Novel expression platform for stable and high-titer large-scale production of recombinant protein

Abstract

An object of the present invention is to provide an expression platform comprising a vector comprising DHFR, at least one promoter, and a DNA element, such as UCOE, IRES, or a combination thereof. Another object of the present invention is to provide a method for producing cells having a unique genetic context using an expression platform, which cells are capable of stably expressing a target protein at high levels, and a method for high yield recovery of the produced target protein.

Description

Technical Field

[0001] The present invention relates to an expression platform, which comprises an expression vector that provides high gene copy number and increases the production of recombinant proteins or peptides.

[0002] In particular, the present invention relates to an expression platform comprising a vector that contains a nucleotide sequence encoding a target protein or peptide (such as an antibody); a nucleotide sequence encoding dihydrofolate reductase (DHFR) that provides a high enhancement of gene copy number; a promoter selected from a CMV promoter, an EF1α promoter, an SV40 promoter, or a combination thereof; and a DNA element, such as a ubiquitous chromatin opening element (UCOE), which improves the random integration efficiency, provides genetic stability, and enables high-titer large-scale production of recombinant proteins by effectively maintaining the activity of the corresponding promoter; and a nucleotide sequence encoding an internal ribosome entry site (IRES) that allows cap-independent translation initiation. Compared with some existing cell expression vectors, the vector platform of the present invention enables methods to effectively produce single cells and clonal cell populations with unique genetic backgrounds, ensures stable production of recombinant proteins or peptides, and achieves high recovery rates in both the upstream and downstream of the production process. Background Art

[0003] Recombinant production of proteins and polypeptides (including antibodies) in eukaryotic cells involves the creation of an expression system. Expression systems for producing recombinant proteins (such as biotherapeutics or biopharmaceuticals) typically 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 host cell's intrinsic machinery is utilized to produce the desired recombinant protein, such as a biotherapeutic. Establishing an efficient and reliable expression system for the production of an approvable biotherapeutic is a complex task involving multiple aspects.

[0004] There are various methods for the design and construction of expression vectors, and this process usually requires a large amount of trial and error to obtain reasonable protein levels. An important consideration in the design process is the use of intron sequences in vector construction.

[0005] One method is to utilize the entire naturally occurring gene sequence, i.e., containing the complete intron and exon sequences. In this case, it is expected that the post-transcriptional splicing mechanism within the cell will excise the intron sequences, leaving behind mature mRNA that contains only the exon sequences of the gene.

[0006] Another method is to utilize only the nucleic acid sequence corresponding to the cDNA of the gene. In this case, no splicing events are expected to occur, and the precursor mRNA sequence is essentially the same as the mRNA sequence in terms of protein-coding content.

[0007] In another method, vector construction involves the selection and placement of introns that are usually not related to the original gene sequence.

[0008] The vector has an "origin of replication", which is a segment of DNA that ensures the host bacterium replicates (copies) the vector. Usually, it also contains a promoter sequence for the expression of the introduced gene (and production of the protein).

[0009] Plasmids are extrachromosomal, self-replicating cytoplasmic DNA elements (usually circular) that exist in prokaryotes (less common in eukaryotes) and can serve as vectors. They come in various forms, such as simple plasmids for direct transformation and F fosmids for phage transduction. There are also other forms of circular DNA, each with different desirable properties (e.g., larger insert fragment size, low copy number, compatible with phages, etc.). They are sometimes called "high-capacity vectors" because they have a larger insert fragment size than simple plasmids.

[0010] Bacterial artificial chromosomes (BACs) contain regions from a special plasmid called the F factor (fertility): this region contains the origin of replication and genes that ensure its precise segregation during bacterial cell division. A major advantage of BAC vectors is their large insert fragment size (100 - 200 kb). However, the large insert fragment size also poses problems because it cannot be manipulated by restriction endonucleases.

[0011] Yeast artificial chromosomes (YACs) have a cloning capacity of up to 3000 kbp. It is introduced into yeast cells by electroporation and then exists as linear DNA like a chromosome. It replicates with the other chromosomes in yeast and maintains a copy number of 1 after cell division.

