Efficient selectivity of recombinant proteins

Abstract

To provide expression of recombinant proteins in mammalian cells in a consistent and efficient manner.SOLUTION: The invention includes methods and compositions for improved expression of proteins in mammalian cells by employing mammalian selection markers. The invention includes methods of enhancing selectivity and enhanced expression copies as well as the protein yield of recombinant proteins in mammalian cells, and methods of using such expression systems. The present invention provides an improved method for production of recombinant proteins in mammalian cell systems utilizing a mammalian Tn-resistance gene, GPT, as a regulatable selection marker. The present invention also provides an improved method for glycosylation of recombinant proteins, i.e., a method for making glycoproteins, in mammalian cell systems in order to provide a consistent quality yield of the desired proteins.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Incorporation by Reference) This application incorporates by reference a Sequence Listing submitted in computer-readable form as a file entitled "8700WO_ST25.txt" (75,769 bytes), created on August 3, 2015.

[0002] The present invention provides for the expression of recombinant proteins in mammalian cells in a consistent and efficient manner. In particular, the present invention includes methods and compositions for improved expression of proteins in mammalian cells by employing mammalian selection markers. The present invention includes methods for enhancing the selectivity and amplified expression copies of recombinant proteins in mammalian cells, as well as methods of using such expression systems.

Background Art

[0003] The development of cell expression systems is an important goal for providing a reliable and efficient source of a given protein for research and therapeutic uses. Recombinant protein expression in mammalian cells is often preferred for producing therapeutic proteins, for example, because mammalian expression systems can appropriately modify recombinant proteins post-translation.

[0004] A variety of vectors are available for expression in mammalian hosts, each containing a selection marker that allows for the easy isolation of cells that express the recombinant protein during cell culture. Selection marker genes (SMGs) are utilized in such systems because they confer a selective advantage to cells that express the protein of interest, but SMGs need to be optimized for, among other reasons, their phenotypic neutrality, efficiency, and versatility.

[0005] Despite the availability of numerous vectors and expression systems that can serve as hosts for SMGs, recombinant protein expression achieved within mammalian systems is often unsatisfactory in terms of quantity, quality, or both. The biological "fingerprint" of a molecule, such as post-translational modifications like glycosylation, is particularly important in defining the utility and efficacy of molecules in the development of recombinant protein therapies (Cumming, DA, 1990, Glycobiology, 1(2):115-130). SMGs that do not adversely affect the biological properties of the expressed target protein are particularly useful. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Cumming, D. A., 1990, Glycobiology, 1(2):115-130. [Overview of the project] [Problems that the invention aims to solve]

[0007] Most SMGs are of bacterial origin, and their use in mammalian systems presents other disadvantages due to increased concerns about the risk of horizontal transfer of bacterial antibiotic resistance genes to environmental bacteria (Breyer, D. et al., 2014, Critical Reviews in Plant Sciences 33:286-330). Eliminating the use of bacterial antibiotic resistance genes may have a positive impact on consumer acceptance and reduce such perceived risks.

[0008] Genetically modified autologous cells are rapidly achieving clinical success (e.g., Kershaw, MH et al., 2013, Nature Reviews: Cancer). (See 13:525-541). In particular, the undesirable introduction of non-human components into human autologous cells can have serious consequences for patient safety, so the selection and design of vectors for gene modification in human autologous cell products is important. (Eaker, et al., 2013, Stem Cells Trans. Med. 2:871-883; first published online in SCTM EXPRESS October 7, 2013). Vector systems containing only mammalian-derived components, rather than bacterial ones, would be useful for patient-specific T cell use in adoptive immunotherapy.

[0009] Therefore, for the production of target mammalian proteins, it is desirable to introduce mammalian-selective genes, particularly those that provide phenotypic or metabolic advantages to transformed cells, into the expression system. Furthermore, cell lines that reliably express sufficiently high levels of therapeutic proteins and that appropriately and consistently modify therapeutic proteins after translation are highly desirable. Thus, there is a need for improved mammalian expression systems in this field. [Means for solving the problem]

[0010] The use of mammalian tunicamycin (Tn) resistance genes as selection markers within mammalian expression systems can increase efficiency and transfectant copy number. It has been observed that the use of Tn resistance genes manipulably linked to the target gene creates selection pressure on a population of mammalian cells, thereby increasing the random incorporation of transfectants (i.e., the target gene). While it is understood that selection marker systems may foster the selection of desired transfectants, the methods of the present invention confer not only an unexpected increase in both efficiency and random incorporation of the target gene, but also the reliable biological quality of the desired protein. Therefore, the compositions and methods of the present invention enable the useful selection of qualitatively preferable post-translational modifications to expressed proteins.

[0011] In one embodiment, the present invention provides isolated cells comprising a mammalian tunicamycin (Tn) resistance gene encoding a protein having at least 93% identity with the amino acid sequence of Sequence ID No. 3, and operably linked to a target gene (GOI) and at least one regulatory element.

[0012] In another aspect, the present invention provides a method for generating a recombinant target protein (POI), the method comprising: providing a mammalian host cell encoding a nucleic acid molecule comprising (i) a mammalian tunicamycin (Tn) resistance gene and (ii) a gene encoding a POI; culturing the cell in the presence of a first concentration of Tn; isolating a cell population expressing at least one copy of the Tn resistance gene; culturing the cell population in the presence of increasing concentrations of Tn, wherein the increasing concentrations of Tn increase the generation of POI; and isolating the POI from the cell culture.

[0013] In another embodiment, the present invention provides a method for glycosylation of an N-glycan proteinaceous substrate, comprising: providing mammalian host cells encoding a nucleic acid molecule including a mammalian tunicamycin (Tn) resistance gene that is manipulably linked to a gene encoding a proteinaceous substrate requiring glycosylation; culturing the cells in the presence of a first concentration of Tn; isolating a cell population expressing at least one copy of the Tn resistance gene; culturing the cell population in the presence of increasing concentrations of Tn, wherein the increasing concentrations of Tn increase the generation of POIs; and isolating the proteinaceous substrate from the cell culture.

[0014] In some embodiments of this method, a Tn resistance gene is operably linked to a gene encoding a POI, and the gene encoding a POI is operably linked to at least one regulatory element.

[0015] In some embodiments, the Tn resistance gene is exogenously added to the cells. In other embodiments, the Tn resistance gene encodes a protein having at least 93% identity with the amino acid sequence of SEQ ID NO: 3. In yet another embodiment, the Tn resistance gene encodes a protein having at least 94% identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Tn resistance gene encodes a protein having at least 93% identity with the amino acid sequence of SEQ ID NO: 4. In yet another embodiment, the Tn resistance gene encodes a protein having at least 94% identity with the amino acid sequence of SEQ ID NO: 4.

[0016] In some embodiments, the mammalian Tn resistance gene includes the Chinese hamster (Cricetulus griseus) Tn resistance gene. In other embodiments, the mammalian Tn resistance gene includes the human Tn resistance gene.

[0017] The Tn resistance gene may also include nucleic acid sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0018] In certain embodiments of the invention described above, the mammalian Tn resistance gene includes a nucleic acid sequence having at least 92% identity with the nucleic acid sequence of Sequence ID No. 2. In some embodiments, the mammalian Tn resistance gene includes a nucleic acid sequence having at least 92% identity with the nucleic acid sequence of Sequence ID No. 12.

[0019] In the present invention, at least one regulatory element operably linked to a Tn resistance gene is provided in isolated cells, the regulatory element including, but not limited to, a promoter, a ribosome-binding site, and an enhancer. In yet another embodiment, the GOI is operably linked to a promoter. In yet another embodiment, the GOI is operably linked to a ribosome-binding site such as an IRES.

[0020] In some embodiments, the isolated cells and methods of the present invention further comprise a second gene of interest (GOI), where the GOI encodes a protein of interest (POI). In one embodiment, the gene of interest (GOI) is an exogenously added GOI. In another embodiment, the exogenously added GOI is a human gene. In yet another embodiment, the regulatory element is an exogenously added regulatory element.

[0021] In other embodiments, the first GOI and / or the second GOI encoding the POI include, but are not limited to, an antibody heavy chain, an antibody light chain, an antigen-binding fragment, and / or an Fc fusion protein.

[0022] In another embodiment, the first GOI and the second GOI are independently selected from the group consisting of a gene encoding an antibody light chain or an antigen-specific fragment thereof, an antibody heavy chain or an antigen-specific fragment thereof, an Fc fusion protein or a fragment thereof, and a receptor or a ligand-specific fragment thereof. In one embodiment, the recombinase recognition site is present between the first GOI and the second GOI. In other embodiments, the present invention further comprises a 5' recombinase recognition site for the first GOI and a 3' recombinase recognition site for the second GOI.

[0023] In yet another embodiment, the GOI encodes a glycoprotein selected from an antibody light chain or an antigen-binding fragment thereof, an antibody heavy chain or an antigen-binding fragment thereof, an Fc fusion protein or a fragment thereof, a ligand, and a receptor or a ligand-binding fragment thereof.

[0024] The isolated non-natural cells of the present invention may be derived from eukaryotic cells. In one embodiment, the cells are mammalian cells. In some embodiments, the isolated cells are ex vivo human cells. In other embodiments, the cells are selected from the group consisting of CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), lymphocytes, stem cells, retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the aforementioned cells. In certain embodiments, the isolated cells of the present invention are CHO-K1 cells, lymphocytes, retinal cells, or stem cells.

[0025] In one embodiment, the first concentration of Tn is 1 μg / mL. In another embodiment, the increasing concentrations of Tn are the second and third concentrations of Tn.

[0026] In some embodiments, the second concentration of Tn is greater than the first concentration, and the third concentration of Tn is greater than the second concentration. In certain embodiments, the second concentration of Tn is 2.5 μg / mL and the third concentration is 5 μg / mL.

[0027] In still other embodiments, the increasing concentrations of Tn include the second concentration of Tn, and the second concentration of Tn is 2.5 μg / mL or 5 μg / mL.

[0028] Unless otherwise specified or apparent from the context, any aspect or embodiment of the present invention can be used in conjunction with any other aspect or embodiment of the present invention.

