Materials and Methods cell culture Suspension Lonza CHOK1SV® GS-KO® cells were maintained in CD-CHO medium (Gibco 10743-029) supplemented with 6 mM L-glutamine (Sigma G8540). They were incubated at 37° C., 140 rpm, and 5% CO 2 The cells were incubated at 0.2 × 10 in 20 ml in a 125 ml Erlenmeyer flask. 6 viable cells / ml were seeded and these were passaged every 3-4 days.

[0164] Reversion assay Lonza CHOK1SV® GS-KO® cells were seeded at 5000 viable cells per well in 200 μl medium in 96-well plates and analyzed for growth after 11 days and 3 weeks. CD CHO without tyrosine and Lonza CM76 (Lonza Biologics plc) without tyrosine but supplemented with 6 mM L-glutamine were used as test media. 7.2×10 6 Viable cells were tested in CM76 tyrosine-free medium at 2.4 × 10 6Cells were tested in CD CHO tyrosine-free medium. Complete medium (CD-CHO+L-glut) was used as a positive control, while medium without L-glutamine (CD-CHO only) was used as a negative control.

[0165] Plasmids and transfection to generate stable cell lines [Table 1]

[0166] The truncated PAH sequence has a deletion of 116 amino acids at the N-terminus that contains the regulatory domain (Daubner SC et al., 1997, ibid.). The various domains of PAH are shown in FIG.

[0167] Plasmids were linearized with PvuI (NEB, R3150L) and purified using an ethanol precipitation protocol. Electroporation was performed in a Biorad Genepulser Xcell electroporator. 100 μl of TE buffer and 1 × 10 7 Live Lonza CHOK1SV GS-KO cells / 20 μg of linearized plasmid in 700 μl of CM76 tyrosine-free (+6 mM L-glut) medium were added to an electroporation cuvette. The DNA cell mix was electroporated at 300 V and 900 μF with a cuvette diameter of 0.4 mm. Immediately after electroporation, 1 ml of pre-warmed medium was added to the cuvette. The cells were then transferred to 2×5 ml of CM76 tyrosine-free (+6 mM L-glut) medium in a T25 flask. The flasks were incubated at 4° C. for 2 h at 37 °C for 1 h at 5% CO. 2Incubated at 37°C in a static incubator with gaseous environment. After 24 hours, an additional 5ml of CM76 tyrosine-free (+6mM L-glut) medium was added to the T25 flask. Cell counts were performed using a ViCell instrument 21 days after transfection to assess transfection success. Further confirmation of successful transfection was performed by visualizing cells growing in the T25 flask under a microscope (Leica MZFLIII with GFP2 filter, ×100 magnification) for eGFP expression.

[0168] Growth curve profiles and culture viability Cells were cultured at 0.2 × 10 in 20 ml in a 125 ml Erlenmeyer flask. 6 200 cells / ml and incubated in 5% CO 2 Ambient shaking at 140 rpm, 37° C. Viable cell concentration and cell diameter were determined using 0.2 ml samples and 0.8 ml pre-warmed PBS. Readings were recorded every 48 hours using a ViCell (Beckman Coulter) instrument for the number of days indicated in the example figures.

[0169] FACS 1×10 5 The cells were pelleted in a centrifuge at 1,000 rpm for 5 minutes and resuspended in 350 μl of PBS. Samples were then loaded onto the probe of a FACScalibur™ (BD biosciences) to measure fluorescence intensity in relation to cell number. Forward scatter (FSC) was measured using an E-1 amplifier, side scatter (SSC) was set to 465, while FL1 recorded cells at 473, and all settings were converted to logarithmic scale.

[0170] SDS-PAGE, Western blot 1×10 6The cells were pelleted in a centrifuge at 1000 rpm for 5 min and resuspended in 20 mM HEPES-NaOH, pH 7.2, 100 mM NaCl, 10 mM Na β-glycerophosphate, 50 mM NaF, 0.5% Nonidet-P40, 1 mM activated Na 3 VO 4 The cells were lysed in 100 μl of ice-cold lysis buffer consisting of 10 μg / ml leupeptin, 2 μg / ml pepstatin, and 0.2 mM PMSF added immediately before use.