[0012] Some viruses (such as adenoviruses, lentiviruses, and baculoviruses) and bacteria (such as Agrobacterium tumefaciens) are reliable vectors for stable transfection of eukaryotic cells. Viral vectors are usually genetically engineered viruses that carry modified viral DNA or RNA that has lost its infectivity but still contains viral promoters and transgenes, allowing the transgenes to be transcribed under the control of the viral promoter. However, since viral vectors usually lack infectious sequences, helper viruses or packaging cell lines are required for large-scale transfection. Viral vectors are usually designed to permanently integrate the insert into the host genome, leaving a unique genetic marker in the host genome after integration of the transgene. For example, retroviruses leave a characteristic retroviral integration pattern after insertion, which is detectable and indicates that the viral vector has integrated into the host genome.

[0013] The choice of expression system for the production of recombinant proteins depends on multiple factors, including cell growth characteristics, required expression levels, intracellular or extracellular expression, post-translational modifications and the biological activity of the target protein, as well as regulatory issues and economic factors in the production of therapeutic proteins. The key advantages of mammalian cells over other expression systems such as bacteria or yeast are their ability to perform proper protein folding, complex N-linked glycosylation and authentic O-linked glycosylation, as well as a range of other post-translational modifications. Due to these advantages, eukaryotic cells, especially mammalian cells, are currently the preferred expression platform for the production of complex therapeutic proteins.

[0014] Cell culture studies are widely used in pharmaceutical, medical and biotechnological research. For the production of recombinant proteins, cell culture conditions should be standardized to ensure the optimal performance and stability of cell cultures. During cell culture, it is necessary to continuously control different parameters and conditions to ensure the normal growth of cells and the optimal production of the required recombinant proteins.

[0015] As mentioned above, mammalian cells have become the main system for the production of recombinant protein products for clinical applications because they are able to correctly fold and assemble proteins and add human-like post-translational modifications. In fact, to date, all cell lines used for biopharmaceutical protein production have been derived from mammals (Non-Patent Document 1). However, the development of mammalian cell lines is usually very time-consuming. In addition, the mammalian cell culture process is also affected by low cell yields and unstable expression (Non-Patent Document 2). Productivity and expression stability are preconditions for the development of a commercially viable process. Therefore, the ultimate goal of cell line development is to obtain clonal cell lines that can secrete the target protein with a high specific productivity (Qp) and maintain a continuous high level of secretion over multiple cell passages, thus enabling scale-up production and cost reduction. Expression vector and cell line engineering are the keys to achieving this goal.

[0016] The difficulty in protein synthesis lies in the high production cost due to low yields. The productivity of clones depends on the selection of multiple factors: external factors such as culture conditions (medium composition, temperature, pH, etc.) and downstream purification processes; internal factors such as the selection of the vector and its regulatory elements, such as promoters, transcriptional or translational enhancer elements, and the proper orientation of other elements, as well as the selection of a suitable host cell. Mammalian cells are the most promising expression system for obtaining high expression of recombinant therapeutic proteins because of their natural glycosylation ability. In addition, the post-translational modifications in this expression system are more similar to those of proteins expressed in human cells, thus ensuring that the recombinant protein has appropriate physiological activity. However, the expression level in eukaryotic cells also highly depends on another internal factor, namely the integration site of the recombinant expression construct containing the gene of interest in the host cell genome.

[0017] As described above, mammalian cell culture is the preferred technique in the industry for overexpressing target proteins. This is because most proteins of industrial value are proteins from humans or animals, and specific protein modification mechanisms (glycosylation, phosphorylation, amidation) are readily achievable in animal cells. High protein production costs and the significant time and expense required for cell line creation are known limitations of this technique (Non-Patent Document 3). Animal cells currently used industrially are Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK), and myeloma cells, and target proteins are expressed by transfecting expression vectors into the cells.

[0018] CHO cells are an epithelial cell line commonly used in biotechnological research and the commercial production of recombinant therapeutic proteins. It can be used for genetics, toxicity screening, nutrition, and gene expression research, as well as for expressing recombinant proteins. CHO cells are the most widely used mammalian host for the industrial production of recombinant protein therapeutics (Non-Patent Document 4).