[0029] Other objects and advantages will become apparent from consideration of the following detailed description of the invention. In certain embodiments, for example, the following are provided: (Item 1) Isolated cells containing a mammalian tunicamycin (Tn) resistance gene encoding a protein having at least 93% identity to the amino acid sequence of Sequence ID No. 3, and operably linked to a target gene (GOI) and at least one regulatory element. (Item 2) The isolated cells described in item 1, to which the Tn resistance gene has been exogenously added. (Item 3) The isolated cell according to item 1 or 2, wherein the Tn resistance gene encodes the protein having at least 95% identity with the amino acid sequence of Sequence ID No. 3. (Item 4) The isolated cell according to any one of items 1 to 3, wherein the Tn resistance gene encodes the protein having at least 98% identity with the amino acid sequence of Sequence ID No. 3. (Item 5) The isolated cell according to item 1 or 2, wherein the Tn resistance gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. (Item 6) The isolated cell according to item 1 or 2, wherein the Tn resistance gene comprises the nucleic acid sequence containing Sequence ID: 12. (Item 7) The isolated cell according to any one of items 1 to 6, wherein the at least one regulatory element is selected from the group consisting of a promoter, a ribosome binding site, and an enhancer. (Item 8) Isolated cells as described in any one of items 1 to 7, further containing a second target gene (GOI). (Item 9) Isolated cells as described in any one of items 1 to 8, wherein the target gene (GOI) is an exogenously added GOI. (Item 10) Isolated cells as described in any one of items 1 to 9, wherein the exogenously added GOI is a human gene. (Item 11) Isolated cells according to any one of items 1 to 10, wherein the regulatory element is an exogenously added regulatory element. (Item 12) Isolated cells according to any one of items 1 to 11, wherein the first and / or second GOI is selected from the group consisting of antibody heavy chains, antibody light chains, antigen-binding fragments, and Fc fusion proteins. (Item 13) Isolated cells according to any one of items 1 to 12, wherein the first GOI and the second GOI are independently selected from the group consisting of genes encoding antibody light chains or antigen-specific fragments thereof, antibody heavy chains or antigen-specific fragments thereof, Fc fusion proteins or fragments thereof, and receptors or ligand-specific fragments thereof. (Item 14) Isolated cells as described in any one of items 1 to 113, wherein the GOI is operably linked to a promoter. (Item 15) The GOI is an isolated cell according to any one of items 1 to 14, encoding a glycoprotein selected from an antibody light chain or its antigen-binding fragment, an antibody heavy chain or its antigen-binding fragment, an Fc fusion protein or its fragment, a ligand, and a receptor or its ligand-binding fragment. (Item 16) Isolated cells according to any one of items 8 to 15, wherein the recombinase recognition site is located between the first GOI and the second GOI. (Item 17) The isolated cell according to item 8, further comprising the recombinase recognition site 5' for the first GOI and the recombinase recognition site 3' for the second GOI. (Item 18) The isolated cells described in any one of items 1 to 17, wherein the aforementioned cells are eukaryotic cells. (Item 19) The isolated cells described in any one of items 1 to 18, wherein the cells are mammalian cells. (Item 20) Isolated cells according to any one of items 1 to 18, wherein the cells are selected from the group consisting of CHO-K1, COS-7, HEK293, tumor cells, lymphocytes, retinal cells, and stem cells. (Item 21) The isolated cells described in any one of items 1 to 20, wherein the aforementioned cells are CHO-K1 cells. (Item 22) A method for generating recombinant target proteins (POIs), a. To provide a mammalian host cell encoding a nucleic acid molecule including a mammalian tunicamycin (Tn) resistance gene and (ii) the gene encoding the POI, b. Culturing the cells in the presence of the first concentration of Tn, c. Isolating a cell population expressing at least one copy of the Tn resistance gene, d. Culturing the cell population in the presence of an increasing concentration of Tn, wherein the increasing concentration of Tn increases the generation of POI, e. A method comprising isolating the POI from the cell culture. (Item 23) The method according to item 22, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence having at least 95% identity with the nucleic acid sequence of sequence number 2. (Item 24) The method according to item 22, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence having at least 98% identity with the nucleic acid sequence of sequence number 2. (Item 25) The method according to any one of items 22 to 24, wherein the mammalian Tn resistance gene includes the Chinese hamster (Cricetulus griseus) Tn resistance gene. (Item 26) The method according to item 22, wherein the mammalian Tn resistance gene includes the human Tn resistance gene. (Item 27) The method according to item 22, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. (Item 28) The method according to any one of items 22 to 27, wherein the Tn resistance gene is operably linked to the gene encoding the POI. (Item 29) The method according to any one of items 22 to 27, wherein the gene encoding the POI is operably linked to at least one regulatory element. (Item 30) The method according to item 29, wherein the at least one regulatory element is selected from the group consisting of a promoter, a ribosome binding site, and an enhancer. (Item 31) The method according to any one of items 22 to 30, further comprising a second gene encoding the aforementioned POI. (Item 32) The method according to any one of items 22 to 31, wherein the aforementioned gene is an exogenously added target gene (GOI). (Item 33) The method according to item 32, wherein the exogenously added GOI is a human gene. (Item 34) The method according to item 29 or 30, wherein the regulatory element is an exogenously added regulatory element. (Item 35) The method according to any one of items 22 to 34, wherein the POI is selected from the group consisting of an antibody heavy chain, an antibody light chain, an antigen-binding fragment, and an Fc fusion protein. (Item 36) The method according to any one of items 22 to 35, wherein the first concentration of Tn is 1 μg / mL. (Item 37) The method according to any one of items 22 to 36, wherein the increasing concentration of Tn includes a second concentration of Tn and a third concentration of Tn. (Item 38) The method according to item 37, wherein the second concentration of Tn is greater than the first concentration of Tn, and the third concentration of Tn is greater than the second concentration of Tn. (Item 39) The method according to item 37 or 38, wherein the second concentration of Tn is 2.5 μg / mL and the third concentration of Tn is 5 μg / mL. (Item 40) The method according to any one of items 22 to 36, wherein the increasing concentration of Tn includes a second concentration of Tn, and the second concentration of Tn is 2.5 μg / mL or 5 μg / mL. (Item 41) A method for glycosylation of an N-glycan protein substrate, a. To provide mammalian host cells encoding nucleic acid molecules including a mammalian tunicamycin (Tn) resistance gene that is manipulably linked to a gene encoding the protein substrate requiring glycosylation, b. Culturing the cells in the presence of the first concentration of Tn, c. Isolating a cell population expressing at least one copy of the Tn resistance gene, d. Culturing the cell population in the presence of an increasing concentration of Tn, wherein the increasing concentration of Tn increases the generation of POI, e. A method comprising isolating the proteinaceous substrate from the cell culture. (Item 42) The method according to item 41, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence having at least 93% identity with the nucleic acid sequence of sequence number 2. (Item 43) The method according to item 39, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence having at least 98% identity with the nucleic acid sequence of sequence number 2. (Item 44) The method according to any one of items 41 to 43, wherein the mammalian Tn resistance gene includes the Chinese hamster (Cricetulus griseus) Tn resistance gene. (Item 45) The method according to item 41, wherein the mammalian Tn resistance gene includes the human Tn resistance gene. (Item 46) The method according to item 41, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. (Item 47) The method according to any one of items 41 to 46, wherein the Tn resistance gene is operably linked to the gene encoding the POI. (Item 48) The method according to any one of items 41 to 47, wherein the gene encoding the POI is operably linked to at least one regulatory element. (Item 49) The method according to item 48, wherein the at least one regulatory element is selected from the group consisting of a promoter, a ribosome binding site, and an enhancer. (Item 50) The method according to any one of items 41 to 47, further comprising a second target gene (GOI). (Item 51) The method according to item 50, wherein the target gene (GOI) is an exogenously added GOI. (Item 52) The method according to item 51, wherein the exogenously added GOI is a human gene. (Item 53) The method according to item 48 or 49, wherein the regulatory element is an exogenously added regulatory element. (Item 54) The method according to any one of items 41 to 53, wherein the POI is selected from the group consisting of an antibody heavy chain, an antibody light chain, an antigen-binding fragment, and an Fc fusion protein. (Item 55) The method according to any one of items 41 to 54, wherein the first concentration of Tn is 1 μg / mL. (Item 56) The method according to any one of items 41 to 55, wherein the increasing concentration of Tn includes a second concentration of Tn and a third concentration of Tn. (Item 57) The method according to item 56, wherein the second concentration of Tn is greater than the first concentration of Tn, and the third concentration of Tn is greater than the second concentration of Tn. (Item 58) The method according to item 56 or 57, wherein the second concentration of Tn is 2.5 μg / mL and the third concentration of Tn is 5 μg / mL. (Item 59) The method according to any one of items 41 to 55, wherein the increasing concentration of Tn includes a second concentration of Tn, and the second concentration of Tn is 2.5 μg / mL or 5 μg / mL. [Brief explanation of the drawing]

