A new expression platform for stable, high-titer mass production of recombinant proteins

Abstract

An object of the present invention is to provide an expression platform comprising a DHFR-containing vector, at least one promoter, and DNA elements such as UCOE, IRES, or a combination thereof. Another object of the present invention is to provide a method for utilizing the expression platform to produce cells having a unique genetic background capable of robustly and high-level expression of a target protein, and a method for recovering the produced target protein in high yield.

Description

[Technical Field]

[0001] The present invention relates to an expression platform comprising an expression vector that provides high gene copy number and high productivity of recombinant proteins or peptides. In particular, the present invention relates to an expression platform comprising a vector containing a nucleotide sequence encoding a target protein or peptide, such as an antibody; a nucleotide sequence encoding dihydrofolate reductase (DHFR), which provides high gene copy number enhancement; a promoter selected from the CMV promoter, EF1α promoter, SV40 promoter, or a combination thereof; and a DNA element, such as a ubiquitous chromatin opening element (UCOE), which enhances random integration efficiency and effectively keeps the corresponding promoter active, thereby providing genetic stability and high-titer mass production of recombinant proteins. It also provides an internal ribosome entry site (IRES) that enables cap-independent translation initiation. Compared to several existing cell expression vectors, the vector platform of the present invention enables an effective method for producing single cells and clonal populations of cells with unique genetic backgrounds, which ensure robust production of recombinant proteins or peptides and high recovery rates upstream and downstream of the production process. [Background technology]

[0002] Recombinant production of proteins and peptides, including antibodies, in eukaryotic cells requires the creation of an expression system. Expression systems for producing recombinant proteins, such as biological therapeutics and biopharmaceuticals, generally consist of a nucleic acid vector construct encoding the desired recombinant protein and a selected host cell. The vector is introduced into the host cell, and the endogenous cellular machinery is utilized to produce the desired recombinant protein, e.g., a biological therapeutic. The complexities of establishing an efficient and reliable expression system for producing an appropriable biological therapeutic are manifold.

[0003] Many approaches exist for the design and construction of expression vectors, and the process usually requires considerable trial and error experimentation until reasonable levels of protein are produced. An important consideration in the design process concerns the use of intron sequences in vector construction.

[0004] One approach is to use the entire gene sequence as it naturally occurs, i.e., complete with both intron and exon sequences, in which case the post-transcriptional splicing machinery in the cell is expected to remove the intron sequences, resulting in a mature mRNA that contains only the exon sequences of the gene.

[0005] Another approach is to utilize nucleic acid sequences corresponding only to the cDNA of a gene, in which no splicing events occur and the pre-mRNA sequence is expected to be substantially identical to the mRNA sequence in its protein-coding content.

[0006] In yet another approach, vector construction involves selecting and placing introns that are not normally present in the original gene sequence. Vectors contain an "origin of replication," a stretch of DNA that allows the vector to be replicated (copied) in the host bacterium. Vectors often also contain promoter sequences, which allow the introduced gene to be expressed (protein produced).

[0007] Plasmids are extrachromosomal, self-replicating cytoplasmic (usually circular) DNA elements found in prokaryotes and, less commonly, in eukaryotes, where they are sometimes used as vectors. They come in a variety of forms, including simple plasmids used for direct transformation and fosmids for phage delivery. There are also many other forms of circular DNA, each with different desirable properties (e.g., large insert size, low copy number, phage compatibility, etc.). These vectors are sometimes called "high-capacity vectors" because of their larger insert size than simple plasmids.

[0008] Bacterial artificial chromosomes (BACs) contain several regions derived from specialized plasmids called F (fertility) factors. These regions contain the origin of replication and genes that ensure accurate segregation during bacterial cell division. A major advantage of BAC vectors is their large insert size (100-200 kb). However, the extremely large insert size presents a problem: it cannot be manipulated using restriction enzymes.

[0009] Yeast artificial chromosomes (YACs) have the capacity to clone fragments up to 3000 kbp. They are introduced into yeast cells by electroporation and are maintained as linear DNA like chromosomes. They are replicated within yeast along with other chromosomes, and the copy number remains at 1 even after cell division.

[0010] Several viruses (e.g., adenovirus, lentivirus, baculovirus) and bacteria (e.g., Agrobacterium tumefaciens) are reliable vectors for stable transfection of eukaryotic cells. Viral vectors are generally genetically engineered viruses that contain modified viral DNA or RNA that are noninfectious yet contain a viral promoter and a transgene, allowing transcription of the transgene under the control of the viral promoter. However, viral vectors often lack infectious sequences, requiring helper viruses or packaging cell lines for large-scale transfection. Because viral vectors are often designed to permanently integrate inserted genes into the host genome, they leave distinct genetic markers in the host genome even after transgene integration. For example, retroviruses leave characteristic retroviral integration patterns after insertion, which are detectable and indicate that the viral vector has integrated into the host genome.

[0011] The choice of expression system for recombinant protein production depends on many factors, including cell growth characteristics, desired expression level, intracellular or extracellular expression, post-translational modifications, and biological activity of the protein of interest, as well as regulatory and economic considerations in the production of therapeutic proteins. The primary advantages of mammalian cells over other expression systems, such as bacteria and yeast, are their ability to perform proper protein folding, complex N-linked glycosylation, authentic O-linked glycosylation, and a wide range of other post-translational modifications. Because of these advantages, eukaryotes, particularly mammalian cells, are now the expression platform of choice for producing complex therapeutic proteins.

[0012] Cell culture research is widely used in pharmaceutical, medical, and biotechnology research. To produce recombinant proteins, cell culture conditions must be standardized to ensure optimal performance and stability of the cell culture. Various parameters and conditions during cell culture must be constantly controlled to ensure proper cell growth and optimal production of the desired recombinant protein.

[0013] As mentioned above, mammalian cells have become the primary system for producing recombinant protein products for clinical applications due to their ability to properly fold, assemble, and modify proteins in a manner similar to human proteins. In fact, all cell lines used to date for the production of biopharmaceutical proteins are derived from mammals (Non-Patent Document 1). However, the development of mammalian cell lines is often very time-consuming. Furthermore, mammalian cell culture processes are hampered by low yields and unstable cell expression (Non-Patent Document 2). Productivity and expression stability are prerequisites for developing commercially viable processes. Therefore, the ultimate goal of cell line development is to obtain a clonal cell line that secretes the protein of interest at a high specific productivity (Qp) and at consistently high levels over extended cell generations, allowing for scale-up and cost-effective production. Expression vectors and cell line engineering are key to achieving this goal.

[0014] The challenge of protein synthesis is the high production costs due to low production yields. The production yield of a clone depends on the selection of several factors, including external factors such as culture conditions (e.g., medium components, temperature, pH) and downstream purification steps, and internal factors such as the selection of vectors and their control elements, including promoters, transcriptional or translational enhancing elements, and their appropriate orientation, as well as the selection of appropriate host cells. Mammalian cells, with their natural glycosylation capabilities, are the most promising expression systems for achieving high expression of recombinant therapeutic proteins. Furthermore, post-translational modifications in such expression systems are likely to be similar to those found in proteins expressed in human cells, thus ensuring the appropriate physiological activity of the recombinant protein. However, expression levels in eukaryotic cells also depend heavily on another internal factor: the site at which the recombinant expression construct constituting the gene of interest is integrated into the host cell genome.