[0171] 10 μg of reduced protein samples or 10 μl of non-reduced supernatant samples were run on 10% SDS-PAGE acrylamide gels and Western transfer onto nitrocellulose was performed as previously described (Roobol, Carden et al., 2009, FEBS J. 276:286-302). Antibodies were supplied by Sigma (anti-GCH1, SAB1405858-50 μg, anti-PAH, HPA031642, anti-GS G2781, anti-B-actin A5441, anti-human IgG (γ-chain specific) I9764), and CRUK (eGFP 3E1). Anti-tubulin (Woods, Sherwin et al., 1989, J. Cell Sci. 93:491-500) was a contribution from Professor Keith Gull, University of Oxford, UK, whereas anti-L7a was generated against the N-terminal sequence of human L7a (Roobol and Carden, 1999, Eur. J. Cell Biol. 78(1):21-32). Secondary antibodies for immunoblot detection of cell lysate proteins were anti-total IgG (mouse or rabbit)-HRP conjugates (Sigma), followed by ECL (GE Healthcare) detection.

[0172] qRTPCR 1 x 10 for RNA extraction 6Viable cells were harvested and mRNA abundance was determined by qRTPCR on an Eppendorf RealPlex cycler instrument using a Qiagen Quantifast kit with the following primer sets; PAH (qrtPAHtotfwd CATCAAGGCATATGGTGCTG (SEQ ID NO: 7) and qrtPAHtotrvs GGGCTGGAACTCTGTGACAT ((SEQ ID NO: 8)), GCH1 (GCH1 forward: CTTCACCAAGGGCTACCAGG (SEQ ID NO: 9); GCH1 reverse: AGGCCAAGGACTTGCTTGTT (SEQ ID NO: 10)), and β-actin (CHObactqF agctgagagggaaattgtgcg (SEQ ID NO: 11) and CHObactqR GCAACGGAACCGCTC ATT (SEQ ID NO: 12).

[0173] Example 2: Reversion assay of GSKO cells grown in either CM76 or CD CHO medium without tyrosine but supplemented with 6 mM L-glutamine This example demonstrates the low reversion rates observed when growing an exemplary cell that is unable to grow in the absence of tyrosine.

[0174] CHOK1SV GS-KO® host cells were seeded in 96-well plates in medium lacking tyrosine but supplemented with 6 mM glutamine. The positive control was CHOK1SV GS-KO® host cells growing in medium supplemented with 6 mM glutamine, and the negative control was medium lacking glutamine. The results are shown in Table 2 below. In medium lacking tyrosine, no revertant colonies / cell growth were observed and the plates looked similar to the negative control. This suggests that the tyrosine auxotrophy marker is a useful selection marker in production cells. [Table 2]

[0175] Example 3: Growth of exemplary production cells in the absence of tyrosine This example shows that cells lacking exogenous nucleic acids encoding PAH and GCH1 enzyme molecules do not grow in the absence of tyrosine, whereas exemplary production cells containing vectors LMM172 or LMM173, which contain exogenous nucleic acids encoding both PAH and GCH1 enzyme molecules, are observed to grow in the absence of tyrosine and express the reporter molecule eGFP. However, when full-length CHO PAH was first tested along with GCH1, it was found that transfected cells recovered very slowly in tyrosine-free medium (CD CHO) or did not recover at all when CM76 medium was used (data not shown). Therefore, a truncated version of PAH was attempted in which the N-terminal 116 amino acids encoding the regulatory domain were removed.

[0176] The vectors used to generate these pools contained truncated forms of PAH (cassette 1, tPAH with the first 116 amino acids deleted, derived from either CHO cells or human, with sequences driven by the SV40 promoter), GCH1 (cassette 3, driven by the SV40 promoter), and eGFP (cassette 2, driven by the CMV promoter). Two controls were included in which the first cassette contained the glutamine synthetase (GS) gene driven by the SV40 promoter (vector LMM170). Transfected controls were grown either in the absence (negative control) or presence of tyrosine (positive control).

[0177] CHOK1SV GS-KO® host cells were transfected via electroporation with the linearized vector and then cultured for 3 weeks in medium lacking tyrosine but supplemented with 6 mM glutamine (except for the positive control, which also contained tyrosine).

[0178] It was shown that CHOK1SV GS-KO® host engineered cells grow well in tyrosine-free medium only when truncated PAH and GCH1 were co-expressed. In addition, when these components were transfected individually, the cells did not survive transfection and did not grow in tyrosine-free medium (data not shown). Thus, both PAH enzyme molecules, including truncated PAH and GCH1 enzyme molecules, are required to support the growth of exemplary CHO production cells in the absence of tyrosine.