[0030] [Figure 1] A schematic diagram of an operational expression cassette within a cloning vector construct used for introducing a nucleic acid sequence encoding a target gene, such as eGFP into the cellular genome, is shown. SV40 promoter: Simian virus 40 promoter; GPT: GlcNAc-1-P transferase (e.g., CHO-GPT, SEQ ID NO: 2; or hGPT, SEQ ID NO: 12); IRES: Internal ribosome entry site; eGFP: Enhanced green fluorescent protein; SV40polyA: Simian virus 40 polyA. [Figure 2A]Figures 2A-2C show the mammalian GPT amino acid sequences compared to the Chinese hamster (GPT_CRIGR; UniProtKB Accn.No.P24140; SEQ ID NO:3), namely, human (GPT_HUMAN; UniProtKB Accn.No.Q9H3H5; SEQ ID NO:4), rhesus monkey (GPT_MACMU; UniProtKB Accn.No.F6TXM3; SEQ ID NO:5), chimpanzee (GPT_PANTR; UniProtKB Accn.No.H2R346; SEQ ID NO:6), dog (GPT_CANFA; UniProtKB Accn.No.E2RQ47; SEQ ID NO:7), guinea pig (GPT_CAVPO; UniProtKB Accn.No.E2RQ47; SEQ ID NO:8), and rat (GPT_RAT; UniProtKB This represents the alignment of Accn.No.Q6P4Z8 (sequence number:9) and mouse (GPT_MOUSE;UniProtKB Accn.No.P42867;sequence number:10). [Figure 2B] Figures 2A-2C show the mammalian GPT amino acid sequences compared to the Chinese hamster (GPT_CRIGR; UniProtKB Accn.No.P24140; SEQ ID NO:3), namely, human (GPT_HUMAN; UniProtKB Accn.No.Q9H3H5; SEQ ID NO:4), rhesus monkey (GPT_MACMU; UniProtKB Accn.No.F6TXM3; SEQ ID NO:5), chimpanzee (GPT_PANTR; UniProtKB Accn.No.H2R346; SEQ ID NO:6), dog (GPT_CANFA; UniProtKB Accn.No.E2RQ47; SEQ ID NO:7), guinea pig (GPT_CAVPO; UniProtKB Accn.No.E2RQ47; SEQ ID NO:8), and rat (GPT_RAT; UniProtKB This represents the alignment of Accn.No.Q6P4Z8 (sequence number:9) and mouse (GPT_MOUSE;UniProtKB Accn.No.P42867;sequence number:10). [Figure 2C]Figures 2A-2C show the mammalian GPT amino acid sequences compared to the Chinese hamster (GPT_CRIGR; UniProtKB Accn.No.P24140; SEQ ID NO:3), namely, human (GPT_HUMAN; UniProtKB Accn.No.Q9H3H5; SEQ ID NO:4), rhesus monkey (GPT_MACMU; UniProtKB Accn.No.F6TXM3; SEQ ID NO:5), chimpanzee (GPT_PANTR; UniProtKB Accn.No.H2R346; SEQ ID NO:6), dog (GPT_CANFA; UniProtKB Accn.No.E2RQ47; SEQ ID NO:7), guinea pig (GPT_CAVPO; UniProtKB Accn.No.E2RQ47; SEQ ID NO:8), and rat (GPT_RAT; UniProtKB This represents the alignment of Accn.No.Q6P4Z8 (sequence number:9) and mouse (GPT_MOUSE;UniProtKB Accn.No.P42867;sequence number:10). [Figure 3A] Figures 3A and 3B illustrate how protein optimization can be achieved using the methods and compositions of the present invention. Figure 3A shows a method for selecting positive cell transfectants from a first cell pool cultured with 1 μg / mL tunicamycin (Tn). Subsequently, a second cell culture was performed to enhance protein expression by increasing the tunicamycin concentration to, for example, 2.5 μg / mL or 5 μg / mL. Figure 3B shows a method for selecting positive cell transfectants from a first cell pool cultured with 1 μg / mL tunicamycin (Tn), and then successively increasing the Tn concentration in subsequent cell cultures to optimize protein expression. [Figure 3B]Figures 3A and 3B illustrate how protein optimization can be achieved using the methods and compositions of the present invention. Figure 3A shows a method for selecting positive cell transfectants from a first cell pool cultured with 1 μg / mL tunicamycin (Tn). Subsequently, a second cell culture was performed to enhance protein expression by increasing the tunicamycin concentration to, for example, 2.5 μg / mL or 5 μg / mL. Figure 3B shows a method for selecting positive cell transfectants from a first cell pool cultured with 1 μg / mL tunicamycin (Tn), and then successively increasing the Tn concentration in subsequent cell cultures to optimize protein expression. [Figure 4A] Figures 4A and 4B show FACS scatter plots representative of various parameters of hygromycin selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration at the YFP site and targeted integration. Randomly integrated cells express both YFP and eGFP. Figure 4A: Cells are transfused using an hpt expression vector containing Cre recombinase vector and eGFP, but cultured without hygromycin. Figure 4B: Cells are transfused using an hpt expression vector containing Cre recombinase vector and eGFP in the presence of 400 μg / mL of hygromycin. [Figure 4B]Figures 4A and 4B show FACS scatter plots representative of various parameters of hygromycin selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration at the YFP site and targeted integration. Randomly integrated cells express both YFP and eGFP. Figure 4A: Cells are transfused using an hpt expression vector containing Cre recombinase vector and eGFP, but cultured without hygromycin. Figure 4B: Cells are transfused using an hpt expression vector containing Cre recombinase vector and eGFP in the presence of 400 μg / mL of hygromycin. [Figure 5A]Figures 5A to 5F show FACS scatter plots representing various parameters of tunicamycin (Tn) selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration and targeted integration at the YFP site. Randomly integrated cells express both YFP and eGFP. Figure 5A: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5B: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5C: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. Figure 5D: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5E: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5F: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. [Figure 5B]Figures 5A to 5F show FACS scatter plots representing various parameters of tunicamycin (Tn) selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration and targeted integration at the YFP site. Randomly integrated cells express both YFP and eGFP. Figure 5A: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5B: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5C: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. Figure 5D: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5E: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5F: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. [Figure 5C]Figures 5A to 5F show FACS scatter plots representing various parameters of tunicamycin (Tn) selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration and targeted integration at the YFP site. Randomly integrated cells express both YFP and eGFP. Figure 5A: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5B: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5C: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. Figure 5D: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5E: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5F: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. [Figure 5D]Figures 5A to 5F show FACS scatter plots representing various parameters of tunicamycin (Tn) selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration and targeted integration at the YFP site. Randomly integrated cells express both YFP and eGFP. Figure 5A: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5B: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5C: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. Figure 5D: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5E: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5F: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. [Figure 5E]Figures 5A to 5F show FACS scatter plots representing various parameters of tunicamycin (Tn) selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration and targeted integration at the YFP site. Randomly integrated cells express both YFP and eGFP. Figure 5A: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5B: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5C: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. Figure 5D: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5E: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5F: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. [Figure 5F]Figures 5A to 5F show FACS scatter plots representing various parameters of tunicamycin (Tn) selectivity. Modified CHO cells contain a YFP gene adjacent to the lox site. Selective markers adjacent to the lox site (antibiotic resistance gene and eGFP) replace YFP using Cre recombinase via Cre recombinase integration and targeted integration at the YFP site. Randomly integrated cells express both YFP and eGFP. Figure 5A: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5B: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5C: Cells are transfused using a CHO-GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. Figure 5D: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP, but cultured without tunicamycin. Figure 5E: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 1 μg / mL Tn. Figure 5F: Cells are transfused using a human GPT expression vector containing Cre recombinase vector and eGFP in the presence of 2.5 μg / mL Tn. [Figure 6A]Figures 6A and 6B show the relative ability of a cell pool expressing GPT to enhance the expression of manipulably linked GOIs such as eGFP, compared to a pool that does not express GPT. Figure 6A: The relative number of CHO-GPT gene copies measured by PCR for the cell pool is illustrated below. Pool-49 cells (no exogenous GPT added), no Tn selection; Pool-49 cells (no exogenous GPT), with 5 μg of Tn selection; Pool-1 cells, spontaneously expressing higher levels of GPT (data not shown), tested without Tn selection; Pool-78 cells (no exogenous GPT), no Tn selection; CHO cells expressing exogenously added hpt and 400 μg / mL hygromycin selection; CHO cells expressing exogenous GPT under 1 μg / mL Tn selection conditions; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool and further cultured in 1 μg / mL Tn; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool and further cultured in 2.5 μg / mL Tn; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool, 5 μg / mL The cells were further cultured in Tn. Figure 6B: Illustrates the relative number of gene copies of the target gene, eGFP, measured by qPCR, relative to the same cell pool (as in Figure 6A). [Figure 6B]Figures 6A and 6B show the relative ability of a cell pool expressing GPT to enhance the expression of manipulably linked GOIs such as eGFP, compared to a pool that does not express GPT. Figure 6A: The relative number of CHO-GPT gene copies measured by PCR for the cell pool is illustrated below. Pool-49 cells (no exogenous GPT added), no Tn selection; Pool-49 cells (no exogenous GPT), with 5 μg of Tn selection; Pool-1 cells, spontaneously expressing higher levels of GPT (data not shown), tested without Tn selection; Pool-78 cells (no exogenous GPT), no Tn selection; CHO cells expressing exogenously added hpt and 400 μg / mL hygromycin selection; CHO cells expressing exogenous GPT under 1 μg / mL Tn selection conditions; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool and further cultured in 1 μg / mL Tn; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool and further cultured in 2.5 μg / mL Tn; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool, 5 μg / mL The cells were further cultured in Tn. Figure 6B: Illustrates the relative number of gene copies of the target gene, eGFP, measured by qPCR, relative to the same cell pool (as in Figure 6A). [Figure 7A] Figures 7A to 7D illustrate the glycosylation characteristics of Fc fusion protein 1 (FcFP1) generated from the following cell cultures. Figure 7A: CHO cells that do not express GPT using the standard protocol (Lot B10002M410), in contrast to Figure 7B: CHO cells expressing CHO-GPT and without Tn selection (Lot 110728). Figure 7C: CHO cells expressing CHO-GPT and with selected Tn of 1 μg / mL (Lot 110728-01), in contrast to Figure 7D: CHO cells expressing CHO-GPT and with selected Tn of 5 μg / mL (Lot 110728-02). Each chromatogram shows a fragment containing sialyzed residues as shown below. 0SA = 0 sialic acid residues; 1SA = 1 sialic acid residue; 2SA = 2 sialic acid residues; 3SA = 3 sialic acid residues; 4SA = 4 sialic acid residues. [Figure 7B] Figures 7A to 7D illustrate the glycosylation characteristics of Fc fusion protein 1 (FcFP1) generated from the following cell cultures. Figure 7A: CHO cells that do not express GPT using the standard protocol (Lot B10002M410), in contrast to Figure 7B: CHO cells expressing CHO-GPT and without Tn selection (Lot 110728). Figure 7C: CHO cells expressing CHO-GPT and with selected Tn of 1 μg / mL (Lot 110728-01), in contrast to Figure 7D: CHO cells expressing CHO-GPT and with selected Tn of 5 μg / mL (Lot 110728-02). Each chromatogram shows a fragment containing sialyzed residues as shown below. 0SA = 0 sialic acid residues; 1SA = 1 sialic acid residue; 2SA = 2 sialic acid residues; 3SA = 3 sialic acid residues; 4SA = 4 sialic acid residues. [Figure 7C] Figures 7A to 7D illustrate the glycosylation characteristics of Fc fusion protein 1 (FcFP1) generated from the following cell cultures. Figure 7A: CHO cells that do not express GPT using the standard protocol (Lot B10002M410), in contrast to Figure 7B: CHO cells expressing CHO-GPT and without Tn selection (Lot 110728). Figure 7C: CHO cells expressing CHO-GPT and with selected Tn of 1 μg / mL (Lot 110728-01), in contrast to Figure 7D: CHO cells expressing CHO-GPT and with selected Tn of 5 μg / mL (Lot 110728-02). Each chromatogram shows a fragment containing sialyzed residues as shown below. 0SA = 0 sialic acid residues; 1SA = 1 sialic acid residue; 2SA = 2 sialic acid residues; 3SA = 3 sialic acid residues; 4SA = 4 sialic acid residues. [Figure 7D]Figures 7A to 7D illustrate the glycosylation characteristics of Fc fusion protein 1 (FcFP1) generated from the following cell cultures. Figure 7A: CHO cells that do not express GPT using the standard protocol (Lot B10002M410), in contrast to Figure 7B: CHO cells expressing CHO-GPT and without Tn selection (Lot 110728). Figure 7C: CHO cells expressing CHO-GPT and with selected Tn of 1 μg / mL (Lot 110728-01), in contrast to Figure 7D: CHO cells expressing CHO-GPT and with selected Tn of 5 μg / mL (Lot 110728-02). Each chromatogram shows a fragment containing sialyzed residues as shown below. 0SA = 0 sialic acid residues; 1SA = 1 sialic acid residue; 2SA = 2 sialic acid residues; 3SA = 3 sialic acid residues; 4SA = 4 sialic acid residues. [Figure 8] The duplicate glycosylation profiles of Fc fusion protein 1 (FcFP1) sampled from (A)Lot B10002M410, (B)Lot 110728, (C)Lot 110728-01, and (D)Lot 110728-02 are illustrated. The glycosylation profiles of each protein generated from the GPT lot are compatible with the reference standard protein, and the main glycosylation species are consistently generated. It is clear that no novel or unique glycosylation species are generated within the GPT lot compared to the reference standard protein of the glycosylation type. [Modes for carrying out the invention]

[0031] Before describing this method, it should be understood that the present invention is not limited to the specific methods and experimental conditions described herein, as such methods and conditions may vary. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are used solely to describe specific embodiments and are not intended to be limiting.

[0032] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly states otherwise. For example, a reference to “one method” includes one or more methods and / or processes of a type described herein and / or which would be apparent to a person skilled in the art upon reading this disclosure.

[0033] Unless otherwise defined or specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains.

[0034] Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but specific methods and materials will be described below. All publications mentioned herein are incorporated herein by reference to their entirety.