[0015] As mentioned above, mammalian cell culture is the preferred technology in industry for overexpressing target proteins. This is because most industrially valuable proteins are derived from humans or animals, and certain protein modification mechanisms (glycosylation, phosphorylation, amidation) are easily carried out in animal cells. Known limitations of this technology include the high cost of protein production and the time and expense required for cell line development (Non-Patent Document 3). Currently, the animal cells used in industry are Chinese hamster ovary (CHO) cells, baby hamster kidney (HBK) cells, and myeloma cells. Target proteins are expressed by transfecting the cells with expression vectors.

[0016] CHO cells are an epithelial cell line commonly used in biotechnology research and commercially for the production of recombinant therapeutic proteins. They are used in genetics, toxicity screening, nutrition, gene expression studies, and recombinant protein expression. CHO cells are the most widely used mammalian host for the industrial production of recombinant protein therapeutics (Non-Patent Document 4).

[0017] CHO cells can produce proteins with complex glycosylation and post-translational modifications (PTMs) similar to those produced in humans. CHO cells are easy to culture on a large scale and have high viability, making them ideal for GMP protein production. In addition, CHO cells tolerate changes in parameters such as oxygen level, pH, temperature, and cell density (Non-Patent Document 5). Most genetic manipulations performed on CHO cells are performed in cells lacking the DHFR enzyme.

[0018] Gene amplification is a strategy routinely used in animal cell expression systems. There are two commonly used amplification systems: DHFR-based amplification and glutamine synthetase-based amplification. Both significantly increase recombinant protein yields in animal cell lines. Despite their advantages in improving protein production, gene amplification systems have the disadvantage of being time-consuming due to the need for multiple rounds of gene amplification and the use of high concentrations of methotrexate (MTX). Prolonged subculture of cell lines can lead to gene loss and unstable expression.

[0019] Despite some challenges, selection schemes based on DHFR-deficient cells remain one of the standard methods for generating transfected CHO cell lines for the production of recombinant therapeutic proteins.

[0020] Non-Patent Document 6 and Patent Document 1 report that when a foreign gene is inserted adjacent to the DHFR gene in an expression vector, the foreign gene is co-expressed at a high level in animal cells. Gene amplification in CHO cells begins with molecular cloning of the gene of interest and the DHFR gene into a single mammalian expression system. The cells are then transfected with plasmid DNA carrying the two genes and grown under selective conditions in thymidine-free medium. Cell lines vary widely in growth rate and recombinant protein production levels. It may be necessary to evaluate hundreds of candidate cell lines to obtain several stably transfected cell lines with the desired phenotypic characteristics (Non-Patent Document 7 and Non-Patent Document 8).

[0021] Current methods for generating mammalian cell lines for expressing recombinant proteins suffer from several drawbacks (Non-Patent Document 9). Episomal systems allow high levels of recombinant protein expression but are often only stable for a short period of time (Non-Patent Document 10). Mammalian cell lines containing integrated foreign genes are somewhat stable, but there is growing evidence that stability depends on the presence of only a few copies, or even one copy, of the foreign gene.

[0022] The efficiency of the system itself depends on a variety of factors, including the design of the vector and the choice of host cell. A strategic combination of regulatory elements, selectable markers, and stability elements within the vector sequence must balance the ease of vector manipulation and application with the need for high-yield production of the desired biological therapeutic. Developing methods to engineer host cells that stably express recombinant proteins at high levels for long periods of time presents a particular challenge.

[0023] Despite these advances, protein expression levels in mammalian cells are relatively low and often unstable during development, making the development and production of therapeutic proteins expensive.

[0024] Current biomanufacturing processes require cell lines that can grow robustly in suspension culture without clumping, stably and productively integrate heterologous DNA, produce high concentrations of recombinant proteins in a given system, and perform desired post-translational modifications with uniform product characteristics (Non-Patent Document 10, Non-Patent Document 11).

[0025] Additionally, the availability of a suitable expression system and the ability to rapidly obtain high-yielding clones may also influence the choice of cell line. To be approved by regulatory authorities, production cell lines must be well characterized and genetically stable. For example, glycosylation of a given protein can vary depending on the mammalian cell type used, and two subclones from the same parent line may have significantly different metabolic requirements.

[0026] Although the characteristics of regulatory elements and internal factors used in expression vectors are well understood, it is not possible to absolutely predict whether a combination of these elements will reliably and efficiently achieve high expression of a target protein. Some combinations result in significantly poorer expression than others. For example, as reported in Non-Patent Document 12, an expression vector consisting of the SRα promoter, an AMY RNA leader sequence, and DHFR achieved erythropoietin (EPO) expression of only 45 IU / ml (equivalent to 0.346 μg / ml). Patent Document 2 reports EPO levels of 750-1470 U / million cells / 48 hours (or 375-735 U / million cells / 24 hours) using an expression vector consisting of a different element combination, i.e., SV40, a polyadenylation (polyA) sequence, and DHFR. Patent Document 3 reports that yet another expression vector consisting of the EF-1 promoter and an apoB SAR element achieved expression of 1500-1700 IU of EPO / million cells / 24 hours. For other recombinant proteins such as TNFR-IgGFc (Enbrel), an expression vector containing a combination of a CMY promoter, TPL, YA I&II, and DHFR has been reported (Patent Document 4). [Prior art documents] [Patent documents]

[0027] [Patent Document 1] U.S. Patent No. 4,656,134 [Patent Document 2] U.S. Patent No. 5,955,422 [Patent Document 3] U.S. Patent No. 5,888,774 [Patent Document 4] U.S. Patent No. 5,605,690 [Non-patent literature]

[0028]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

[0029] Surprisingly, despite the vast knowledge generated in this field over the past two decades, even today, those skilled in the art cannot simply select a combination of internal factors and regulatory elements to design an expression vector that guarantees high expression of a protein or peptide of interest. Combining certain elements with others may not have a significant additive or synergistic effect on the vector's expression potential. Therefore, the process of developing novel expression vectors that result in high levels of protein expression is challenging, requiring empirical testing of many possibilities. Therefore, there is a need for improved cell culture processes for recombinant protein production, especially for the large-scale production of therapeutic proteins that are stable over long periods of time. [Means for solving the problem]

[0030] In the development of cell lines for producing biological products, three parameters are particularly important: stability, productivity, and quality. These three parameters can be controlled by the mammalian expression vector used to introduce a gene of interest into a host cell. Therefore, the present invention aims to design and construct a mammalian expression vector containing elements that will provide good results in accordance with these parameters.

[0031] We developed an expression platform that can obtain large amounts of target proteins using a recombinant vector containing the human DHFR gene operably linked to the mouse DHFR promoter. This system can effectively amplify target genes at lower concentrations of MTX.

[0032] The vectors of the present invention enable efficient selection of cell line clones containing the amplified DHFR gene and target gene under very low concentrations of MTX, compared to existing animal cell expression vectors. Indeed, it has been determined that some scale-up processes according to the present invention do not require the use of MTX. In the examples provided herein, MTX was used only in the limiting dilution cloning (LDC) step. Thus, the cell culture process can be carried out in the presence of low levels of methotrexate (MTX) or in the absence of MTX. The cell culture process refers to cell line production, followed by process and medium optimization in small-scale systems, including shaker flasks and bench-scale bioreactors, and the scale-up process.

[0033] Some vectors of the present invention contain an IRES element, which allows expression of two proteins from a single mRNA, which may be important for protein yield. The CMV promoter is susceptible to gene silencing in CHO cells by epigenetic events such as DNA methylation and histone modifications.