[0019] CHO cells can produce proteins with complex glycosylation and other post-translational modifications (PTMs), similar to proteins produced by humans. They are easy to culture on a large scale and have a high survival rate, so they are very suitable for GMP protein production. In addition, CHO cells are tolerant to changes in parameters such as oxygen level, pH value, temperature, or cell density (Non-Patent Document 5). Most gene manipulations in CHO cells are carried out in cells lacking the DHFR enzyme.

[0020] Gene amplification is a commonly used strategy in animal cell expression systems. There are two commonly used amplification systems. They are DHFR-based amplification and glutamine synthetase-based amplification. Both of these amplification systems can significantly increase the recombinant protein yield of animal cell lines. Although gene amplification systems have the advantage of increasing protein production, the disadvantages are that they require multiple rounds of gene amplification and the use of high concentrations of methotrexate (MTX), which is very time-consuming. Prolonged subculture of cell lines can lead to gene loss and unstable expression.

[0021] Despite some challenges, the screening protocol based on DHFR-deficient cells remains one of the standard methods for generating transfected CHO cell lines for the production of recombinant therapeutic proteins.

[0022] Non-Patent Document 6 and Patent Document 1 reported that in an expression vector, when a foreign gene is inserted near the DHFR gene, this gene can be co-expressed at a high level in animal cells.

[0023] Gene amplification in CHO cells begins with the molecular cloning of the target gene and the DHFR gene into a single mammalian expression system. The plasmid DNA carrying these two genes is then transfected into the cells, and the cells are selectively cultured in a thymidine-free medium. The growth rates and the levels of recombinant protein production vary widely among each cell line. 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 Documents 7 and 8).

[0024] Current methods for creating mammalian cell lines to express recombinant proteins have some drawbacks (Non-Patent Document 9). The episomal system can achieve high expression levels of recombinant proteins, but usually only maintains stability for a short period of time (Non-Patent Document 10). Mammalian cell lines containing integrated foreign genes are slightly more stable, but there is increasing evidence that stability depends on the foreign gene being present in only a few copies, or even one copy.

[0025] The efficiency of the system itself depends on various factors, including the design of the vector and the choice of host cell. The strategic placement of regulatory elements, selection markers, and stabilizing elements in the vector sequence must balance the ease of vector manipulation and application with the need to produce the required biotherapeutic agent efficiently. Developing methods that enable host cells to stably express recombinant proteins at high levels for a long time is a particular challenge.

[0026] Despite the progress, the protein expression levels in mammalian cells are relatively low and tend to be unstable during development, resulting in high development and production costs for therapeutic proteins.

[0027] Current biomanufacturing processes require cell lines to be able to achieve clump-free and robust growth in suspension culture, stably and efficiently integrate heterologous DNA, produce high concentrations of recombinant proteins in a specific system, and have the required post-translational modifications with uniform product characteristics (Non-Patent Documents 10 and 11).

[0028] In addition, the availability of a suitable expression system and the speed of obtaining high-yield clones also affect the choice of cell line. To obtain regulatory approval, the production cell line must be fully characterized and genetically stable; therefore, using cell types familiar to the regulatory authorities can reduce the rigor of their review. Some cell line-specific differences can significantly affect the performance of the production system; for example, the glycosylation of a specific protein can vary depending on the mammalian cell type used, and even two subclones from the same parental cell line may have significant differences in their metabolic requirements.

[0029] Although the properties of certain regulatory elements and internal factors used in expression vectors have been well understood, it is impossible to absolutely predict whether they can achieve high expression of the target protein reliably and efficiently when combined together. The expression effects of some combinations are much worse than those of other combinations. For example, as described in Non-Patent Document 12, when using an expression vector composed of an SRα promoter, an AMY RNA leader sequence, and DHFR, the expression level of erythropoietin (EPO) can only reach 45 IU / ml (equivalent to 0.346 μg / ml). Patent Document 2 reported the use of another expression vector composed of an element combination of SV40 and polyadenylation (polyA) sequences and DHFR, and the EPO level was 750 to 1470 U / million cells / 48 hours (or 375 to 735 U / million cells / 24 hours). Another expression vector reported in Patent Document 3 is composed of a combination of an EF-1 promoter and an apoB SAR element, and it is reported that this vector achieved an expression of 1500 to 1700 IU EPO / million cells / 24 hours. For other recombinant proteins, TNFR-IgGFc (Enbrel) was reported, which is an expression vector containing a combination of a CMY promoter, TPL, YA I and II, and DHFR (Patent Document 4). Summary of the Invention