[0035] Various genes well known in the art may confer a selectable phenotype to mammalian cells in culture. Generally, selective marker genes express proteins, usually enzymes, that confer resistance to various antibiotics in cell culture. Under certain selective conditions, cells expressing fluorescent protein markers become visible and therefore selectable. Examples in the art include beta-lactamase (bla; beta-lactam antibiotic resistance gene or ampR; ampicillin resistance gene), bls (blastosidine-resistant acetyltransferase gene), hygromycin phosphotransferase (hpt; hygromycin resistance gene), and others.

[0036] The methods described herein rely on the use of tunicamycin and an enzyme (marker) that enables cells to resist the growth of tunicamycin in cell culture. Tunicamycin (Tn) is a mixture of antibiotics that act as inhibitors of bacterial and eukaryotic N-acetylglucosamine transferases, interfering with the formation of N-acetylglucosamine lipid intermediates and the glycosylation of newly synthesized glycoproteins. (King, IA, and Tabiowo, A., 1981, Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro. Biochem. J., 198(2):331-338). Tn is cytotoxic because it specifically inhibits UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT), an enzyme that catalyzes the initial steps in the biosynthesis of dolichol-linked oligosaccharides. In the presence of tunicamycin, asparagine-linked glycoproteins produced in the endoplasmic reticulum (ER) may not be glycosylated by N-linked glycine and therefore may not fold correctly in the ER, potentially becoming targets for degradation (Koizumi, et al., 1999, Plant Physiol. 121(2):353-362). Therefore, Tn is a noteworthy inducer of the denaturing protein response (UPR) that leads to apoptosis in bacterial and eukaryotic cells.

[0037] The uridine diphosphate GPT gene (also known as GlcNAc-1-P transferase) is identified as being overexpressed under certain cellular conditions to confer resistance to Tn (Criscuolo and Krag, 1982, J Biol Chem, 263(36):19796-19803; Koizumi, et al., 1999, Plant Physiology, Vol.121, pp.353-361). The gene encoding GPT, also described in GenBank Accn.No.M36899 (Sequence ID: 2), was isolated from a Tn-resistant Chinese hamster ovary cell line and encodes a 408-amino acid protein (Sequence ID: 3) (Scocca and Krag, 1990, J Biol Chem 265(33):20621-20626; Lehrman, M. et al., 1988, J Biol Chem 263(36):19796-803). Hamster GPT is overexpressed in yeast cells (S. pombe) and confers Tn resistance to these cells; it also provides a convenient source for purifying the GPT enzyme (Scocca JR, et al., 1995, Glycobiology, 5(1):129-36). The transcriptional levels of GPT were analyzed within hybridoma cells (IgG-expressing B cells vs. resting B cells). While IgG-producing cells did not exhibit an increase in GPT transcription or activity levels, a small increase in GPT was still observed during the transition from resting to active B cells. In conclusion, GPT levels may correspond to the early development of the proliferative response to LPS (antigen) stimulation within B cells (Crick, DC et al., 1994, J Biol Chem 269(14):10559-65).

[0038] Furthermore, it was previously unknown whether the presence or absence of Tn in the cell expression system alters GPT expression, which would affect the glycosylation of the protein product and therefore its quality. It is understood that optimal and consistent glycosylation is a crucial protein characteristic for the production of therapeutic glycoproteins.

[0039] The present invention provides an improved method for the generation of recombinant proteins within mammalian cell lines utilizing the mammalian Tn resistance gene, GPT, as a controllable selection marker, while an increase in the copy number of a target gene manipulably linked to GPT correlates with an increase in the random incorporation of the GPT expression cassette into the cell.

[0040] This technology identifies the fact that the production of therapeutic proteins (particularly glycoproteins) relies on mammalian-type expression systems that mimic the innate glycosylation of such proteins. (See Bork, K. et al, 2009, J Pharm Sci. 98(10):3499-3508 for reference.) For example, the terminal monosaccharides of certain glycoproteins, such as N-linked complex glycans, are typically occupied by sialic acid. Sialization can affect the pharmacokinetic properties of glycoproteins (such as absorption, serum half-life, and clearance), or other physicochemical or immunogenic properties of glycoproteins. Overexpressed recombinant glycoproteins often have incomplete or inconsistent glycosylation. Reliable methods are crucial for process consistency and the quality of therapeutic glycoproteins produced within mammalian cell lines.

[0041] The present invention also provides an improved method for glycosylation of recombinant proteins, namely a method for producing glycoproteins in mammalian cell lines to provide a consistent quality yield of the desired protein.

[0042] (Definition of terms) DNA regions are manipulatively linked when they are functionally related to each other. For example, a promoter is manipulatively linked to a coding sequence if it has the ability to participate in the transcription of the sequence; a ribosome binding site is manipulatively linked to a coding sequence if it is positioned to allow translation. Generally, manipulative linking may include, but does not require, continuity. For sequences such as secretion leaders, continuity and proper placement within the reading frame are typical features. Generative enhancement sequences such as promoters are manipulatively linked to a target gene (GOI) if they are functionally related to the GOI, for example, if their presence results in increased GOI expression.

[0043] Thus, the phrase "manipulatively linked" in the context of DNA expression vector construction means that, for example, a regulatory sequence such as a promoter, operator, or marker is appropriately positioned relative to the coding sequence, thereby directing or allowing the production of the target polypeptide / protein encoded by the coding sequence. For example, if a selective marker is required for cell survival under certain culture conditions, the target gene is manipulatively linked to the selective marker gene, since expression will not occur without the presence of a manipulable selective marker protein.

[0044] As used herein, “promoter” refers to a DNA sequence sufficient to direct the transcription of a DNA sequence that is operablely linked, i.e., linked in such a way that it allows the transcription of a target gene and / or a selected marker gene when an appropriate signal is present. Gene expression may be regulated under any promoter or enhancer element known in the art.

[0045] In the context of the present invention, the “expression vector” may be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a nucleic acid molecule encoding an Fc fusion protein or polypeptide is contained within a bare DNA or RNA vector, for example, a linear expression element (e.g., as described in Sykes and Johnston, 1997, Nat Biotech 12, 355-59), a compressed nucleic acid vector (e.g., as described in U.S. Patent No. 6,077,835 and / or International Patent Application No. 00 / 70087), or a plasmid vector (such as pBR322, pUC 19 / 18, or pUC 118 / 119). Such nucleic acid vectors and their uses are well known in the art (see, for example, U.S. Patent Nos. 5,589,466 and 5,973,972).

[0046] As used herein, “operator” refers to a DNA sequence introduced within or near a gene in such a way that the gene may be modified by the binding of a repressor protein to the operator, and as a result, the transcription of a GOI, i.e., a polypeptide or nucleotide encoding a target protein, is prevented or enabled.

[0047] The ribosome binding site may include an "internal ribosome entry site" (IRES) or a 5' cap. Numerous IRES sequences are well known in the art. IRESs represent translational regulatory sequences, and the IRES site typically positions the 5' of the target gene and enables RNA translation in a manner independent of the cap. Transcribed IRESs may directly bind to ribosomal subunits so that the mRNA start codon is properly oriented within the ribosome for translation. IRES sequences are typically located within the 5' UTR of mRNA (just upstream of the start codon). IRESs functionally replace the needs of various protein factors that interact with the eukaryotic translation mechanism.

[0048] When used to describe protein expression, the terms “enhanced” or “improved” include an increase in the quantity and / or quality consistency of the proteins (i.e., gene products) produced by the expression system or method of the present invention. Thus, this includes at least about 1.5-fold to at least about 3-fold enhancement of expression, and is typically observed by random integration into the genome compared to, for example, a pool of integrations using a different selection marker construct. Thus, the multiplier of expression enhancement observed for the protein of interest was compared to the expression level of the same gene measured under substantially identical conditions, either in the absence of an expression cassette or cells of the present invention containing the GPT gene, or in the presence of an expression cassette or cells containing a different selection marker. Expression enhancement may also be measured by the number of resulting random integration events. Enhanced recombination efficiency includes enhancement of the recombination ability of a locus (e.g., employing a recombinase recognition site). Enhancement refers to a measurable efficiency on random recombination, which is typically 0.1%. Under certain conditions, the enhanced recombination efficiency is approximately 10 times that of random recombination, or approximately 1%. Unless otherwise specified, the claimed invention is not limited to a specific recombination efficiency. Enhanced expression can also be measured by the number of gene copies obtained as a result of quantitative polymerase chain reaction (qPCR) or other well-known techniques.

[0049] Enhanced or improved products also refer to more consistent quality, such as post-translational modifications observed in the GPT expression system of the present invention. Consistent quality includes, for example, having a desirable glycosylation profile after the replication generation line. Consistency in quality refers to the degree of homogeneity and standardization, while the replication generation batch is essentially unchanged. The calculation of Z numbers for measuring consistency is taught herein. Other statistical measures for measuring consistency are known in the art.

[0050] The term "selective pressure" refers to a force or stimulus applied to a living organism (e.g., a cell) or system (e.g., as an expression system) that alters the behavior and survival (such as the ability to survive) of the organism or system within a given environment.

[0051] The term "gene amplification" refers to an increase in the number of identical copies of a gene sequence. Certain cellular processes are characterized by the generation of multiple copies of a particular gene(s) that amplify the phenotype (e.g., antibiotic resistance) that the gene imparts to the cell.

[0052] When the phrase “exogenously added gene” or “exogenously added GOI” is used in reference to an expression cassette, this phrase refers to any gene that does not exist naturally within the cell genome as it would be found, or an additional gene copy incorporated into the genome (at a different locus). For example, an “exogenously added gene” in the CHO genome (e.g., a selection marker gene) could be a hamster gene not found naturally within a particular CHO locus (i.e., a hamster gene from another locus in the hamster genome), a gene from any other species (e.g., a human gene), a chimeric gene (e.g., human / mouse), or a hamster gene not found naturally within the CHO genome (i.e., a hamster gene with less than 99.9% identity to a gene from another locus in the hamster genome), or any other gene not found naturally within the CHO natural genome.

[0053] Random integration events differ from targeted integration events in that gene insertions into the cell's genome are not site-specific during random integration events. An example of targeted integration is homologous recombination. Random (non-homologous) integration means that the location (locus) of the resulting integration is known or specified. Random integration is thought to occur by non-homologous end joining (NHEJ), but is not limited to this method.

[0054] Selection efficiency refers to the percentage of surviving cells that express the selection marker and, where applicable, the target protein under the control of the selection marker.

[0055] When describing Tn-resistant proteins, the percentage of identity means that the homologous sequence exhibits identity along a region of continuous homology, but the presence of gaps, deletions, or insertions that do not have homology within the compared sequence is not considered when calculating the percentage of identity. Refer to the following amino acid sequence comparison to illustrate the use of “percent of identity” in this context.

[0056] [ka]

[0057] As used herein, the hamster homolog does not have homologous sequences to compare in alignment; therefore, the determination of the "percentage of identity" between the above "GPT_CRIG" sequence (for Chinese hamster GPT) and the mouse homolog ("GPT_MOUSE") does not involve a comparison of hamster amino acids 10 and 11 (i.e., mouse GPT may have an insertion at that point, or the hamster homolog may have a gap or deletion). Thus, in the above comparison, the comparison of the percentage of identity extends from "MWA" at the 5' end to "ESQ" at the 3' end. In this case, the mouse homolog differs only in that it has an "R" at hamster GPT position 51. Since the comparison covers 58 consecutive bases within a 60-base pair interval, a difference of only one amino acid (not a gap, deletion, or insertion) results in over 98% identity between the two sequences (hamster and mouse) from hamster GPT position 1 to hamster GPT position 58 (because the "percentage of identity" does not include penalties for gaps, deletions, and insertions). While the above example is based on amino acid sequences, it is understood that the percentage of identity of nucleic acid sequences will be calculated in the same manner.