[0179] Figure 2 shows a histogram of mean fluorescence from cell populations after transfection and 3 weeks of recovery obtained using flow cytometry, confirming the expression of eGFP in cells growing in tyrosine-free medium. Mean fluorescence from exemplary producer cells containing truncated CHO cell-derived PAH sequence and GCH1 (vector LMM172) was similar to that from the GS positive control (vector LMM170+ve). Producer cells containing the CHO truncated PAH sequence (LMM172) showed higher GFP expression compared to the human truncated PAH sequence (LMM173). This is a model where recombinant proteins can replace eGFP when the combined PAH and GCH1 system is used as a selection marker.

[0180] Example 4: PAH protein and mRNA abundance This example shows that an exemplary production cell containing vector LMM173, which includes exogenous nucleic acid encoding human PAH and GCH1 enzyme molecules, exhibits PAH protein and mRNA expression as well as eGFP protein expression, and this example further shows that an exemplary production cell containing vector LMM172, which includes exogenous nucleic acid encoding CHO PAH and GCH1 enzyme molecules, exhibits PAH mRNA expression and eGFP protein expression.

[0181] Western blot analysis of lysates from cell pools in Figure 2 was performed. Controls were grown in medium containing 6 mM glutamine and tyrosine, and LMM170 cells were grown in medium containing tyrosine but not glutamine. LMM172 and LMM173 were grown in tyrosine-free medium supplemented with 6 mM glutamine. Tubulin and L7a were used as loading controls. The PAH antibody detected only human cleaved PAH (bands at approximately 37 and 50 kDa), but not CHO cleaved PAH (LMM172) (data not shown). eGFP was confirmed to be expressed in cell pools in which the transfected vector contained the eGFP gene in cassette 2.

[0182] Figure 3 shows qRT-PCR data detecting expression of truncated CHO PAH and truncated human PAH mRNA. The truncated CHO PAH mRNA was expressed to much greater amounts than the truncated human PAH. Both were increased above the control, confirming exogenous PAH mRNA expression in the exemplary production cells.

[0183] Example 5: Growth profiles and culture viability in the absence of tyrosine This example shows that exemplary production cells containing vector LMM172, which contains exogenous nucleic acid encoding CHO PAH and GCH1 enzyme molecules, are capable of growing to higher viable cell concentrations in the absence of tyrosine than similar cells without exogenous nucleic acid, and have extended culture survival times.

[0184] FIG. 4 shows the growth data of an exemplary production cell pool generated as described in Examples 3 and 4. The cell pool was cultured in 125 ml Erlenmeyer flasks for 18 days in the absence of tyrosine or glutamine. Cells were sampled every 2 days and viable cell counts and culture viability were assessed using a ViCell instrument. No additional feed was introduced. FIG. 4 shows (A) viable cell concentration, and (B) culture viability. The CHO cell truncated PAH cell pool (LMM172) grew to a higher cell number and had a longer culture viability than the human truncated PAH cell pool (LMM173).

[0185] Example 6: Exemplary production cell growth profiles and viable cell concentrations when grown in the absence of tyrosine but supplemented with additional phenylalanine This example illustrates the growth and culture viability characteristics of exemplary production cells containing exogenous nucleic acids encoding PAH and GCH1 enzyme molecules.

[0186] FIG. 5 shows the growth data of an exemplary producer cell pool. Cultures were grown for 18 days in 125 ml Erlenmeyer flasks and supplemented with phenylalanine (Sigma P5482) where indicated. Cells were sampled every 2 days and no further feed was introduced. The cells were analyzed for cell growth and culture viability. The exemplary producer cells expressing the truncated human PAH grew and reached higher viable cell concentrations in a shorter time than the exemplary producer cells expressing the truncated CHO PAH when supplemented with 6 mM phenylalanine. This experiment also showed that the cell line was truly prototrophic as the GSKO control died.

[0187] Example 7: Exemplary production cell growth profile and viable cell concentration when grown in commercial CD-CHO medium in the absence of tyrosine but supplemented with additional phenylalanine This example evaluates cell growth and culture viability. Figure 6 shows growth data for an exemplary production cell transfected and grown in commercial CD-CHO (ThermoFisher Scientific) medium lacking tyrosine but supplemented with 6 mM glutamine. Transfected CHOK1SV GS-KO™ host cells recovered faster after transfection in CD CHO medium compared to CM76 medium. The recovery rate was reduced from 21 days to 18 days when the cells were ready to be transferred to shake flasks after transfection. In addition, cells transfected with a plasmid DNA construct containing a truncated human PAH recovered in a similar time and to a similar viable cell number after transfection as observed when using a vector containing a truncated CHO PAH cell. This indicated that CD CHO is a better transfection medium for this system.