[0030] [Technical Problem]

[0031] Surprisingly, despite the large amount of knowledge accumulated in this field over the past two decades, even today, those proficient in this technology cannot simply select a combination of internal factors and regulatory factors to design an expression vector to ensure high expression of the target protein or peptide. When a special element is added to a combination of other elements, it may not provide any significant additional or synergistic effect on the expression potential of the vector. Therefore, the process of developing a new expression vector capable of achieving high-level protein expression is challenging and still requires experimental testing of multiple possibilities. Therefore, there is a need for a cell culture method for improving the production of recombinant proteins, especially for the large-scale production of long-term stable therapeutic proteins.

[0032] [Problem Solution]

[0033] During the cell line development process, three particularly important parameters need to be considered for the production of biologics. They are stability, productivity, and quality. These three parameters can be controlled by a mammalian expression vector that can transfer the target gene into host cells. Therefore, the object of the present invention is to design and create a mammalian expression vector carrying elements that can provide good results according to these parameters.

[0034] The inventors of the present invention have developed an expression platform that allows the use of recombinant vectors containing a human DHFR gene operably linked to a murine DHFR promoter to obtain large amounts of target proteins. This system can effectively amplify target genes at lower concentrations of MTX.

[0035] Compared with existing animal cell expression vectors, the vectors of the present invention can efficiently select cell line clones containing the DHFR gene and target genes amplified at extremely low concentrations of MTX. In fact, according to the present invention, it has been determined that MTX is not required in certain scale-up processes. In the examples provided herein, MTX is only used for the limiting dilution cloning (LDC) step. Therefore, the cell culture process can be carried out in the presence of low-dose methotrexate (MTX) or without MTX. The cell culture process refers to cell line production, followed by process and medium optimization and scale-up processes in small-scale systems, including shake flasks and benchtop bioreactors.

[0036] Certain vectors of the present invention contain an IRES element, which allows the expression of two proteins from a single mRNA, which is important for protein production. Due to epigenetic events such as DNA methylation and histone modification, the CMV promoter is prone to gene silencing in CHO cells.

[0037] On the one hand, the present disclosure provides a vector design system for improving the expression and / or production of target proteins or peptides. In some aspects, the protein or peptide is a recombinant protein or peptide. In some aspects, the target protein is an antibody.

[0038] On the other hand, the present disclosure provides recombinant nucleic acids encoding target proteins or peptides and genetic elements required for expressing target proteins or peptides in host cells.

[0039] On the other hand, the present disclosure provides expression vectors for producing proteins or peptides, including antibodies.

[0040] On the other hand, the present invention provides an expression vector for generating stable cells expressing recombinant proteins or peptides.

[0041] On the other hand, the present disclosure provides a host cell, such as a eukaryotic host cell, which contains one or more of the above nucleic acid molecules and / or vectors, such as expression vectors. The host cell can be transiently or stably transfected with the nucleic acid sequences of the present invention. The cell can be a mammalian cell, such as a CHO cell.

[0042] On the other hand, the present disclosure provides a system for culturing such cells according to the present disclosure for large-scale production of recombinant proteins or peptides.

[0043] On the other hand, the present disclosure provides a screening system for selecting host cells that express a target protein or peptide, which system is capable of obtaining high yields of the target protein or peptide.

[0044] On the other hand, the present disclosure provides an expression system for producing recombinant products (such as proteins, polypeptides, antibodies), which system comprises a host cell transfected with a vector according to the present disclosure.

[0045] The vector of the present disclosure can be an expression vector and can comprise one or more of the above-described modified nucleic acid elements. The vector can also comprise a nucleotide sequence that enhances one or more of the following in the host cell: replication, selection, mRNA transcription, mRNA stability, protein expression, or protein secretion. For example, the vector can comprise 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 (such as DHFR), an internal ribosome entry site sequence (IRES), and a polyadenylation sequence.