[0058] The term "cell" includes any cell suitable for the expression of recombinant nucleic acid sequences. Examples of cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of Escherichia coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., budding yeast, fission yeast, Pichia pastrius, Pichia methenolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, nettle moth, etc.), non-human animal cells, mammalian cells, human cells, or cell fusions such as hybridomas or quadromas. In certain embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In other embodiments, the cells are eukaryotic and selected from the following: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293) Cell lines derived from the aforementioned cells, including EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cells derived from the aforementioned cells. In some embodiments, the cells include one or more viral genes, for example, retinal cells expressing viral genes (e.g., PER.C6® cells).

[0059] The term “integral cell density” or “ICD” refers to the cell density in a culture medium taken as an integral over a period of time, and is expressed as cells-days / mL. In some embodiments, the ICD is measured around day 21 of cell culture.

[0060] The phrase "glycosylation" or "glycosylating a protein" involves the formation of glycoproteins, while oligosaccharides are attached to either asparagine (Asn) residues (i.e., N-linked) side chains or serine (Ser) / threonine (Thr) residues (i.e., O-linked) of proteins. Glycans can be homopolymers or heteropolymers of monosaccharide residues, which can be linear or branched. N-linked glycosylation is well known to initiate primarily within the endoplasmic reticulum, while O-linked glycosylation has been shown to initiate in either the ER or the Golgi apparatus.

[0061] "N-glycan proteins" or "N-glycan protein substrates" include proteins that can contain or accept N-linked oligosaccharides. N-glycans can consist of N-acetylgalactosamine (GalNAc), mannose (Man), fucose (Fuc), galactose (Gal), neuraminic acid (NANA), and other monosaccharides, however, N-glycans typically have a common core pentasaccharide structure containing three mannose molecules and two N-acetylglucosamine (GlcNAc) sugars. Proteins having a continuous amino acid sequence (i.e., sequence) Asn-X-Ser or Asn-X-Thr (where X is any amino acid other than proline) can provide attachment sites for N-glycans.

[0062] (overview) This invention is at least in part based on the discovery that recombinant proteins can be produced within cells under certain conditions, and the protein-coding gene is manipulably linked to the Tn resistance gene, GPT, and also increases random integration events within the cellular genome, thus shaping the copy number of the target gene and ultimately the form of a select group of cells that produce the protein in order to increase protein production.

[0063] The present invention is at least partially based on the finding that protein-producing cells can be optimized to express proteins using consistent and reliable post-translational modifications. GPT expression cassettes can also be incorporated into the cellular genome, such as in an expression constructor, via an expression vector using various gene editing techniques known in the art. Expression vectors containing GPT can be incorporated into the genome by random recombination or targeted recombination (e.g., Cre-lox-mediated recombination), such as homologous recombination or recombinase-mediated recombination that identifies specific recombination sites.

[0064] Homologous recombination in eukaryotic cells can be facilitated by introducing breaks at integration sites within chromosomal DNA. Model systems have demonstrated that the frequency of homologous recombination during gene targeting increases when double-strand breaks are introduced into target sequences of chromosomes. This may be accomplished by targeting a specific site of integration within a certain nucleus. DNA-bound proteins that identify DNA sequences at target loci are known in the art. Gene targeting vectors are also employed to facilitate homologous recombination. In the absence of gene targeting vectors for homology to be repaired, cells frequently close double-strand breaks by non-homologous end joining (NHEJ), which may result in multiple nucleotide deletions or insertions at the cleavage site. Gene targeting vector construction and nuclease selection are within the scope of the skills of those skilled in the art to which this invention relates.

[0065] In some embodiments, zinc finger nucleases (ZFNs) having a modular structure and containing individual zinc finger domains identify specific 3-nucleotide sequences within a target sequence (e.g., targeted embedding sites). In some embodiments, ZFNs can be utilized with combinations of individual zinc finger domains that target multiple target sequences.

[0066] Transcriptional activator-like (TAL) effector nucleases (TALENs) may also be used for site-directed genome editing. The TAL effector protein DNA-binding domain is typically used in combination with the non-specific cleavage domain of a restriction nuclease such as FokI. In some embodiments, a fusion protein containing the TAL effector protein DNA-binding domain and the restriction nuclease cleavage domain is employed to identify and split DNA at a target sequence within the locus of the present invention (Boch J et al., 2009 Science 326:1509-1512).

[0067] RNA-induced endonucleases (RGENs) are programmable genome engineering tools developed from bacterial adaptive immune mechanisms. In this system, namely the clustered, regularly arranged, short palindromic repetition (CRISPR) / CRISPR-associated (Cas) immune response, the protein Cas9, when complexed with two RNAs, forms a sequence-specific endonuclease, one of which induces target selection. RGENs consist of component (Cas9 and tracrRNA) and a target-specific CRISPRRNA (crRNA). Both the efficiency of DNA target cleavage and the location of the cleavage site vary based on the position of a protospacer-adjacent motif (PAM), an additional requirement for target recognition (Chen, H. et al., J. Biol. Chem. published online March 14, 2014, manuscript M113.539726).

[0068] Those skilled in the art will know of other homologous recombination methods available, such as BuD-derived nucleases (BuDNs) with strictly DNA-bound specificity (Stella, S. et al., Acta Cryst. 2014, D70, 2042-2052). A precise genome modification method is selected based on available tools compatible with specific target sequences within the genome, thereby avoiding interference with the cellular phenotype.

[0069] Cells and methods are provided for the stable integration of nucleic acid sequences (target genes) into mammalian cells, and the nucleic acid sequences have enhanced expression capabilities thanks to integration using GPT sequences. Compositions and methods are also provided for using GPT in conjunction with expression constructs (e.g., expression vectors) and for adding exogenous GPT into target mammalian cells. Cells and methods are also provided for use in methods for producing consistent and robust glycoproteins, particularly therapeutic glycoproteins.

[0070] Building a GPT Selective Marker Cassette An expression vector comprising an operational GPT expression cassette is provided herein. The expression cassette contains regulatory elements necessary to allow and drive the transcription and translation of mammalian GPT and the desired gene product.

[0071] Various combinations of the genes and regulatory sequences described herein can also be developed. Examples of other combinations of suitable sequences described herein that can be developed include sequences containing multiple copies of the GPT gene disclosed herein, or sequences induced by combining the disclosed GPT with other nucleotide sequences to achieve an optimal combination of regulatory elements. Such combinations can be linked or sequenced sequentially to provide optimal spacing of GPTs oriented to the target gene and regulatory elements.

[0072] Homologous sequences of the gene encoding GPT are known to exist not only in cells from other mammalian species (e.g., humans, see Figure 2) but also in cell lines derived from other mammalian tissue types, and can be isolated by techniques well known in the art. An exemplary list of mammalian GPT amino acid sequences is provided in Figure 2. To allow for optimal expression of the corresponding GPT proteins shown in SEQ ID NOs: 3-10, nucleotide sequences, such as codon optimization, can be modified for the nucleotide sequences shown in SEQ ID NOs: 2 and SEQ ID NOs: 11-17. Furthermore, the amino acid sequences shown in SEQ ID NOs: 3-10 can be modified by altering the nucleotide sequences encoding GPT. Such techniques, including but not limited to site-directed or random mutagenesis techniques, are well known in the art.

[0073] Subsequently, as described herein, the resulting GPT variants can be tested for GPT activity, for example, for resistance to tunicamycin. GPT proteins whose amino acid sequences are at least about 93%, at least about 95%, at least about 96%, at least about 97%, or at least about 98% identical to SEQ ID NO: 3, which possesses GPT activity, can be isolated by the specified experiments and are expected to exhibit the same Tn resistance, selectivity efficiency, and post-translational benefits as those for SEQ ID NO: 3. Thus, mammalian homologs of GPT and variants of GPT are also encompassed by embodiments of the present invention. Figures 2A–2C show alignments of various mammalian GPT amino acid sequences (i.e., SEQ ID NOs: 3–10). Mammalian GPT sequences (nucleic acids and amino acids), among others, are conserved in the genomes of hamster, human, mouse, and rat. Table 1 identifies exemplary mammalian GPT proteins and their degree of homology. [Table 1A] [Table 1B]

[0074] The GPT / tunicamycin method provided herein is used to enhance the target protein. It is possible to develop cell populations that express a specific level of expression. The absolute level of expression will vary depending on how efficiently the protein is processed by the cell, and will differ for each particular protein.

[0075] Therefore, the present invention also includes nucleotide sequences expressing GPT selected from the group consisting of SEQ ID NO: 2 and SEQ ID NOs: 11-17. The present invention also includes nucleotide sequences expressing GPT that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to nucleotide sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NOs: 11-17.

[0076] The present invention includes a vector comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 12. Examples of vectors comprising the mammalian GPT gene and voluntary regulatory elements include transient transfection vectors or stable transfection vectors.

[0077] In one embodiment, as illustrated in Figure 1, a GPT gene is employed to enhance GOI expression. Figure 1 shows a GOI operably linked to an IRES sequence and a GPT selection marker. The GPT cassette further comprises a promoter sequence (e.g., SV40 promoter) and a polyadenylation (poly(A)) sequence (e.g., SV40 poly(A)).

[0078] The expression-enhancing cassette (containing GPT and upstream promoter) is optimally integrated into the cell genome. Using the method of the present invention, GOIs are expressed in the GPT expression cassette under culture conditions based on increasing Tn concentration (Figure 3A or Figure 3B). FACS readouts, as shown in Figures 5B, 5C, 5E, and 5F, illustrate the distribution of expression within a population of stably transfected cells and, in particular, the dramatic increase in selection efficiency using mammalian Tn resistance selection markers, CHO-GPT and hGPT. Mammalian GPT expression further enhances the expression of the target gene product, e.g., the production of the fluorescent protein, eGFP. Successive cultures with increasing Tn concentrations result in approximately 2-fold enhanced expression compared to GOIs expressed in the expression system using GPT under Tn concentration-based culture conditions, as illustrated in Figure 6B.

[0079] The present invention comprises mammalian cells containing such a GPT gene, the GPT gene being exogenous and incorporated into the cell genome by the method of the present invention. Cells containing such a GPT gene having at least one exogenously added target gene (GOI) are either upstream or downstream of the GPT gene.

[0080] In various embodiments, GOI expression can be enhanced by placing the GOI under the control of the mammalian selection marker GPT. In other embodiments, random incorporation events of the GOI can be enhanced by placing the GOI under the control of the mammalian selection marker GPT and by providing cell culture conditions consisting of a Tn concentration greater than 0.5 μg / mL. In some embodiments, the cell culture conditions consist of a Tn concentration greater than 1 μg / mL. The regulatory element may be operably linked to the GOI, and the expression of the GOI (at a selected distance from the GOI and GPT (in the 5' or 3' direction)) retains the ability to enhance GOI expression over expression typically observed due to random incorporation events. In various embodiments, the enhancement is at least about 1.5 to about 2 times, or more. The enhancement of expression compared to random incorporation or random expression is about 1.5 to about 2 times, or more.