[0034] In some embodiments, the present disclosure provides a vector design system for improving expression and / or production of a protein or peptide of interest. In some embodiments, the protein or peptide is a recombinant protein or peptide. In some embodiments, the protein of interest is an antibody.

[0035] In another aspect, the disclosure provides recombinant nucleic acids encoding proteins or peptides of interest, and the genetic elements necessary for expression of the proteins or peptides of interest in host cells.

[0036] In another aspect, the disclosure provides expression vectors for producing proteins or peptides, including antibodies. In another aspect, the present invention provides expression vectors for generating stable cells that express recombinant proteins or peptides.

[0037] In another aspect, the present disclosure provides a host cell, e.g., a eukaryotic host cell, comprising one or more of the aforementioned nucleic acid molecules and / or vectors, e.g., expression vectors. The host cell can be transiently or stably transfected with the nucleic acid sequence of the present invention. The cell can be a mammalian cell, e.g., a CHO cell.

[0038] In another aspect, the present disclosure provides a system for culturing such cells according to the present disclosure for large-scale production of recombinant proteins or peptides. In another aspect, the present disclosure provides a selection system for selecting host cells that express a protein or peptide of interest, thereby allowing for high yields of the protein or peptide of interest.

[0039] In another aspect, the present disclosure provides an expression system for producing a recombinant product, such as a protein, peptide, antibody, etc., comprising a host cell disrupted with a vector according to the present disclosure. The vector of the present disclosure may be an expression vector and may contain one or more of the modified nucleic acid elements described above.The vector may further contain a nucleotide sequence that enhances one or more of replication, selection, mRNA transcription, mRNA stability, protein expression, or protein secretion in a host cell.For example, the vector may contain a nucleotide sequence responsible for replication or enhancer expression, an enhancer promoter element, a nucleotide sequence encoding a leader sequence, a gene encoding a selectable marker (e.g., DHFR), an internal ribosome entry site sequence (IRES), and a polyadenylation sequence.

[0040] In some embodiments, vectors (eg, expression vectors) are modified to reduce or eliminate mis-splicing and / or intron read-through by-products and / or to enhance expression of recombinant proteins.

[0041] In another aspect, the present disclosure provides vector designs comprising nucleic acid sequences encoding antibodies (e.g., recombinant antibodies) or fragments thereof with reduced (e.g., substantially free) of mis-splicing products and / or intron read-through products for long-term, stable, large-scale production of therapeutic proteins.

[0042] In another aspect, the present disclosure utilizes novel element combinations to develop novel vector platforms, resulting in the synergistic effects of these elements leading to high expression of desired proteins.

[0043] In some embodiments, the expression vector of the present disclosure comprises the following operably linked elements: a nucleotide sequence encoding one or more proteins or peptides of interest; one or more terminator sequences; one or more promoters; a nucleotide sequence encoding a chromatin opening element (UCOE), an internal ribosome entry site (IRES), or a combination thereof; and A nucleotide sequence encoding dihydrofolate reductase (DHFR) as a selectable marker.

[0044] In some embodiments, the expression vector is a bicistronic or dual promoter vector, where the promoters may be the same or different from each other.

[0045] In some embodiments, the protein or peptide of interest is a monoclonal antibody. In some embodiments, the protein or peptide of interest is an antibody light chain (LC) and an antibody heavy chain (HC). In some embodiments, the expression of the LC is controlled by a first promoter, and the expression of the HC is controlled by a second promoter.

[0046] In some embodiments, the DHFR is human DHFR and the nucleotide sequence encoding human DHFR is operably linked to a DHFR promoter from a mouse. In some embodiments, the first promoter and the second promoter are each independently a CMV promoter or an EF1-α promoter.

[0047] In some embodiments, the UCOE comprises the sequence of SEQ ID NO:4. In some embodiments, the nucleotide sequence encoding the IRES comprises the sequence of SEQ ID NO:6.

[0048] In some embodiments, the DHFR comprises the sequence of SEQ ID NO:8. In some embodiments, the expression vector further comprises a first multiple cloning site (MCSI) and a second multiple cloning site (MCSII).

[0049] In some embodiments, the one or more terminator sequences are a polyA signal sequence and a polyA SV40 terminator sequence. In some embodiments, the elements in the expression vector are arranged in the following order from 5' to 3': UCOE, EF1-α promoter, MCSI, a nucleotide sequence encoding an IRES, MCSII, a polyA signal sequence, an SV40 terminator sequence, a nucleotide sequence encoding DHFR, and a polyA signal sequence.

[0050] In some embodiments, the elements in the expression vector are arranged in the following order from 5' to 3': UCOE, CMV promoter, MCSI, a nucleotide sequence encoding an IRES, MCSII, a polyA signal sequence, an SV40 terminator sequence, a nucleotide sequence encoding DHFR, and a polyA signal sequence.

[0051] In some embodiments, the SV40 terminator sequence comprises the sequence of SEQ ID NO:1. In some embodiments, the present disclosure provides a cell or population of cells transfected with an expression vector of the present disclosure. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell.

[0052] In some embodiments, the present disclosure provides mammalian cell culture processes for growing cells of the present disclosure, carried out in the presence of methotrexate (MTX) or in the absence of MTX.

[0053] In some embodiments, the present disclosure provides methods of producing a protein or peptide of interest by culturing a cell of the present disclosure under conditions that allow expression of the protein or peptide of interest in a culture medium.

[0054] In some embodiments, the cells of the present disclosure are clones that stably express a protein or peptide of interest. In some embodiments, the protein or peptide of interest is a monoclonal antibody, an antibody heavy chain, an antibody light chain, or a combination thereof.

[0055] definition In the context of the present invention, words such as "comprises" are to be interpreted in an inclusive rather than an exclusive sense.

[0056] As used herein, the term "stable," when used in reference to a genome, means that the information content of the genome is stably maintained from one generation to the next, from one passage to the next. Thus, a genome is considered stable if no significant changes (e.g., gene deletions or chromosomal translocations) occur in the genome. The term "stable" does not exclude subtle changes that may occur in the genome, such as point mutations.

[0057] "Operably linked" refers to the juxtaposition of two or more components, wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter and / or enhancer is operably linked to a coding sequence when it acts in cis to control or regulate the transcription of the linked sequence. Generally, "operably linked" DNA sequences are contiguous, and, where necessary to join two protein-coding regions, such as a secretory leader and a polypeptide, contiguous and in reading frame.

[0058] The "selectable marker" expressed by the introduced polynucleotide allows for the selection of host cells expressing said selectable marker under appropriate selective culture conditions. The selectable marker is preferably a biomolecule, particularly a polypeptide.

[0059] A "vector" according to the present invention is a polynucleotide capable of carrying at least one polynucleotide fragment. The term "expression vector" includes a specific type of vector in which a nucleic acid construct is optimized for high-level expression of a desired protein product. Expression vectors often contain transcriptional regulatory elements, such as promoters and enhancer elements, optimized for high-level transcription in a specific cell type and / or optimized for constitutive expression based on the use of a specific inducer. Expression vectors also contain sequences that provide for proper and / or enhanced translation of the protein. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses, and retroviruses. The term "expression cassette" refers to a separate component of vector DNA consisting of genes and regulatory sequences that are expressed by a transformed cell. Upon successful transformation, the expression cassette directs the cellular machinery to make RNA and protein. Vectors function like molecular carriers, delivering each nucleic acid fragment of a polynucleotide into a host cell. A vector may contain at least one expression cassette consisting of regulatory sequences for proper expression of a polynucleotide incorporated therein. A polynucleotide to be introduced into a cell (eg, encoding a product of interest or a selectable marker) can be inserted into an expression cassette of a vector for expression therefrom.