[0046] In certain aspects, the vector (such as an expression vector) is modified to reduce or eliminate mis-splicing and / or intron read-through by-products, and / or enhance the expression of the recombinant protein.

[0047] On the other hand, the present disclosure provides a vector design that comprises a nucleic acid sequence encoding an antibody (such as a recombinant antibody) or a fragment thereof, which sequence has reduced (such as substantially no) mis-splicing and / or intron read-through products, for the large-scale production of a therapeutic protein that remains stable over a long period of time.

[0048] On the other hand, the present disclosure develops a novel vector platform using a novel combination of various elements, which platform can exert the synergistic effect of these elements to efficiently express the desired protein.

[0049] In certain aspects, the expression vector of the present disclosure comprises the following operably linked elements:

[0050] A nucleotide sequence encoding one or more target proteins or peptides;

[0051] One or more terminator sequences;

[0052] One or more promoters;

[0053] An unmethylated CpG island element (UCOE), a nucleotide sequence encoding an internal ribosome entry site (IRES), or a combination thereof; and

[0054] A nucleotide sequence encoding dihydrofolate reductase (DHFR) as a selectable marker.

[0055] In some aspects, the expression vector is a bicistronic vector or a bipromoter vector, where the promoters can be the same as or different from each other.

[0056] In some aspects, the target protein or peptide is a monoclonal antibody. In some aspects, the target protein or peptide is an antibody light chain (LC) and an antibody heavy chain (HC). In some aspects, the expression of LC is controlled by a first promoter and the expression of HC is controlled by a second promoter.

[0057] In some aspects, DHFR is a human DHFR and the nucleotide sequence encoding human DHFR is operably linked to a murine DHFR promoter.

[0058] In some aspects, the first promoter and the second promoter are independently a CMV promoter or an EF1-α promoter, respectively.

[0059] In some aspects, the UCOE includes the sequence of SEQ ID NO:4.

[0060] In some aspects, the nucleotide sequence encoding IRES includes the sequence of SEQ ID NO:6.

[0061] In some aspects, DHFR includes the sequence of SEQ ID NO:8.

[0062] In some aspects, the expression vector further includes a first multiple cloning site (MCSI) and a second multiple cloning site (MCSII).

[0063] In some aspects, one or more terminator sequences are a polyA signal sequence and a polyA SV40 terminator sequence.

[0064] In some aspects, the elements in the expression vector are arranged in the 5' to 3' direction in the following order:

[0065] UCOE, EF1-α promoter, MCSI, nucleotide sequence encoding IRES, MCSII, polyA signal sequence, SV40 terminator sequence, nucleotide sequence encoding DHFR, and polyA signal sequence.

[0066] In some aspects, the elements in the expression vector are arranged successively in the 5' to 3' direction as follows:

[0067] UCOE, CMV promoter, MCSI, nucleotide sequence encoding IRES, MCSII, polyA signal sequence, SV40 terminator sequence, nucleotide sequence encoding DHFR, and polyA signal sequence.

[0068] In some aspects, the SV40 terminator sequence contains the sequence of SEQ ID NO:1.

[0069] In some aspects, the present disclosure provides cells or cell populations transfected with the expression vectors of the present disclosure. In some aspects, the cells are mammalian cells. In some aspects, the cells are Chinese hamster ovary (CHO) cells.

[0070] In some aspects, the present disclosure provides a method for culturing mammalian cells for propagating the cells described in the present disclosure in the presence or absence of methotrexate (MTX).

[0071] In some aspects, the present disclosure provides a method for producing a target protein or peptide by culturing the cells of the present disclosure under conditions that express the target protein or peptide in a culture medium.

[0072] In some aspects, the cells of the present disclosure are clones that stably express a target protein or peptide.

[0073] In some aspects, the target protein or peptide is a monoclonal antibody, an antibody heavy chain, or an antibody light chain or a combination thereof.

[0074] [Definitions]

[0075] In the context of the present invention, words such as "comprising", "including", etc. should be understood in an inclusive sense, rather than an exclusive sense, that is, the meaning of "including but not limited to".