[0188] Cells assessed for growth in CD CHO medium were sampled every 2 days and no further feed was introduced. Cultures were analyzed for cell growth and culture viability. When tyrosine prototrophic cell pools were grown in CD CHO medium (Figure 6), the truncated human PAH cell pool benefited most from the additional 6 mM phenylalanine.

[0189] Example 8: Pre-adapting cells to phenylalanine supplementation reduces the growth lag phase. This example shows that pre-adapting an exemplary production cell pool to additional supplemented phenylalanine before performing batch culture improves growth (Figure 7). Human truncated PAH expressing cells respond better to phenylalanine supplementation than CHO type. Human truncated PAH expressing cells (LMM173) were pre-adapted by passaging the cells with 6 mM phenylalanine before starting the growth curve. The cells were cultured in 125 ml Erlenmeyer flasks for 16 days. The cells were analyzed for cell growth measured by viable cell concentration and culture viability. Cell growth was enhanced by the addition of phenylalanine, but the growth lag phase was further reduced when the cells were pre-adapted to growth without CD CHO tyrosine supplemented with 6 mM L-glutamine and 6 mM phenylalanine (Sigma P5482). GS-KO host cells were unable to grow in CD CHO medium supplemented or not with 6 mM phenylalanine.

[0190] Example 9: Dual metabolic selection markers with recombinant protein production This example evaluates how truncated PAH / GCH1 combinatorial selection can be utilized when combined with cell lines producing recombinant proteins under glutamine synthetase selection. The strength of the promoter was varied to drive GCH1 expression to determine if this affected the subsequent cells that emerged in terms of growth profile. To achieve maximum growth (highest viable cell concentration), different plasmids with promoters and truncated PAH in combination with GCH1 were the best combination. The vectors used to generate these pools contained truncated versions of either CHO or human PAH (cassette 1, SV40 promoter), GCH1 (cassette 3 driven by either PGK, SV40, or mCMV promoter), and eGFP (cassette 2 driven by CMV promoter). See Table 1.

[0191] These were linearized and transfected into a cell line expressing the model monoclonal antibody cB72.3 under GS selection. Transfections were performed in CD-CHO without glutamine and without tyrosine in T25 static flasks. After transfection and selection, once the cells had recovered and grown, they were transferred to shake flasks in CM76 without glutamine and without tyrosine.

[0192] Figure 8 shows qRT-PCR data of truncated CHO PAH expression and truncated human PAH mRNA expression in the resulting cell pools, comparable to the findings in Example 4. Expression of GCH1 was also detected, with levels reflecting the strength of the promoter that was driving the cassette. The analysis confirms the mRNA overexpression of PAH in the truncated PAH cell pool. As previously observed, truncated CHO PAH was expressed at much higher levels than human PAH. GCH1 mRNA expression levels correlated with the promoter strength driving the gene.

[0193] Western blot analysis of lysates from the above cell pools was performed. All double-selected cell pools were grown in CM76 without glutamine and without tyrosine. CHOK1SV GS-KO™ control samples were taken from cells grown in complete medium (containing glutamine and tyrosine). Cleaved PAH, GCH1, and GS were detected in all double-selected marker expressing cell lines, except for CHO PAH, as the antibody did not detect it (data not shown), according to Example 4. Using heavy chain antibodies, we also confirmed that the recombinant protein (cB72.3) was secreted into the supernatant (data not shown). Tubulin, β-actin, and L7a served as loading controls.

[0194] Figure 9 shows the growth data of an exemplary production cell pool. The cell pool was cultured in 125 ml Erlenmeyer flasks for 18 days in the absence of tyrosine and glutamine and supplemented with additional phenylalanine as indicated. Cells were sampled every 2 days and the number of live cells and culture viability were assessed using a ViCell instrument. No additional feed was introduced. The greatest growth (highest live cell concentration achieved) was observed for LMM186 (SV40 human PAH, SV40 GCH1) when pre-adapted to additional 6 mM phenylalanine-supplemented medium.

[0195] Because two selection markers are utilized simultaneously, cells were cultured in CM76 without tyrosine or glutamine. The best growing cell pool was generated from LMM186 when supplemented with 6 mM phenylalanine (SV40 PAH human and SV40 GCH1).