[0060] As used herein, the term "DHFR (dihydrofolate reductase)" refers to an enzyme that reduces dihydrofolate to tetrahydrofolate, and is a key enzyme for nucleic acid synthesis and essential for cell growth.

[0061] As used herein, the term "intron" includes a segment of DNA that is transcribed but removed from the RNA transcript by splicing together the sequences on either side of it (exons). Introns are considered to be intervening sequences within the protein-coding region of a gene and generally do not contain information that is expressed in the protein produced from the gene.

[0062] The term "antibody" refers to a protein having a four polypeptide chain structure consisting of two heavy chains and two light chains, the chains being stabilized, for example, by interchain disulfide bonds, and the immunoglobulin or antibody having the ability to selectively or specifically bind to an antigen.

[0063] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope.

[0064] The term "commercially available" may also refer to commercially available CHO cell lines (e.g., CHO cell lines manufactured by CATALENT). The term "region" can also refer to portions or parts of an antibody chain or antibody chain domain (e.g., portions or parts of a heavy or light chain, or constant or variable domain, as defined herein), as well as more discrete portions or parts of said chains or domains. For example, light and heavy chain or light and heavy chain variable domains contain "complementarity determining regions" or "CDRs" interspersed among "framework regions" or "FRs", as defined herein.

[0065] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, and includes the progeny of such a cell. The term "SV40 terminator sequence" refers to the SV40 polyA region. The SV40 polyA is a region of the SV40 (simian virus 40) genome where transcription from both directions terminates. Thus, this region functions as a transcription terminator and a polyA signal. [Effects of the Invention]

[0066] The present invention describes recombinant protein expression vectors for generating stable cells. This unique modification is particularly suited for the production of recombinant proteins, particularly monoclonal antibodies, but can be used for any protein of interest. Introns and exons are nucleotide sequences within a gene. Introns are removed by RNA splicing as the RNA matures and are no longer expressed in the final messenger RNA (mRNA) product, whereas exons are covalently linked to each other to create the mature mRNA. The nucleic acid molecule reduces or eliminates intron-leading-edge (IRT) byproducts of the desired protein or peptide compared to naturally occurring sequences. Furthermore, expression vectors of the present invention with introns and exons whose natural operational relationships have been altered not only reduce or eliminate IRT byproducts, but also exhibit increased stability and protein expression levels compared to vectors designed using standard, art-recognized techniques.

[0067] The features and advantages of the present invention can be summarized as follows: (1) The vector of the present invention is unique in the combination of regulatory elements it contains, thus ensuring optimal production of recombinant proteins.

[0068] (2) Compared with currently available animal cell expression vectors, the vector of the present invention enables effective selection of cell line clones carrying an amplified DHFR gene and a foreign gene under very low concentrations of MTX.

[0069] (3) The present invention has the advantageous effect of allowing the use of lower concentrations of MTX, thereby reducing costs by improving cell growth rates and productivity. (4) Unlike existing commercially available products, the present invention provides good results in terms of biosimilarity, which is not always possible with existing products.

[0070] (5) In terms of stability, the risk of this invention is very low. (6) Existing commercial systems may require production using proprietary media and supplements. This situation impacts costs and makes it difficult to create a process that fits within a working budget. However, the present invention overcomes this drawback and allows the use of various media and supplements.

[0071] In the present invention, even if random integration occurs, the UCOE improves integration efficiency and stability. [Brief explanation of the drawings]

[0072] [Figure 1] Schematic diagram of a dual promoter vector. [Figure 2] Schematic diagram of bicistronic vectors. [Figure 3] Secondary structure of the IRES complex. [Figure 4] CMV promoter sequence (SEQ ID NO:3). [Figure 5] Structure of the A2UCOE. [Figure 6] A2UCOE sequence (SEQ ID NO: 4). [Figure 7] IRES sequence (SEQ ID NO: 5). [Figure 8] A sequence fragment showing the ATG-11 and ATG-12 translation initiation sites of the IRES (SEQ ID NO: 7). [Figure 9] Charge profile (CEX) graph comparing the change profiles of the originator molecule, the molecule produced in the Catalent cell line, and the molecule produced in Example-1E. [Figure 10] Charge profile (cIEF) graphs without and with CpB compare the change profiles of the originator molecule and the molecule prepared in Example-1E. [Figure 11] EF1α sequence (SEQ ID NO: 2). [Figure 12] SV40 terminator sequence (SV40) (SEQ ID NO: 1). [Figure 13] DHFR sequence (SEQ ID NO: 6). DETAILED DESCRIPTION OF THE INVENTION

[0073] [Host cell] The mammalian host cells of the present invention can be any of those commonly used in the art to express recombinant proteins, polypeptides, or peptides. For example, the host cells can be Chinese hamster ovary (CHO) cells, such as CHO-K1, CHO-DG44 DHFR-, and CHO-S. These include both adherent and suspension cell lines.

[0074] Chinese hamster ovary cell lines are commonly used as hosts for recombinant protein production in both research and the biotechnology industry. Recombinant cell lines are generated by randomly integrating a multicistronic plasmid vector containing a gene of interest and a selectable marker gene into the host genome. Recombinant cell lines require several rounds of limiting clonal dilution to isolate stable, high-expressing clones. Stable, high expression of a gene of interest is a rare and desirable trait in recombinant clonal cell lines. Despite being clonal, cell lines can eventually become heterogeneous and lose productivity.

[0075] [vector] Any suitable expression vector can be used in the present invention. Preferably, dual promoter and bicistronic vectors are used in the present invention.

[0076] A schematic diagram of a dual promoter vector is shown in Figure 1. The expression of two different target genes is controlled by two different promoters. When a dual promoter vector is used to express an antibody, the expression of the heavy and light chains may be controlled by two different promoters.

[0077] A schematic diagram of a bicistronic vector is shown in Figure 2. The expression of two different target genes is controlled by the same promoter. When expressing an antibody using a bicistronic vector, the expression of the heavy and light chains is controlled by the same promoter region. To control the heavy and light chains with a single promoter, an IRES (internal ribosome entry site) sequence can be used in the bicistronic vector model.

[0078] [Vector design incorporating regulatory elements] The following section describes some of the DNA elements that can be used in the expression system of the present invention. Other suitable DNA elements with the same or similar function known to those of skill in the art can also be used in embodiments of the present invention.

[0079] [promoter] Below are some examples of promoters that may be used in the present invention: As will be appreciated by those skilled in the art, other suitable promoters may also be used.

[0080] Simian virus 40 promoter Mammalian expression plasmids are primarily used to produce mRNA. Commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) contain the sequence motif AAUAAA, which promotes both polyadenylation and termination. Among these, SV40 late poly(A) and rbGlob poly(A) are thought to be more efficient at terminating transcription due to the presence of additional helper sequences. Poly(A) sequences generally promote transcript stability and degradation in eukaryotes and prokaryotes, respectively. On the other hand, SV40 sequences are terminator sequences that signal the end of the transcription unit. Terminator sequences are also involved in RNA processing and stability. To achieve high levels of expression, the gene of interest is usually placed downstream of a strong viral promoter, such as the simian virus 40 (SV40) terminator sequence (SEQ ID NO: 1) or the cytomegalovirus (CMV) promoter (SEQ ID NO: 3). The CMV promoter is a popular choice because it is a relatively strong promoter compared to the SV40 terminator sequence and is common in commercially available plasmids, although other promoters, such as the human elongation factor 1 alpha (EF-1α) promoter and the Chinese hamster elongation factor 1 (CHEF-1) promoter, are stronger.