[0076] As used herein, the term "stable" when referring to the genome means the stable maintenance of the genomic information content from one generation to the next, or in the case of a particular cell line, from one passage to the next. Thus, the genome is considered stable if there are no major changes (such as gene deletions or chromosomal translocations). The term "stable" does not exclude minor changes that may occur in the genome, such as point mutations.

[0077] "Operably linked" refers to the juxtaposition of two or more components, where the relationship between the components allows them to operate in an intended manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if it controls or regulates the transcription of the linked sequence in a cis-acting manner. Usually (but not necessarily), operably linked DNA sequences are contiguous, and when necessary, two protein-coding regions (such as a secretory leader region and a polypeptide region) are contiguous and in frame.

[0078] A "selectable marker" expressed by an introduced polynucleotide allows for the selection of host cells that express the selectable marker under appropriate screening culture conditions. The selectable marker is preferably a biomolecule, particularly a polypeptide.

[0079] According to the present invention, a "vector" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. The term "expression vector" includes a specific type of vector in which the nucleic acid construct is optimized for high-level expression of a desired protein product. Expression vectors typically have transcriptional regulators such as promoter and enhancer elements that are optimized for high-level transcription in a specific cell type and / or are optimized so that expression is constitutive in the presence of a specific inducer. Expression vectors also have sequences that provide proper and / or enhanced translation of the protein. As is known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses, and retroviruses. An "expression cassette" is a distinct part of the vector DNA and consists of a gene and regulatory sequences that are expressed by the transfected cell. In each successful transformation, the expression cassette directs the cellular machinery to make RNA and protein. The vector acts like a molecular carrier to deliver the nucleic acid fragment, polynucleotide, to the host cell respectively. The vector may contain at least one expression cassette that contains regulatory sequences for proper expression of the polynucleotide contained therein. In order to be expressed, the polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a product of interest or a selectable marker) can be inserted into the expression cassette of the vector.

[0080] As used herein, the term "DHFR (dihydrofolate reductase)" refers to the enzyme that reduces dihydrofolate to tetrahydrofolate, which is a key enzyme for nucleic acid synthesis and is also essential for cell growth.

[0081] As used herein, the term "intron" includes a transcribed DNA fragment that is removed from the RNA transcript by splicing the sequences (exons) on both sides of it. Introns are considered to be interspersed sequences within the protein-coding region of a gene and generally do not contain any information represented by the protein produced by the gene.

[0082] The term "antibody" refers to a protein having a four-polypeptide chain structure consisting of two heavy chains and two light chains, which are stabilized, for example, by interchain disulfide bonds, and in which the immunoglobulin or antibody has the ability to selectively or specifically bind an antigen.

[0083] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies making up the group are identical and / or bind the same epitope.

[0084] The term "commercial product" may also refer to a commercially available CHO cell line (e.g., a CHO cell line produced by CATALENT).

[0085] The term "region" can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain as defined herein, or a part or portion of a constant or variable domain), as well as more discrete parts or portions of said chain or domain. For example, the light and heavy chains or the variable domains of the light and heavy chains include "complementary determining regions" or "CDRs", which are interspersed between "framework regions" or "FRs", as defined herein.

[0086] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell.

[0087] 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 transcripts from both directions terminate. Thus, it can act as a transcription terminator and poly A signal in either direction.

[0088] [Advantages of the Invention]

[0089] The present invention describes an expression vector for the production of a stable recombinant protein in cells. This unique modification is particularly suitable for the production of recombinant proteins, especially monoclonal antibodies, but can also be used for any protein of interest. Introns and exons are nucleotide sequences in genes. As RNA matures, introns are removed by RNA splicing, meaning they are not expressed in the final messenger RNA (mRNA) product, while exons continue to covalently bind to each other to form mature mRNA. The nucleic acid molecule reduces or eliminates the intron readthrough (IRT) by-products of the desired protein or peptide relative to the native sequence. In addition, the introns and exons of the expression vector of the present invention have an altered natural operative association, which not only reduces or eliminates IRT by-products compared to vectors designed using recognized standard techniques, but also improves stability and protein expression levels.