[0081] In another embodiment, homogeneously glycosylated proteins can be achieved using the methods and compositions of the present invention. As shown in Table 4, batches of GPT / GOI recombinant proteins treated with Tn can replicate batches having equivalent glycosylation profiles. Thus, enhanced protein expression, such as a consistent glycosylation profile, can be directly compared by calculating the Z number, as taught herein. The Z number formula takes into account not only the relative number of peaks on the chromatogram representing the sialic acid (SA) moiety, but also the relative shape and intensity of each peak. The Z number is based on the area occupied by each peak and may also be used as a unit of measurement for consistency for complexed glycoproteins (see, for example, Figures 7A–7D, Figure 8, and Example 3 described herein).

[0082] For example, optimization of protein expression for each GOI can also be achieved, including orientation of the expression cassette or codon optimization. Protein optimization may also be achieved by varying Tn concentrations in the cell culture method.

[0083] Recombinant expression vectors may include synthetic or cDNA-derived DNA fragments encoding proteins operably linked to suitable transcriptional and / or translational regulators derived from mammalian, viral, or insect genes. Such regulators, as described in detail herein, include transcription promoters, enhancers, sequences encoding suitable mRNA-ribosome binding sites, and sequences controlling transcriptional and translational arrest. Mammalian expression vectors may also include non-transcriptional elements such as the origin of replication, other 5' or 3' adjacent non-transcriptional sequences, and 5' or 3' untranslated sequences such as splice donor and acceptor sites. Further selective marker genes (such as fluorescent markers) to facilitate transfectant recognition may also be incorporated.

[0084] In another embodiment, the vector comprises a nucleic acid molecule (or target gene) encoding a target protein, which includes an expression vector containing the nucleic acid molecule (gene) to be described, and the nucleic acid molecule (gene) is operably linked to an expression control sequence.

[0085] A vector containing the target gene (GOI) is provided, and the GOI is manipulatively linked to an expression regulatory sequence suitable for expression in mammalian host cells.

[0086] Useful promoters that may be used in the present invention include the SV40 early promoter region, the promoter contained within the 3' long terminal repeat of Rous sarcoma virus, the regulatory sequence of the metallothionein gene, and the mouse or human cytomegalovirus IE promoter (Gossen et al., (1995) Proc. Nat. Acad. Sci. USA). 89:5547-5551), the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose 2-phosphate carboxylase, promoter elements from yeast or other fungi (such as the Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphonoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions), where the animal transcriptional control regions are tissue-specific and utilized in transgenic animals (e.g., elastase I; insulin; immunoglobulin; mouse mammary tumor virus; albumin; α-fetoprotein; α1-antitrypsin; β-globin; and myosin light chain-2).

[0087] The nucleic acid molecules of the present invention may also be manipulably linked to an effective poly(A) termination sequence (e.g., SV40 poly(A)), a replication origin for plasmid products in E. coli, and / or a convenient cloning site (e.g., a polylinker). The nucleic acids may also include moduloable inductive promoters (inductive, reproducible, developmentally modulated) as opposed to structural promoters such as CMV IE (those skilled in the art will recognize that such terms are actually descriptors of the degree of gene expression under certain conditions).

[0088] The present invention provides a method for generating a target protein, while also providing an expression vector containing a target gene (GOI). Such an expression vector may be used for the recombinant generation of any target protein. Transcriptional and translational regulatory sequences within the expression vector, useful for translocation into vertebrate cells, may be provided by viral sources. For example, commonly used promoters and enhancers are derived from viruses such as polyoma, adenovirus 2, Simian virus 40 (SV40), and human cytomegalovirus (CMV). Viral genomic promoters, regulatory sequences, and / or signal sequences may be used to drive expression, and such provided regulatory sequences are compatible with selected host cells. Depending on the cell type in which the recombinant protein is expressed, non-viral cell promoters (e.g., β-globin and EF-1α promoters) may also be used.

[0089] To provide other gene elements useful for the expression of heterologous DNA sequences, DNA sequences derived from the SV40 viral genome, such as SV40 origin, early and late promoters, enhancers, splices, and polyadenylation sites, may be used. Both the early and late promoters are readily obtainable as fragments from the SV40 virus and are particularly useful because they also contain replicated SV40 virus origin (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used. Typically, Hind It contains a nearly 250 bp sequence extending from site III toward site BglI, which is located at the SV40 origin of replication.

[0090] Two-cistronic expression vectors used for multiple transcription expression have been previously described (Kim SK and Wold BJ, Cell 42:129, 1985; Kaufman et al., 1991, supra) and can be used in combination with GPT expression systems. Other types of expression vectors are also useful, such as those described in U.S. Patent No. 4,634,665 (Axel et al.) and U.S. Patent No. 4,656,134 (Ringold et al.).

[0091] A recombinase recognition site can be immobilized at the 5' or 3' position of an integration site, for example, a gene sequence encoding a POI. One example of a preferred integration site is the lox p site. Another example of a preferred integration site is two recombinase recognition sites selected from the group consisting of the lox p site, lox, and lox 5511 sites.

[0092] Gene amplification cassette and expression vector Previously described or useful regulatory elements known in the art may also be included in the nucleic acid construct used to transfect mammalian cells. Figure 1 illustrates an operational cassette within a GPT vector further comprising a promoter sequence, an IRES sequence, a target gene, and a poly(A) sequence.

[0093] The expression vector in the context of the present invention may be any suitable vector, including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid molecule encoding the antibody is contained within a bare DNA or RNA vector, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), a compressed nucleic acid vector (e.g., as described in U.S. Patent No. 6,077,835 and / or International Patent Application No. 00 / 70087), or a plasmid vector (such as pBR322, pUC 19 / 18, or pUC 118 / 119). Such nucleic acid vectors and their uses are well known in the art (see, for example, U.S. Patent Nos. 5,589,466 and 5,973,972).

[0094] The expression vector may alternatively be a vector suitable for expression within a yeast system. Any vector suitable for expression within a yeast system may be used. Suitable vectors include, for example, vectors containing structural or induced promoters such as yeast alpha factor, alcohol oxidase, and PGH (F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley). InterScience New York (1987), and Grant et al. (This has been examined in al., Methods in Enzymol 153, 516-544 (1987).)

[0095] In certain embodiments, the vector comprises a nucleic acid molecule (or target gene) encoding a target protein, which includes an expression vector containing the nucleic acid molecule (gene) to be described, and the nucleic acid molecule (gene) is manipulably linked to an expression regulatory sequence suitable for expression in a host cell.

[0096] Expression regulatory sequences are modified to control and drive the transcription of the target gene and the subsequent expression of the protein in various cell lines. A plasmid combines an expressible target gene with an expression regulatory sequence (i.e., an expression cassette) containing desired regulatory elements such as promoters, enhancers, selection markers, operators, etc. In the expression vector of the present invention, the GPT and the target protein (such as a nucleic acid molecule encoding an antibody) may contain, or be associated with, any suitable promoter, enhancer, operator, repressor protein, poly(A) termination sequence, and other expression promoters.

[0097] The expression of a target gene, such as an antibody-encoding nucleotide sequence, may be regulated under any promoter or enhancer element known in the art. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer or CMV major IE (CMV-MIE) promoter, as well as the RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoters).

[0098] In some embodiments, the vector includes a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC, and HIV LTR.

[0099] The nucleic acid molecules of the present invention may also be manipulably linked to effective poly(A) termination sequences, origins of replication for plasmid products in E. coli, antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., polylinkers). The nucleic acids may also include modulable inductive promoters (inductive, reproducible, developmentally modulated) as opposed to structural promoters such as CMV IE (those skilled in the art will recognize that such terms are actually descriptors of the degree of gene expression under certain conditions).

[0100] Selection markers are well-known elements in the art. In some situations, additional selection markers may be used in addition to GPT, such markers that make cells visible. Positive or negative selection may be used.

[0101] In some embodiments, the vector includes one or more select marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (eCFP), yellow fluorescent protein (YFP), or enhanced yellow fluorescent protein (eYFP).

[0102] For the purposes of the present invention, gene expression in eukaryotic cells is tightly regulated using a strong promoter controlled by an operator, which is then regulated by a regulatory fusion protein (RFP). The RFP essentially consists of a transcription-blocking domain and a ligand-binding domain that modulates its activity. Examples of such expression systems are described in U.S. Patent No. US20090162901A1, which is incorporated herein by reference in its entirety.

[0103] Numerous operators in prokaryotic cells and bacteriophages have been well characterized (Neidhardt, ed. Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol 2 ASM Press, Washington DC 1996). These include, but are not limited to, the operator region of the E. coli LexA gene that binds the LexA peptide, as well as lactose and tryptophan operators that bind repressor proteins encoded by the E. coli LacI and trpR genes. These include lambda P that binds repressor proteins encoded by lambda cI and P22arc. R This also includes bacteriophage operators from the phage P22 ant / mnt gene. In some embodiments, when the transcription-blocking domain of the repressor protein is a restriction enzyme such as NotI, the operator is a recognition sequence for that enzyme. Those skilled in the art will recognize that the operator needs to be adjacent to the promoter or positioned 3' relative to the promoter so that it has the ability to control transcription by the promoter. For example, U.S. Patent No. 5,972,650, incorporated herein by reference, specifies that the tetO sequence is within a certain distance from the TATA box. In certain embodiments, the operator is preferably positioned immediately downstream of the promoter. In other embodiments, the operator is positioned within 10 base pairs of the promoter.

[0104] In certain embodiments, the operator is selected from the group consisting of a tet operator (tetO), a NotI recognition sequence, a LexA operator, a lactose operator, a tryptophan operator, and an Arc operator (AO). In some embodiments, the repressor protein is selected from the group consisting of TetR, LexA, LacI, TrpR, Arc, lambda C1, and GAL4. In other embodiments, the transcription blocking domain is derived from a eukaryotic repressor protein (e.g., a repressor domain derived from GAL4).

[0105] In an exemplary cell expression system, cells are modified to express a tetracycline repressor protein (TetR), and the target protein is placed under the transcriptional control of a promoter, with its activity regulated by TetR. Two tandem TetR operators (tetO) are placed immediately downstream of the CMV-MIE promoter / enhancer in the vector. Transcription of the gene encoding the target protein, directed by the CMV-MIE promoter in such a vector, may be blocked by TetR in the absence of tetracycline or certain other suitable inducers (e.g., doxycycline). In the presence of an inducer, the TetR protein lacks the ability to bind tetO, thus resulting in transcription and subsequent translation (expression) of the target protein. (See, for example, U.S. Patent No. 7,435,553, which is incorporated herein by reference in its entirety.)