[0081] Human EF1α promoter Human EF1α (EF-1α, gene symbol EEF1A1) (SEQ ID NO: 2) is a constitutively active promoter in a wide variety of cell types. Several studies have shown that promoters of endogenous mammalian genes, such as EEF1A1, may be more resistant to silencing than viral promoters. The EF-1α promoter used with the flanking regions of the CHO EF-1α gene is more active in CHO cells compared to the CMV and SV40 promoters (Non-Patent Document 3).

[0082] CMV promoter The CMV promoter (SEQ ID NO: 3) is the most widely used promoter in the biopharmaceutical industry in customized and commercially available vectors due to its continuous and high expression. However, the CMV promoter can be silenced over time in some cell types, causing heterogeneity among transfected cells (Non-Patent Documents 4 and 5). To prevent transcriptional repression by methylation, a core CpG island element (IE) is incorporated into the promoter (Non-Patent Document 7).

[0083] [Ubiquitous Chromatin Opening Element (UCOE)] UCOEs are cis-acting epigenetic regulatory elements derived from the promoter regions of housekeeping genes. These are methylation-free CpG islands that reduce DNA methylation, preventing heterochromatin formation and transgene silencing (Non-Patent Document 8).

[0084] The A2UCOE region from the human HNRPA2B1-CBX3 locus is one of the most efficient UCOEs, and its incorporation into an expression vector has been shown to increase the expression level of transgenes in mammalian cells (Non-Patent Document 13 and Non-Patent Document 16). The A2UCOE having the sequence of SEQ ID NO: 4 is an example of an A2UCOE that can be used in the present invention. Other suitable chromatin opening elements can also be used in embodiments of the present invention.

[0085] [IRES (internal ribosome entry site)] The EMCV (encephalomyocarditis virus) IRES is a non-coding RNA fragment that can initiate high levels of cap-independent protein synthesis in mammalian cells and cell-free extracts. The advantage of using an IRES element is that it allows the expression of two genes from a single mRNA: the first gene is expressed by cap-dependent translation, and the second gene is expressed by cap-independent binding of ribosomes to the IRES sequence. The IRES sequence creates a complex secondary structure that allows mammalian ribosomes to bind and initiate translation. The secondary structure of the IRES is shown in Figure 3.

[0086] An IRES having the sequence of SEQ ID NO: 6 is one example of an IRES that may be used in the present invention. Other suitable IRES sequences may also be used in embodiments of the present invention. [Selection System] The selectable marker incorporated into the plasmid vector along with the recombinant protein gene is usually either glutamine synthetase (GS) or dihydrofolate reductase (DHFR). These are two well-characterized genetic selection approaches commonly used in CHO cell lines. The plasmid vector carrying the recombinant gene and the selectable marker is introduced into cells by transfection, and the cells are grown under selective conditions (in the absence of hypoxanthine and thymine (-HT) for DHFR, or in the absence of glutamine for GS). Each surviving clone has at least one copy of the selectable marker gene integrated into its genome along with the recombinant protein gene.

[0087] [Dihydrofolate reductase (DHFR)] The DHFR gene is widely used as a selectable marker in mammalian expression systems because it provides a method for amplifying transgenes. DHFR is a key enzyme in folate metabolism. It contributes to the de novo mitochondrial thymidylate biosynthetic pathway and catalyzes de novo glycine and purine synthesis, reactions required for DNA precursor synthesis. DHFR is a common selectable marker gene used in mammalian cells, more specifically, DHFR-deficient Chinese hamster ovary (CHO) cells such as DG44 and DXB11.

[0088] DHFR of SEQ ID NO: 6 is one example of a DHFR that may be used in the present invention. Other suitable DHFR sequences may also be used in embodiments of the present invention. The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited thereto. Those skilled in the art may make various changes or modifications based on the description of the present invention, and these changes or modifications are also included in the present invention.

[0089] [Example] Example 1: Vector design The parameters targeted in the mammalian cell line development process and the elements used to achieve these goals are listed in Table 1.

[0090] [Table 1]

[0091] The designed expression vector model was obtained by incorporating the obtained sequence into template DNA. The vector was designed using the GeneART Webportal online vector design program. GeneArt Webportal contains sequence information for elements found on its portal.

[0092] [Table 2]

[0093] Example 1A: The Example 1A vector mapped above contains two CMV promoters (SEQ ID NO: 3). A UCOE element was placed at the 5' end of both promoters. This model is the simplest design, but since it contains two identical elements and promoters, it may undergo homologous recombination within the genome.

[0094] Example 1B: The Example 1B vector has two EF1α promoters (SEQ ID NO: 2). Since it contains two identical elements and promoters, there is a possibility of homologous recombination.

[0095] Example-1C: Two different promoters, CMV and EF1α, were used in the Example-1C vector. Example 1D: Two different promoters, CMV and EF1α, were used in the Example 1D vector. The CMV promoter is prone to silencing. EF1α contains an intron region, making it more resistant to silencing. Therefore, in the Example 1D vector model, a UCOE element was used only in front of the CMV promoter, assuming that EF1α would remain active.

[0096] Example 1E: The Example 1E vector contains one CMV promoter (SEQ ID NO: 3). An IRES element was inserted between two multiple cloning sites (MCS1 and MCS2). An antibody was used as the target protein. Both the heavy chain (HC) and light chain (LC) were expressed under the control of the CMV promoter. The LC was cloned into MCS1 and the HC into MCS2, with an LC:HC ratio of 1 or greater.

[0097] Example 1F: The Example 1F vector contains one EF1α promoter (SEQ ID NO: 2). Using an IRES element, both the HC and LC were expressed under the control of the EF1α promoter. An antibody was used as the target protein. LC was cloned into MCS1 and HC into MCS2, with the LC:HC ratio at 1 or greater.

[0098] A2UCOE The A2UCOE sequence, SEQ ID NO: 4, was obtained from the NCBI Epigenome Browser (accession number: NC_000007.13)

[10] . The structure of A2UCOE is shown in Figure 5. A2UCOE (1.5 kb) contains regions +309 bp from the transcription start site of CBX, +475 bp from the transcription start site of HNRPA2B1, and the intron region between the CBX and HNRPA2B1 genes.

[0099] The A2UCOE of SEQ ID NO: 4 was used in the examples provided herein. IRES The EMCV wild-type IRES (IRESwt) of SEQ ID NO: 5 is a sequence corresponding to nucleotides 260 to 848 of the EMCV-R genome (Genbank: M81861, NC_001479; Non-Patent Document 15). In this example, the IRESwt sequence of SEQ ID NO: 5 was used.

[0100] [Getting the designed vector model] The antibody HC was inserted after the IRESwt using the NcoI restriction site in the 5' region. Translation can be initiated from either ATG-11 or ATG-12 (SEQ ID NO: 7). However, when translation is initiated from ATG-11, four extra amino acids are added to the N-terminus of the signal sequence, forming the "MAAT" site, which does not affect cleavage of the signal peptide from the correct position. This was confirmed using Nucleofector Kit V and program U-24 on the Nucleofector I system (Lonza, Cologne, Germany). Specifically, mAb heavy chain analysis was performed on the serum albumin proprotein signal sequence in the MAAT region. The results showed that cleavage occurred from the correct position, before the first two amino acids of the mAb heavy chain, QV.