[0090] The features and advantages of the present invention can be summarized as follows:

[0091] (1) The vector of the present invention is unique in terms of the combination of regulatory elements it contains. Thus, it is capable of ensuring optimal production of recombinant proteins.

[0092] (2) Compared with existing animal cell expression vectors, the vector of the present invention can effectively select cell line clones with amplified DHFR genes and foreign genes at extremely low concentrations of MTX.

[0093] (3) The present invention has the beneficial effect of using a lower MTX concentration and reducing costs due to increased cell growth rate and productivity.

[0094] (4) Different from existing commercial products, the present invention provides good biosimilarity. Existing products may not necessarily achieve biosimilarity.

[0095] (5) The risk of the present invention in terms of stability is very low.

[0096] (6) Existing commercial systems may need to use media and supplements specific to these systems for production. This will affect the cost and make it difficult to create a process suitable for the working budget. However, in the present invention, this drawback has been eliminated, and various media and supplements can be used.

[0097] In the present invention, even if random integration occurs, UCOE can improve the integration efficiency and stability. Brief Introduction of the Drawings

[0098] Figure 1 Schematic diagram of the dual-promoter vector.

[0099] Figure 2 Schematic diagram of the dicistronic vector.

[0100] Figure 3 Secondary structure of the IRES complex.

[0101] Figure 4 CMV promoter sequence (SEQ ID NO: 3).

[0102] Figure 5 A2UCOE structure.

[0103] Figure 6 A2UCOE sequence (SEQ ID NO: 4).

[0104] Figure 7 IRES sequence (SEQ ID NO: 5).

[0105] Figure 8 Sequence fragment showing the ATG-11 and ATG-12 translation initiation sites in IRES (SEQ ID NO: 7).

[0106] Figure 9 Charge distribution (CEX) graph, which compares the change curves of the original molecule with the molecule produced by the Catalent cell line and the molecule produced by Example-1E.

[0107] Figure 10 Charge distribution (cIEF) graph without CpB and with CpB, comparing the change curves of the original molecule and the molecule produced by Example-1E.

[0108] Figure 11 ​​​​​​​​​​​EF1α sequence (SEQ ID NO: 2).

[0109] Figure 12 SV40 terminator sequence (SV40) (SEQ ID NO: 1).

[0110] Figure 13 DHFR sequence (SEQ ID NO: 6). Detailed implementation

[0111] [Host cell]

[0112] The mammalian host cells of the present invention can be any cells commonly used in the art for expressing 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 cells include adherent cell lines and suspension cell lines.

[0113] Chinese hamster ovary cell lines are commonly used as hosts for recombinant protein production in the scientific research and biotechnology industries. Recombinant cell lines are formed by randomly integrating a polycistronic plasmid vector containing the gene of interest and a selectable marker gene into the host genome. Recombinant cell lines need to undergo several rounds of limiting dilution cloning to isolate stable high-expressing clones. Stable high expression of the gene of interest is a rare and desirable feature in recombinant cloned cell lines. Although cloned, cell lines may eventually become heterogeneous and lose productivity.

[0114] [Vector]

[0115] Any suitable expression vector can be used in the present invention. Preferably, a dual-promoter and bicistronic vector is used in the present invention.

[0116] Figure 1 The schematic diagram of the dual-promoter vector is depicted in. The expression of two different target genes is controlled by two different promoters. If the dual-promoter vector is used to express an antibody, the expression of the heavy chain and the light chain can be controlled by two separate promoters.

[0117] Figure 2 The schematic diagram of the bicistronic vector is depicted in. The expression of two different target genes is controlled by the same promoter. If the bicistronic vector is used to express an antibody, the expression of the heavy chain and the light chain is controlled by the same promoter region. To control the heavy chain and the light chain by a single promoter, an IRES (Internal Ribosome Entry Site) sequence can be used in the bicistronic vector model.

[0118] [Vector design for introducing regulatory elements]

[0119] ​​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 functions, well-known to those of ordinary skill in the art, can also be used in the embodiments of the present invention.

[0120] [Promoter]

[0121] The following are some examples of promoters that can be used in the present invention. It will be understood by those of ordinary skill in the art that other suitable promoters can also be used.