[0106] Another exemplary cell expression system is TetR-ER LBD It includes regulatory fusion proteins such as the T2 fusion protein, in which the transcription blocking domain of the fusion protein is TetR, and the ligand-bound domain is the estrogen receptor ligand-binding domain (ER) containing the T2 mutation. LBD ) is (ER LBD T2; Feil et al. (1997) Biochem. Biophys. Res. Commun. 237:752-757). When the tetO sequence is placed downstream and proximal to a strong CMV-MIE promoter, transcription of the target nucleotide sequence from the CMV-MIE / tetO promoter is blocked in the presence of tamoxifen and is no longer blocked upon removal of tamoxifen. In another example, the fusion protein Arc2-ER LBD T2, 15 amino acid linker and ER LBDThe use of a fusion protein consisting of a single-stranded dimer composed of two Arc proteins linked by T2(supra) involves Arc operators (AOs), more specifically two tandem Arc operators immediately downstream of the CMV-MIE promoter / enhancer. The cell line Arc2-ER LBD The process may be regulated by T2, and cells expressing the target protein are driven by the CMV-MIE / ArcO2 promoter and are induced by tamoxifen removal. (See, for example, U.S. Patent No. 20090162901A1, incorporated herein by reference.)

[0107] In some embodiments, the vector of the present invention includes a CMV-MIE / TetO or CMV-MIE / AO2 hybrid promoter.

[0108] The vector of the present invention may also employ a Cre-lox tool for recombination techniques to facilitate replication of the target gene. The Cre-lox strategy requires at least two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites, and 2) a loxP site (e.g., an 8-bp core sequence consisting of a specific 34-base pair sequence and two adjacent 13-bp inverted repeats, if recombination occurs) or a mutant lox site. (See, for example, Araki et al. PNAS 92:160-4 (1995); Nagy, A. et al. Genesis 26:99-109 (2000); Araki et al. Nuc Acids Res 30 (19):e103 (2002); and U.S. Patent No. US20100291626A1, all of which are incorporated herein by reference). In an alternative recombination strategy, a yeast-derived FLP recombinase may be used in conjunction with the common sequence FRT (see, for example, Dymecki, S. PNAS 93(12):6191-6196(1996)).

[0109] In another embodiment, a gene (i.e., a nucleotide sequence encoding the recombinant polypeptide of the present invention) is inserted upstream or downstream of the GPT gene in an expression cassette and is optionally manipulatively linked to a promoter, the promoter-linked gene being adjacent at 5' by a first recombinase recognition site and adjacent at 3' by a second recombinase recognition site. Such recombinase recognition sites enable Cre-mediated recombination within the host cell of the expression system. In some cases, the gene linked to the second promoter is downstream (3') of the first gene and adjacent at 3' by a second recombinase recognition site. In yet another case, the gene linked to the second promoter is adjacent at 5' by a second recombinase site and adjacent at 3' by a third recombinase recognition site. In some embodiments, the recombinase recognition sites are selected from the loxP site, lox511 site, lox2272 site, and FRT site. In other embodiments, the recombinase recognition sites are different. In further embodiments, the host cell contains a gene capable of expressing Cre recombinase.

[0110] In one embodiment, the vector includes a first gene encoding the light chain or heavy chain of the antibody of the present invention, and a second gene encoding the light chain or heavy chain of the antibody of the present invention.

[0111] In some embodiments, the vector further includes an X-box binding protein 1 (mXBP1) gene, which has the ability to further enhance protein synthesis / secretion by regulating the expression of genes involved in protein folding within the endoplasmic reticulum (ER). (See, for example, Ron D, and Walter P. Nat Rev Mol Cell Biol. 8:519-529 (2007)).

[0112] Any cell type is suitable for expressing the recombinant nucleic acid sequence of the present invention. Examples of cells used in the present invention include non-human cells, human cells, or mammalian cells such as cell fusions (e.g., hybridomas or quadromas). In certain embodiments, the cells are human, monkey, hamster, rat, or mouse cells. In other embodiments, the cells are eukaryotic and selected from the following: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells include one or more viral genes, e.g., retinal cells expressing a viral gene (e.g., PER.C6(trademark) cells).

[0113] In further embodiments, the present invention relates to recombinant mammalian host cells, such as transfectomas, that produce immunoglobulins, such as antibodies or bispecific molecules. Examples of such host cells include genetically modified mammalian cells, such as CHO cells or HEK cells. For example, in one embodiment, the present invention provides cells comprising nucleic acids stably incorporated into a cell genome, including sequence encoding for the expression of an antibody comprising the recombinant polypeptide of the present invention. In another embodiment, the present invention provides cells comprising unincorporated (i.e., episomal) nucleic acids, such as plasmids, cosmids, phagemids, or linear expression elements, including sequence encoding for the expression of an antibody comprising the recombinant polypeptide of the present invention. In yet another embodiment, the present invention provides cell lines produced by stably transfecting host cells with a plasmid comprising the expression vector of the present invention.

[0114] Accordingly, in one embodiment, the present invention provides cells containing (a) a recombinant polynucleotide encoding an exogenously added mammalian GPT gene, and (b) a polynucleotide encoding a multi-subunit protein. In some embodiments, the exogenously added GPT gene is 90% identical to the nucleic acid sequence of SEQ ID NO: 2, a non-limiting example thereof is provided in SEQ ID NOs: 11-17, and the multi-subunit protein is an antibody. In other embodiments, the cells also contain an exogenously added GPT gene and regulatory elements. In one embodiment, the cells are mammalian cells such as CHO cells used in the manufacture of biopharmaceuticals.

[0115] In another embodiment, the present invention provides cell lines derived from cells described in a prior embodiment. By "derived from," it means that the cell population is clonally transmitted from individual cells to offspring and possesses certain selective qualities, such as the ability to produce an active protein at a given titer or the ability to grow to a specific density. In some embodiments, cell lines derived from cells parasitizing a recombinant polynucleotide encoding the mammalian GPT gene and a polynucleotide encoding a multi-subunit protein have the ability to produce the multi-subunit protein at a titer of at least 3 grams (g / L), at least 5 g / L, or at least 8 g / L per liter of culture medium. In some embodiments, the cell lines can achieve an integrated cell density (ICD) that is at least 30%, at least 50%, at least 60%, or at least 90% greater than the integrated cell density (ICD) achievable by cell lines derived from essentially identical cells but without the recombinant polynucleotide encoding GPT.

[0116] A method for amplifying GOIs is provided. The exemplary method involves applying an increasing concentration of tunicamycin to a eukaryotic GPT expression system, thereby amplifying a gene copy of the GOI that is manipulatively linked to an exogenously added mammalian GPT gene.

[0117] Target protein The nucleic acid sequence encoding the target protein can be conveniently incorporated into a cell containing a Tn resistance marker gene and IRES, and can optionally be adjacent to a recombinase recognition site. Any target protein suitable for expression in mammalian cells can be used, but glycoproteins in particular benefit from the method of the present invention. For example, the target protein can be an antibody or its antigen-binding fragment, a bispecific antibody or its fragment, a chimeric antibody or its fragment, an ScFv or its fragment, an Fc-tagged protein (e.g., Trap protein) or its fragment, a growth factor or its fragment, a cytokine or its fragment, or the extracellular domain or its fragment of a cell surface receptor.

[0118] Glycoproteins containing asparagine-linked (N-linked) glycans are ubiquitous in eukaryotic cells. The biosynthesis of these glycans and their translocation to polypeptides takes place within the endoplasmic reticulum (ER). N-glycan structures are further modified by the number of glycosidases and glycosyltransferases within the ER and Golgi complex. Protein production using this invention aims to achieve consistency in native N-glycan structures to eliminate immunogenic epitopes ("glycotopes").

[0119] Using the method of the present invention, recombinant protein lots exhibit desirable properties. As illustrated in Figures 7-8 herein, HPLC (with fluorescence detection) of replicated protein-producing batches demonstrated that the glycoproteins had homogeneous expression and glycosylation patterns. A method for glycosylation of an N-glycan proteinogenic substrate is provided, on the one hand, mammalian host cells encoding a nucleic acid molecule containing a mammalian tunicamycin (Tn) resistance gene that is manipulably linked to a gene encoding a proteinogenic substrate that requires glycosylation, on the other hand, cells are cultured in the presence of a first concentration of Tn, a population of cells expressing at least one copy of the Tn resistance gene is isolated, the population of cells is cultured in the presence of increasing concentrations of Tn, and the N-glycan proteinogenic substrate is isolated from the cell culture. The N-glycan content of the proteinogenic substrate may be assessed for the presence of monosaccharides and oligosaccharides by any method known in the art.

[0120] Detailed structural analysis of glycan-linked proteins can correlate with the functional characteristics of the protein. Such analysis characterizing protein glycosylation typically involves several steps: i) enzymatic or chemical release of the attached glycan; ii) derivatization of the released glycan via reductive amination or complete methylation using aromatic or aliphatic amines; and iii) analysis of the glycan. Numerous variations of patterns for analyzing glycosylation are known to those skilled in the art. Glycoproteins may have several types of glycosylation structures occupying various sites in specific amounts, and thus the complexity of these structures can make reproduction by a certain synthesis method difficult. Consistency in the type and quantity of glycosylation structures is measurable and represents a desirable outcome for therapeutic protein synthesis.

[0121] Host cells and transfection The mammalian host cells used in the methods of the present invention are eukaryotic host cells, typically mammalian cells, and include, for example, CHO cells and mouse cells. In one embodiment, the present invention provides cells comprising a nucleic acid sequence encoding a Tn resistance marker protein or its homolog or variant derived from Cricetulus griseus (Chinese hamster) (indicated by SEQ ID NO: 3). In some embodiments, the cells comprise multiple copies of the Tn resistance marker gene. In other embodiments, the present invention provides nucleic acid sequences encoding a Tn resistance marker protein derived from human (SEQ ID NO: 4), rhesus monkey (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), dog (SEQ ID NO: 7), guinea pig (SEQ ID NO: 8), rat (SEQ ID NO: 9), or mouse (SEQ ID NO: 10).

[0122] The present invention comprises mammalian host cells transfected with the expression vector of the present invention. The transfected host cells include cells transfected with an expression vector containing a sequence encoding a target protein or polypeptide. The expressed protein is typically secreted into the culture medium depending on the selected nucleic acid sequence, but may be retained intracellularly or deposited within the cell membrane. Various mammalian cell culture systems can be employed to express recombinant proteins. Examples of suitable mammalian host cell lines include the monkey kidney cell line COS-7 (described in Gluzman (1981) Cell 23:175) and other cell lines capable of expressing suitable vectors (e.g., including CV-1 / EBNA (ATCC CRL 10478), L cells, C127, 3T3, CHO, HeLa, and BHK cell lines). Other cell lines developed for specific selection or amplification schemes may also be useful in the methods and compositions provided herein. In one embodiment of the present invention, the cells are a CHO cell line designated as K1 (i.e., CHO K1 cells). To achieve the goal of mass production of recombinant proteins, the host cell line should be pre-fitted to the bioreactor medium in appropriate cases.

[0123] Several transfection protocols are known in the art and have been discussed in Kaufman (1988) Meth. Enzymology 185:537. The chosen transfection protocol will depend on the host cell type and the nature of the GOI, and can be selected based on prescribed experiments. The basic requirements of any such protocol are to first introduce the DNA encoding the target protein into a suitable host cell, and then to identify and isolate the host cell into which the heterologous DNA has been incorporated in a relatively stable expression.