[0101] UCOE composition in the vector: Non-Patent Document 14 tested different configurations of the UCOE element depending on the position of the heavy and light chains in the vector. The 5'LC 5'HC and 3'HC 5'LC 5'HC configurations were shown to provide the highest efficiency. Based on this information, the UCOE element was inserted at the 5' end of the promoter.

[0102] DHFR: In this example, DHFR of SEQ ID NO: 6 was used. Example 2. Transformation, vector transfer, and protein isolation Host cells that have not successfully integrated a vector or vector combination according to the present invention preferably die or have impaired growth under selective culture conditions compared to host cells that have successfully integrated a vector or vector combination according to the present invention. During selection, host cells that have successfully integrated a vector or vector combination can be enriched as a pool from a population of transfected host cells. Individual host cells can be isolated from a population of transfected host cells during selection and expanded, for example, by clonal selection.

[0103] In the cell culture process, the target protein expressed in the host cells is secreted into the culture medium. Large amounts of the target protein can be obtained by purifying the secreted protein. The purification step in the present invention can include conventional purification methods known to those skilled in the art, such as solubility fractionation using ammonium sulfate or PEG, ultrafiltration, molecular weight fractionation, fractionation by various chromatographic methods (e.g., based on size, charge, hydrophobicity, or affinity), or a combination thereof.

[0104] Once the target amino acids of the gene sequence were designed, the codons were optimized for CHO tRNA preference and submitted to Twist Bioscience for synthesis. In this example, an antibody was used as the target protein. The weights of the heavy and light chains and the absorbance of the Twist vector were measured by gel electrophoresis. The vector was cleaved at the selected restriction sites (incubated with the restriction enzyme at 37°C for 1 hour and inactivated at 80°C for 2 hours). First, the light chain was cloned and extracted from the gel. Ligation was performed. The ligation products and controls were transformed into competent Top10 E. coli. Different vector clones were pooled and inoculated into separate culture vessels with 10 ml of growth medium. Cultures were grown overnight at 37°C and 225 rpm. Miniprep plasmid isolation was performed. After miniprep plasmid isolation, the concentration and absorbance of each clone were measured. Plasmids isolated from transformed colonies were digested with the appropriate restriction enzymes. The insert and digestion products were confirmed by agarose gel electrophoresis. PCR using specific primers was used as a secondary control. Finally, diagnostic digestion with appropriate restriction enzymes was performed. For large-scale production, selected clones were grown overnight in 100% growth medium at 37°C with shaking at 225 rpm. After this, the plasmid was isolated by maxiprep and its concentration was measured.

[0105] Example 3: Transfection and random integration Vectors carrying nucleic acid sequences encoding the heavy and light chains of monoclonal antibodies were introduced into E. coli cells by bacterial transformation. The transformed bacterial cells were grown by culturing in selective agar medium. This process was performed separately for each vector. The vectors expressing the monoclonal antibodies were linearized by digestion with the appropriate restriction enzymes. The appropriate restriction sites were located in the ampicillin resistance gene and are no longer needed after transformation.

[0106] Example 4: Selection of cells expressing an antibody of interest HT selection and MTX gene amplification HT selection and MTX gene amplification are performed using methods known in the art. MTX can be used to increase the gene copy number of DHFR, which often results in co-amplification of the transgene for the recombinant protein of interest, thereby increasing overall protein productivity. MTX amplification can be performed in a single step or multiple times by gradually increasing the concentration of MTX added to the selection medium.

[0107] DHFR-deficient CHO cells are transfected with recombinant DNA containing a gene of interest closely linked to a nucleotide sequence encoding DHFR. To select for CHO cells producing the protein of interest, an MTX selection system is used. MTX is a folate-like drug that binds to DHFR and inhibits the production of tetrahydrofolate, a compound required for the de novo synthesis of purines and pyrimidines. During gene amplification, CHO cells are cultured in increasingly higher concentrations of MTX. CHO cells carrying increasing numbers of copies of the DHFR gene combined with the gene of interest are selected. CHO cells with insufficient levels of DHFR are deprived of nucleoside precursors (hypoxanthine and thymidine) and die. Once selected, transfected cell lines derived from CHO DHFR-negative hosts do not require MTX in the culture medium.

[0108] DHFR-deficient strains require supplementation with glycine, hypoxanthine, and thymidine. These strains were used to demonstrate that an exogenous DHFR gene can be stably transfected and selected for in otherwise DHFR-deficient cells using glycine / hypoxanthine / thymidine-deficient (GHT-minus) medium. This selection method has become the standard for establishing stable transfection in CHO cell lines intended for the production of therapeutic proteins. The gene expressing the protein of interest and the DHFR gene are combined in a single mammalian expression vector or placed in separate vectors. The plasmid or plasmids are then transfected into CHO cells, and the cells are cultured in GHT-minus medium to provide a selective environment. As a result, all surviving cells have integrated copies of both the DHFR gene and the gene of interest into their genomes. DHFR of SEQ ID NO: 8 was used in this example.

[0109] Glutamine synthetase (GS) selection and methionine sulfoxamine (MSX) amplification CHO cells lacking GS can be transfected with recombinant DNA containing a gene of interest closely linked to the GS gene. CHO cells producing the protein of interest are then selected using the MSX selection system. MSX is a glutamate-like drug that binds to GS and inhibits the production of glutamine, which is necessary for cell growth. During gene amplification, CHO cells are cultured in high concentrations of MSX. CHO cells carrying increased copies of the GS gene containing the gene of interest are selected. CHO cells with insufficient levels of GS die. Once selected, transfected cell lines derived from CHO GS-negative hosts do not require MSX in the culture medium.

[0110] Example 5: Limiting dilution cloning CHO production cell lines are typically clonal populations derived from a single cell to ensure consistent quality of the protein produced. This is achieved by physically separating single cells from a heterogeneous, stably transfected cell population, placing them in separate culture vessels, and growing these cells into clonal cell populations. The challenge with single-cell expansion is that mammalian cells grow slowly or do not survive when cultured at low cell densities in protein-free medium.

[0111] Described herein is a single-cell cloning and expansion procedure used to select a single clone from a pool of CHO DG44 cells that stably produces a protein of interest.

[0112] Cells were seeded into 96-well plates. Because the initial cell density was too high, the cells were first diluted 1:100. The necessary volume for 30 x 100-well plates was prepared. 50 μl of the 1:100 cell suspension was added to control wells for a total volume of 200 μl. After 4 hours, imaging was performed. This step determines the focus, which is crucial for CSI imaging experiments. To achieve good focus, multiple cells, not just a single cell, are required. Therefore, a control well in the 96-well plate containing approximately 500 cells was included at the initial seeding. Once the focus was determined, it was used for all 30 wells of the 96-well plate. The same focus was also used for imaging on days 7 and 14. It is very important to select an appropriate, general focus value for all wells. Two to three days after seeding, a predetermined amount of medium (usually 60 μl) was collected from selected wells for a productivity test. This productivity test was performed at this stage for rapid screening of selected clones. If the protein concentration was higher than 10 μg / ml, the medium was diluted (usually 1:1) with sample diluent.

[0113] Example 6: Process Development and Titer CHO cells are a popular expression system for the large-scale production of complex biopharmaceuticals, but industrial strategies for upstream development are based on empirical results due to a lack of fundamental understanding of intracellular processes.

[0114] Cell culture media and culture methods play a critical role in determining optimal growth and productivity in any culture system. To ensure optimal performance across a wide variety of CHO cell lines, media and feeds were derived from a library of over 70 chemically defined media and over 40 chemically defined feeds based on past performance and nutritional diversity assessed by multivariate data analysis. Using these criteria, four basal media and four novel feeds were identified that supported superior growth and productivity in diverse clones.