[0196] These results show that two different amino acid-based selection systems can be combined without any adverse effect on the performance of the cell line. The resulting cells show superior growth characteristics. This provides greater flexibility for expression, since, for example, one selection system can be used to create and maintain an engineered stable cell line with a gene product that modifies the performance of the cell line, while another selection system can be used to introduce and maintain a sequence that codes for a product that is desired to be produced.

[0197] The results also support the findings of Example 7 that superior performance is achieved using human PAH with phenylalanine supplementation.

[0198] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to certain aspects, it is apparent that other aspects and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. Features and embodiments in different sections may be combined mutatis mutandis.

Claims

1. 1. A method for selecting a eukaryotic cell that contains a nucleic acid sequence encoding a product of interest, comprising the steps of: i) contacting a cell population that is unable to survive or grow in the absence of tyrosine with a vector system, said vector system comprising: a) a first nucleic acid sequence comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of said PAH in a host cell; (b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence enabling expression of GCH1 in a host cell; and (c) a third nucleic acid sequence comprising a sequence encoding a product of interest operably linked to a third control sequence enabling expression of said product in a host cell, said third sequence being present in the same vector as (a) and / or (b) under conditions enabling uptake of said vector system by said cell; ii) culturing said cells under conditions in which the level of said tyrosine is lower than the level required for survival or growth of cells that do not express the PAH and GCH1 enzymes encoded by said vector system; iii) selecting one or more cells capable of growing under such conditions to obtain one or more cells containing said nucleic acid sequence encoding said product.

2. 2. The method of claim 1, wherein (a), (b), and (c) are present in the same vector.

3. 3. The method of claim 1 or 2, wherein (c) comprises two vectors, the two vectors being present in the same vector as (a) or (b).

4. The method according to any one of claims 1 to 3, wherein the eukaryotic cell is a mammalian cell.

5. The method of claim 4, wherein the mammalian cell is a CHO cell.

6. The method of any one of claims 1 to 5, wherein the PAH has a deletion of the N-terminal regulatory domain.

7. 7. The method of any one of claims 1 to 6, wherein the cell culture medium lacks tyrosine and is optionally supplemented with phenylalanine.

8. 1. A eukaryotic host cell comprising: a) a first exogenous nucleic acid encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of said PAH in a host cell; b) a second exogenous nucleic acid encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence enabling expression of GCH1 in the host cell; and c) a third exogenous nucleic acid encoding a product of interest operably linked to a third control sequence enabling expression of said product in said host cell, wherein said third exogenous nucleic acid is present in the same exogenous nucleic acid sequence as said first and / or second exogenous nucleic acid.

9. The host cell of claim 8, wherein the PAH has a deletion of the N-terminal regulatory domain.

10. The host cell according to claim 8 or 9, wherein the PAH is a PAH derived from a Chinese hamster or a human.

11. The host cell according to any one of claims 8 to 10, which is a mammalian cell.

12. The host cell described in claim 11, wherein the mammalian cell is a CHO cell.

13. The host cell according to any one of claims 8 to 12, wherein the first, second and third nucleic acid molecules are integrated into the genome of the host cell.

14. 13. The host cell of any one of claims 8 to 12, wherein the activity of endogenous genes encoding PAH and / or GCH1 of said cell is reduced or eliminated.

15. A vector system comprising one or more nucleic acid vectors, a) a first nucleic acid sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of said PAH in a host cell; b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence allowing expression of said GCH1 in a host cell; and c) a multiple cloning site for inserting a sequence encoding a product of interest operably linked to a third control sequence allowing expression of said product in a host cell, wherein said multiple cloning site and the third control sequence are present in the same vector as (a) and / or (b).

16. A vector system comprising one or more nucleic acid vectors, a) a first nucleic acid sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of said PAH in a host cell; b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence allowing expression of said GCH1 in a host cell; and c) a third nucleic acid sequence comprising a sequence encoding a product of interest operably linked to a third control sequence enabling expression of said product in a host cell, said third nucleic acid sequence being present in the same vector as (a) and / or (b).

17. 13. A method of making a product, said method comprising culturing a host cell according to any one of claims 8 to 12 under conditions suitable for expressing said product, recovering said product, and optionally subjecting said recovered product to one or more processing or purification steps.

18. The method of claim 17, wherein the cells are cultured under conditions in which the level of tyrosine is lower than the level required for survival or proliferation of cells that do not express the PAH and GCH1 enzymes encoded by the vector system of claim 15 or 16.

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