[0124] Certain reagents useful for introducing heterologous DNA into mammalian cells include Lipofectin® Reagent and Lipofectamine® Reagent (Gibco BRL, Gaithersburg, Maryland, USA). Both of these reagents are commercially available and used to form lipid-nucleic acid complexes (or liposomes), which, when applied to cultured cells, facilitate the uptake of nucleic acids into the cells.

[0125] The chosen transfection protocol and the elements selected for use therein will depend on the type of host cell used. Those skilled in the art are familiar with numerous different protocols and host cells and can select an appropriate system for the expression of the desired protein based on the requirements of the cell culture system used. In a further aspect, the present invention relates to expression vectors encoding polypeptides, including but not limited to antibodies, bispecific antibodies, chimeric antibodies, ScFv, antigen-binding proteins, or Fc fusion proteins. Such expression vectors may be used for recombinant synthesis of polypeptides using the methods and compositions of the present invention.

[0126] Other features of the present invention will become apparent in the course of the following description of exemplary embodiments, which are given for illustrative purposes and are not intended to limit the present invention. [Examples]

[0127] The following examples are provided to those skilled in the art to how to prepare and use the methods and compositions described herein and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy to the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is the average molecular weight, and temperature is in degrees Celsius (°C) at or near atmospheric pressure.

[0128] (Example 1) Selection efficiency of transfectant cell expression GPT Modified CHO K1 cells were transfused using plasmid vectors containing CHO-GPT (SEQ ID NO: 2), human GPT (SEQ ID NO: 12), or hygromycin phosphotransferase (Hpt, hygromycin resistance gene), for example, by transcriptionally linking a selected marker gene (CHO-GPT or hpt) to the downstream eGFP gene within each of these vectors via an IRES sequence. For example, each plasmid was constructed to contain the following gene sequence in the 5'-3' direction: Lox site, late SV40 promoter, either CHO-GPT (or Hpt), IRES, enhanced green fluorescent protein (eGFP), and a second Lox site. The purified recombinant plasmids were transfused into modified CHO host cell lines along with a plasmid expressing Cre recombinase, resulting in cell lines containing the lox site, YFP, and second lox site in the 5'-3' direction at the transcriptionally active locus. As a result, host CHO cells can be isolated as green-positive or yellow-negative cells by flow cytometry. When a recombinant plasmid expressing eGFP (transcriptionally modified by the GPT or hpt gene) is transfected together with a plasmid expressing Cre recombinase, Cre recombinase-mediated recombination results in site-specific integration of the GPT / eGFP cassette at chromosomal loci containing lox site replacements, producing the YFP gene (i.e., green-positive cells). If eGFP is integrated randomly, both green-positive and yellow-positive cells will result.

[0129] Cell populations were incubated with either 0, 1 μg / mL, 2.5 μg / mL, or 5 μg / mL of tunicamycin (Tn), or 400 μg of hygromycin (Hyg), as outlined in Table 2. Observed recombinant populations (ORPs) were measured by fluorescence-activated cell sorting (FACS) analysis. Cells were sorted to quantify each cell population, and the selection efficiency was calculated for cells expressing only GFP and not YFP (Figure 4 or Figure 5).

[0130] The selection efficiency (percentage of viable cells expressing GFP) was compared between cell pools resistant to either Tn or Hyg (Table 2). [Table 2]

[0131] Tunicamycin selection was observed to be as efficient as hygromycin selection. Both CHO-GPT and human GPT were efficient in selecting the recombinant in the presence of 1 μg / mL or 2.5 μg / mL tunicamycin.

[0132] (Example 2) Amplification of gene products Elevational selection was performed by applying increasing concentrations of tunicamycin to the GPT expression system. CHO K1 cells were transfused using a plasmid vector containing the CHO-GPT gene (SEQ ID NO: 2) as described above. The plasmid contains a first Lox site, a late SV40 promoter, the CHO-GPT gene, IRES, eGFP, and a second Lox site in the 5'-3' direction. The CRE-lox site directs the integration of the target gene into the genome, resulting in a stable transfectant pool of cells with at least one GPT insertion per cell. (More enucleations may occur due to random integration, as shown above). CHO cells were initially cultured in the presence of 1 μg / mL tunicamycin (Tn). Transfectants were then selected from the stable pool (designated cell pool 2) and subsequently expanded in the presence of 1 μg / mL, 2.5 μg / mL, or 5 μg / mL of Tn. Selection rounds were performed to identify cell populations with enhanced eGFP expression capabilities (multiple copies). Randomly incorporated events increased significantly in the presence of 2.5 μg / mL or 5 μg / mL of Tn.

[0133] The copy number of the gene product of either CHO GPT, eGFP, or mGapdh (normalized control) was measured using a standard qPCR method. The copy number of eGFP in cells from a 1 μg / mL Tn-resistant pool further incubated with 2.5 μg / mL Tn was at least twice the copy number of eGFP in cells from a 1 μg / mL Tn-resistant pool further incubated with 1 μg / mL Tn. When the 1 μg / mL Tn-treated pool was further incubated with 5 μg / mL Tn, the gene copy number increased further. The increase in gene copy number for eGFP correlated with the increase in gene copies for CHO-GPT. (See Figures 6A and 6B.)

[0134] To determine whether an increase in gene copy number leads to increased protein expression, mean fluorescence intensity (MFI) was measured by FACS for the same cell pool expressing GPT and eGFP, treated with multiple rounds of Tn selection, i.e., 1, 2.5, or 5 μg of Tn (see, for example, samples 7, 8, and 9 in Figure 6B). A comparison of eGFP expression for these cell pools is shown in Table 3.

[0135] The GPT-expressing cell pool that underwent a second round of selection with 5 μg of Tn resulted in a productivity output of more than 2.5 times greater for eGFP production compared to 1 μg of Tn treatment, and more than 1.5 times greater than 2.5 μg of Tn treatment (Table 3). [Table 3]

[0136] While not bound by any single theory, a gradual increase in Tn concentration amplifies selective pressure on cells in a controlled manner, thereby increasing productive output.

[0137] In the further experiments described below, a Tn resistance-expressing vector is used to test the effect of Tn selection on glycosylation patterns.

[0138] (Example 3) Exemplary dimeric protein expression and glycosylation profiles CHO cells expressing the "trap" protein (Fc fusion protein-1, hereafter referred to as FcFP1) are transfused with an expression vector containing GPT. The plasmid has a Lox site, an SV40 late promoter, a Tn resistance gene (CHO-GPT), IRES eGFP, SV40 polyA, and a second Lox site in the 5'-3' direction. 1 μg / mL Tn or 5 μg / mL Tn was used for GPT selection marker selection. Selected pooled cells are expanded in suspension culture in serum-free production medium. GPT transfection is confirmed by eGFP expression by FACS analysis. Pellets recovered from the selected pool were sent for copy number analysis for GPT expression, and a 12-day productivity assay was set up to determine the expression level of FcFP1 in the selected pool using different concentrations of tunicamycin.

[0139] FcFP1 was selected due to its complex glycosylation pattern, which includes an abundance of glycosylation sites. To determine the glycosylation profile, cells expressing the FcFP1 protein were expanded in cell cultures under a standard protocol (without Tn) or under Tn treatment conditions shown in Table 4, and the proteins were subsequently isolated and purified. [Table 4]

[0140] Detailed glycan analysis was performed using chromatography based on well-known methods for HPLC and fluorescent anthranilic acid (AA) tagging (Anumula, and Dhume, Glycobiology, 8(7):685-694, 1998) to determine whether Tn adversely affects the glycosylation profile of each lot of glycoprotein. Production lots were also compared to a reference standard representing a therapeutically acceptable batch of the protein. Representative glycan analyses are shown in Figures 7A–7D. Compared to the reference lot, each lot consistently produced the same number of peaks, relative shapes, and relative intensities. The superposition of each chromatogram (Figure 8) shows that no characteristic or abnormal peaks were revealed.

[0141] Oligosaccharide profiling for the FcFP1 protein was performed on a reference standard lot using a well-known HPLC method. The level of sialylation was measured for the FcFP1 trap protein lot, and a Z number was calculated for each lot (three iterations). The Z number represents the measurement of lot-to-lot variation. The Z number takes into account not only the relative number of peaks, but also the relative shape and intensity of each peak. For example, the area of ​​each peak 0SA, 1SA, 2SA, 3SA, and 4SA in Figures 7A to 7D is quantified as shown in Table 5. [Table 5]

number

[0142] The Z-numbers calculated for each lot were within an acceptable range compared to the reference lot, and therefore each protein lot was understood to achieve the same material as the therapeutic molecule. Since the presence of Tn is known to adversely affect N-linked glycoprotein glycosylation, it was unexpected that assuming the condition of increased selective pressure due to Tn would result in protein production that was not only reliable and consistent but also highly productive.

[0143] The present invention may be embodied in other specific embodiments without departing from its spirit or essence.

Claims

1. A method for employing tunicamycin (Tn) as a selective marker in mammalian cell culture, (a) A step of providing a population of mammalian host cells, (b) A step of introducing nucleic acids into the cell population of step (a) by transfection, wherein the nucleic acids include (i) a mammalian tunicamycin (Tn) resistance gene encoding a protein having at least 93% identity with the amino acid sequence of SEQ ID NO: 3, and (ii) a first target gene (GOI) encoding a first target protein (POI), (c) Culturing the cell population from step (b) in the presence of Tn, thereby selecting a population of cell transfectants, each containing the nucleic acid, (d) The steps of expressing the first POI from the first GOI in the selected population of cell transfectants and isolating the first POI A method comprising the first POI being an N-glycosylated protein, wherein the N-glycosylated protein is selected from an antibody light chain or its antigen-binding fragment, an antibody heavy chain or its antigen-binding fragment, or an Fc fusion protein.

2. A method for employing tunicamycin (Tn) as a selective marker in mammalian cell culture, (a) A step of providing a population of mammalian host cells, (b) A step of introducing nucleic acids into the cell population of step (a) by transfection, wherein the nucleic acids include (i) a mammalian tunicamycin (Tn) resistance gene encoding a protein having at least 93% identity with the amino acid sequence of SEQ ID NO: 3, and (ii) a first target gene (GOI) encoding a first target protein (POI), (c) Culturing the cell population from step (b) in the presence of a first concentration of Tn, thereby selecting a first population of cell transfectants, each containing the nucleic acid; (d) The step of culturing the selected first population in the presence of a second concentration of Tn higher than the first concentration, thereby selecting a second population of cell transfectants, each containing the nucleic acid, (e) expressing the first POI from the first GOI in the second population of cell transfectants and isolating the first POI; A method comprising, wherein the first POI is an N-glycosylated protein.

3. The method according to claim 2, wherein the culture in step (c) is cultured at a first concentration of Tn of 1 μg / mL, and the second concentration of Tn is 2.5 μg / mL or 5 μg / mL.

4. The method according to claim 1 or 2, wherein the Tn resistance gene encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

10.

5. The method according to claim 1 or 2, wherein the Tn resistance gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:

17.

6. The method according to any one of claims 2 to 5, wherein the N-glycosylated protein is selected from an antibody light chain or its antigen-binding fragment, an antibody heavy chain or its antigen-binding fragment, or an Fc fusion protein.

7. The method according to any one of claims 1 to 6, wherein the mammalian host cell population is a CHO cell population.

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