[0115] Once cells were adapted to a serum-free environment, they were easily adapted to suspension growth. Competent Top10 E. coli was prepared by CaCl2 transformation. Two colonies were selected and inoculated into 10 ml of LB broth. Restriction digestion with two enzymes was performed, followed by agarose gel electrophoresis. Plasmids were isolated in this manner.

[0116] Suspension adaptation of CHO cells was achieved by using chemically defined media used in production, such as PF CHO and Excell Advanced medium. Suspension adaptation of CHO cells was tested by seeding them directly in serum-free medium in shake flasks (approximately 1 month). Suspension adaptation of CHO cells was checked sequentially, with increasing amounts of CD medium added at each step. CHO DG44 cells were frozen at P20 under 5%, 2.5%, and 1% FBS adaptation conditions.

[0117] The mAb-producing CHO DG44 cell line was cultured in 3-, 15-, and 50-L stirred-tank glass bioreactors using an independent fed-batch process. Daily sampling was performed to measure cell density, cell viability, and metabolite concentrations (glucose, lactate, glutamate, glutamine, ammonium, amino acids, and monoclonal antibodies).

[0118] After transfection, the stable pools for production were subjected to MTX gene amplification. The cells were thawed, passaged, and transferred to 250 mL Erlenmeyer flasks to obtain enough cells for banking and several rounds of MTX amplification. The purpose of this step was to select cells with many copies of the expression cassette containing the sequence encoding the target protein (in this case, an antibody). The cells were seeded in medium containing 50, 100, 200, or 300 nM MTX and passaged every 3–4 days until confluent and reached a viability of 80% or higher. Results were analyzed using ELISA, glycosylation analysis, resin slurry protein A, SDS-PAGE, CEX VCD, and viability assays. Biological similarity analysis was performed. Batch culture was employed to produce sufficient quantities for analysis. One vial from each of the two selected pools was thawed for limiting dilution cloning (LDC) and passaged again to achieve a viability of 95% or higher. LDC was performed by diluting cells 1:100 (1:10 and 1:10) and seeding 0.5 cells / well. Cloning medium was filtered and sterilized using a 0.22 μm Corning bottle-top filter. Cloning medium must be filtered because particles in FBS are invisible to the naked eye but visible under a microscope, which interfere with imaging of single cells in 96-well plates.

[0119] Different concentrations of FBC were used to evaluate the LDC clones. Elisa, glycosylation, resin slurry protein A, SDS-PAGE, and CEX VCD & Viability were performed and the results were analyzed.

[0120] Overall, the selected media and feeds demonstrated superior growth and productivity in all clones tested, and when compared to competitors, the selected media and feeds performed equally or better.

[0121] Example 7: Genetic stability of clones Cell line and process stability are crucial in the manufacturing of pharmaceutical biologics. The core concept of cloning is that the genetic characteristics of a given clone are passed on intact or nearly so to the descendants of the cloned cells, benefiting from the advantageous characteristics identified in the clone. Initially, the emerging population remains closely related to the CHO MS (clonal cells) of one member of the preceding CHO quasispecies family; perhaps selected for superior specific productivity, but their growth rate may be unsatisfactory. The time frame required to expand this initial population to the generation of a master cell bank, seed train culture, scale-up culture, and subsequent production phase is significant. During these phases, many different culture conditions are applied, including different media compositions, before the cells reach stirred-tank bioreactors with full control of pH, oxygen, osmolality, etc. During these phases, cells of this new CHO quasispecies family evolve and may diverge from the initial CHO MS. cGMP stability studies conducted from the master cell bank over several months can provide some insight into such trends, but cannot reliably prevent them.

[0122] Considering process and method variability between P0 and P21, a 22% titer difference was observed. Clone stability cannot be assumed without quality data, which is a critical regulatory factor for maintaining consistent quality.

[0123] Culturing cells for 60 generations is important for genetic stability. Generation 0 is called the research cell bank (RCB) generation, the first 12 generations are called the master cell bank (MCB) generation, and the next 12 generations are called the working cell bank (WCB) generation.

[0124] Comparative Example Several different transcriptional genetic elements and their combinatorial platforms developed within the scope of this invention were compared with reference innovator molecules and molecules obtained using Catalent cell lines.

[0125] [Table 3]

[0126] First, prior to vector design and transfection, transfected cells were purchased from Catalent, a commercial company. Using the biosimilar antibody obtained from these transfected cells, we performed experimental studies on the glycan profile, charge profile by CEX, and charge profile by cIEF. However, as shown below, the biosimilar antibody produced from the Catalent cell line was unsuitable for use due to its extremely poor glycan profile and yield. Subsequently, we received CHO cells developed by Dr. Chasin's laboratory (www.biology.columbia.edu / freeform / chasin-lab). Cloning, vector design, transfection, and transformation using the provided cells were performed as described above.

[0127] [Table 4]

[0128] Two-stage clarification refers to the use of two types of filters: one with a large nominal pore size (0.8-0.4 μm) and one with a small nominal pore size (0.3-0.1 μm). One-stage clarification refers to the use of only one type of filter. There was no difference in cost or time between the production using the Catalent cell line and the production using the Example-1E cell line. The tested cell lines (Catalent cell line and Example-1E) have similar capabilities, but the only difference is the filter layout used, i.e., the filter installation design.

[0129] One-step clarification is more convenient. For the biosimilar produced with the Catalent cell line, downstream processing was performed as a three-chromatography mAb platform process with a recovery rate of less than 20%. A two-chromatography mAb platform process is applicable to the biosimilar produced with the Example-1E cell line. All quality attributes were achieved. Meanwhile, one chromatography step was eliminated, reducing process costs and time, and achieving high recovery rates (>90%).

[0130] Among the quality criteria for biosimilars, the glycan and charge variant profile have the greatest impact on the activity and stability of biosimilar products. Evaluation of the glycan profile revealed no significant difference between the two clones, and the glycan profile of the product was similar to that of the original product.

[0131] Another quality criterion is the charge variant profile. A cloning platform was developed to obtain a product similar to the originator product without the need for optimization. Catalent vectors did not produce a biosimilar profile, and basic variants not present in the originator were present. These basic variants not present in the originator could not be removed by optimizing upstream and downstream processes. The newly designed cloning platform produced a biosimilar charge profile. cIEF results showed that the biosimilar contained higher levels of basic variants than the originator. Carboxypeptidase B enzymatic cleavage demonstrated the presence of a high concentration of basic variants derived from the C-terminal lysine. It is widely known in the literature that C-terminal lysine is cleaved by circulating extracellular carboxypeptidases after infusion of the drug product, and therefore does not impair the drug's activity or biosimilarity (Non-Patent Documents 13 and 16).

[0132] To improve the charge profile, which plays an important role in the activity and stability of biosimilar products, process optimization was performed on products developed using the Catalent vector. However, the charge profile of the biosimilar product was not obtained with the originator product. The charge variant profile obtained with the Catalent vector was considered to be a risk factor for problems during the clinical phase due to different variants not found in the originator, so process optimization studies were continued.

[0133] On the other hand, the product developed using the vector platform of this invention was able to obtain a charge variant profile of a biosimilar profile without process optimization, improving the efficiency of downstream processes by approximately five times, resulting in significant cost and time savings.

[0134] [References list] [Patent Documents] Patent Document 1: Ringold, U.S. Patent No. 4,656,134 Patent Document 2: Lin, U.S. Patent No. 5,955,422 Patent Document 3: U.S. Patent No. 5,888,774 to Delcuve Patent Document 4: Jacobs and Smith, U.S. Patent No. 5,605,690 [Non-patent literature] Non-Patent Document 1: Coco-Martin JM., and Harmsen MM. A review of therapeutic protein expression by mammalian cells. BioProcess Int. 2008. 6:S28-S33 Unauthorized literature 2: Kwaks TH, Otte AP. Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells. Trends Biotechnol. 2006 Mar; 24(3): 137-42. doi: 10.1016 / j.tibtech.2006.01.007. Epub 2006 Feb 7. PMID: 16460822 Unauthorized Literature 3: Esmer Duruel HE et al. (2021). Hucre Kulturlerine Genel Bakis. Selcuk Universitesi Fen Fakultesi Fen Dergisi. 47. 136-149 Non-patented reference 4: Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004 Nov;22(l 1): 1393-8. doi: 10. 1038 / nbtl026. PMID: 15529164 Unauthorized Literature 5: Wurm F, Wurm M. Cloning of CHO cells, productivity and genetic stability - a discussion. Processes 2017, 5 (4): 20. doi: 10.3390 / pr5020020 Unauthorized literature 6: Schimke RT et al. Amplification of dihydrofolate reductase genes in methotrexate-resistant cultured mouse cells. Cold Spring Harb Symp Quant Biol. 1978;42 Pt 2:649-57. doi: 10. 1101 / sqb. 1978.042.01.067. PMID: 277312 Non-patent document 7: Lee F et al. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228-32. doi: 10.1038 / 294228a0. PMID: 6272123 Non-patent document 8: Kaufman RJ, Sharp PA. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. J Mol Biol. 1982 Aug 25;I59(4):60I-2I. doi: 10.1016 / 0022-2836(82)90103-6. PMID: 6292436 Non-patent document 9: Mielke C, Maass K, Tummler M, Bode J. Anatomy of highly expressing chromosomal sites targeted by retroviral vectors. Biochemistry. 1996 Feb 20;35(7):2239-52. doi: 10.102 l / bi952393y. PMID: 8652565 Non-patent document 10: Klehr and Bode. Mol. Genet. (Life Sci. Adv.) 1988;7:47-52 Non-Patent Document 11: Chu L, Robinson DK. Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol. 2001 Apr; 12(2): 180-7. doi: 10. 1016 / s0958- 1669(00)00197-x. PMID: 11287235 Non-Patent Document 12: Jang HP et al. In vitro and in vivo modifications of recombinant and human IgG antibodies. MAbs. 2014;6(5): 1145-54. doi: 10.4161 / mabs.29883. Epub 2014 Oct 30. PMID: 25517300; PMCID: PMC4622420 Non-Patent Document 14: Saunders F. et el. Chromatin function modifying elements in an industrial antibody production platform- comparison of UCOE, MAR, STAR and cHS4 elements. PLoS One. 2015 Apr 7; 10(4):e0120096. doi: 10. 1371 / joumal.pone.0120096. PMID: 25849659; PMCID: PMC4388700 Non-Patent Document 15: Koh EY et al. An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells. PLoS One. 2013 Dec 9;8(12):e82100. doi: 10.1371 / joumal.pone.0082100. PMID: 24349195; PMCID: PMC3857217 Non-Patent Document 16: Cai B et al. C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo. Biotechnol Bioeng. 2011 Feb;108(2):404-12. doi: 10. 1002 / bit.22933. PMID: 20830675 The contents of all patent and non-patent literature or references explicitly cited herein are hereby incorporated by reference.

Claims

1. An expression vector comprising the following elements: a nucleotide sequence encoding one or more proteins or peptides of interest; one or more terminator sequences; one or more promoters; a nucleotide sequence encoding a chromatin opening element (UCOE), an internal ribosome entry site (IRES), or a combination thereof; and a nucleotide sequence encoding dihydrofolate reductase (DHFR) as a selectable marker; wherein these elements are operably linked.

2. The expression vector of claim 1 , wherein the expression vector is a bicistronic vector.

3. The expression vector of claim 1 , wherein the expression vector is a dual promoter vector.

4. The expression vector according to any one of claims 1 to 3, wherein the one or more target proteins or peptides are one or more monoclonal antibodies.

5. The expression vector according to any one of claims 1 to 3, wherein the one or more target proteins or peptides are one or more of an antibody light chain (LC) and an antibody heavy chain (HC).

6. The expression vector according to any one of claims 1 to 5, wherein the one or more promoters are a first promoter and a second promoter that are different from each other.

7. The expression vector of claim 6 , wherein the expression of the LC is controlled by the first promoter and the expression of the HC is controlled by the second promoter.

8. 8. The expression vector according to claim 1, wherein the DHFR is human DHFR, and the nucleotide sequence encoding the human DHFR is operably linked to a mouse-derived DHFR promoter.

9. the first promoter and the second promoter are selected from the group consisting of a CMV promoter and an EF1-α promoter, respectively; the UCOE comprises the sequence of SEQ ID NO: 4, the nucleotide sequence encoding the IRES comprises the sequence of SEQ ID NO:6; and 8. The expression vector of claim 6 or 7, wherein the DHFR comprises the sequence of SEQ ID NO:

8.

10. The expression vector according to any one of claims 1 to 5, further comprising a first multicloning site (MCSI) and a second multicloning site (MCSII), the promoter is an EF1-α promoter; the one or more terminator sequences are a polyA signal sequence and a polyA SV40 terminator sequence; The elements in the expression vector are in the following order from 5' to 3': the UCOE, the promoter, MCSI, a nucleotide sequence encoding the IRES, MCSII, the polyA signal sequence, an SV40 terminator sequence, a nucleotide sequence encoding the DHFR, and the polyA signal sequence An expression vector, which is arranged as follows:

11. The expression vector according to any one of claims 1 to 5, further comprising a first multicloning site (MCSI) and a second multicloning site (MCSII), the promoter is a CMV promoter; the one or more terminator sequences are a polyA signal sequence and a polyA SV40 terminator sequence; The elements in the expression vector are in the following order from 5' to 3': the UCOE, the promoter, MCSI, a nucleotide sequence encoding the IRES, MCSII, the polyA signal sequence, the SV40 terminator sequence, a nucleotide sequence encoding the DHFR, and the polyA signal sequence An expression vector, which is arranged as follows:

12. 12. The expression vector of claim 11, wherein the SV40 terminator sequence comprises the sequence of SEQ ID NO:

1.

13. A cell or cell population transformed with the expression vector according to any one of claims 1 to 12.

14. 14. The cell or cell population of claim 13, which is a mammalian cell.

15. 15. The cell or cell population of claim 14, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

16. The cell or cell population of any one of claims 13 to 15, wherein the mammalian cell culture process is carried out in the presence or absence of methotrexate (MTX).

17. A method for producing a target protein or peptide, comprising culturing the cell or cell population according to any one of claims 13 to 16 in a culture medium under conditions for expressing the target protein or peptide.

18. 18. The method of claim 17, wherein the cell or cell population is a clone that stably expresses the protein or peptide of interest.

19. 19. The method of claim 17 or 18, wherein the cell or cell population is a mammalian cell.

20. 20. The method of claim 17 or 19, wherein the protein or peptide of interest is a monoclonal antibody, an antibody heavy chain, an antibody light chain, or a combination thereof.

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