[0122] Simian virus 40 promoter

[0123] Mammalian expression plasmids are mainly used to produce mRNA. Commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) include the sequence motif AAUAAA, which promotes polyadenylation and termination. Among those listed, SV40 late polyA and rbGlob polyA are considered more effective in terminating transcription due to the presence of additional auxiliary sequences. PolyA sequences generally promote the stability or degradation of eukaryotic and prokaryotic transcripts, respectively. The SV40 sequence is a terminator sequence that indicates the end of the transcription unit. Terminator sequences also play a role in RNA processing and stability. The target gene is usually placed downstream of a strong viral promoter, such as the simian virus 40 (SV40) terminator sequence (SEQ ID NO: 1) and the cytomegalovirus (CMV) promoter (SEQ ID.NO: 3), to obtain high levels of expression. The CMV promoter is a commonly used 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 1α (EF-1α) and Chinese hamster elongation factor 1 (CHEF-1) promoters are stronger.

[0124] Human EF1α promoter

[0125] The human EF1α (EF-1α, gene symbol EEF1A1) (SEQ ID NO: 2) is a promoter that is constitutively active in a variety of cell types. Some studies have shown that the promoters of endogenous mammalian genes (such as EEF1A1) may be more resistant to silencing than viral promoters. The EF-1α promoter, in combination with the flanking regions of the CHO EF-1α gene, is more active in CHO cells than the CMV and SV40 promoters (Non-Patent Document 3).

[0126] CMV promoter

[0127] The CMV promoter (SEQ ID NO: 3) has become the most widely used promoter in customized and commercial vectors in the biopharmaceutical industry due to its continuous and high expression characteristics. However, in some cell types, the CMV promoter may be silenced over time, resulting in heterogeneity among transfected cells (Non-Patent Documents 4 and 5). To prevent transcriptional silencing caused by methylation, the main CpG island element (core CpG island element (IE)) is integrated into the promoter (Non-Patent Document 7).

[0128] [Ubiquitous chromatin opening element (UCOE)]

[0129] UCOEs are cis-epigenetic regulatory elements derived from the promoter regions of housekeeping genes. They are CpG islands without methylation, which prevent heterochromatin formation and transgene silencing by reducing DNA methylation (Non-Patent Document 8).

[0130] The A2UCOE region from the human HNRPA2B1-CBX3 locus is one of the most effective UCOEs, and integrating it into expression vectors has been shown to increase the transgene expression level in mammalian cells (Non-Patent Documents 13 and 16). The A2UCOE with the sequence of SEQ ID NO: 4 is an example of the A2UCOE that can be used in the present invention. Other suitable chromatin opening elements can also be used in the embodiments of the present invention.

[0131] [IRES (Internal Ribosome Entry Site)]

[0132] The EMCV (Encephalomyocarditis virus) IRES is a non-coding RNA fragment that can initiate high-level cap-independent protein synthesis in mammalian cells and cell-free extracts. The advantage of using the IRES element is that two genes can be expressed from a single mRNA. The first gene is expressed through cap-dependent translation, and the second gene is expressed through cap-independent binding of ribosomes to the IRES sequence. The IRES sequence forms a complex secondary structure that enables mammalian ribosomes to bind and initiate translation. Figure 3 The secondary structure of the IRES is shown.

[0133] The IRES with the serial number of SEQ ID NO: 6 is one of the examples of the IRES that can be used in the present invention. Other suitable IRES sequences can also be used in the embodiments of the present invention.

[0134] [Selection system]

[0135] Selectable markers integrated into plasmid vectors together with recombinant protein genes are usually glutamine synthetase (GS) or dihydrofolate reductase (DHFR). These are two well-characterized gene selection methods commonly used in CHO cell lines. The plasmid vector carrying the recombinant gene and the selectable marker is delivered into cells by transfection, and the cells are grown under selective conditions (for DHFR, growth in the absence of hypoxanthine and thymidine (-HT); for GS, growth in the absence of glutamine). Each surviving clone has at least one copy of the selectable marker gene, with the recombinant protein gene integrated into its genome.

[0136] [Dihydrofolate reductase (DHFR)]

[0137] 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. De novo mitochondrial thymidylate contributes to the biosynthetic pathway and catalyzes reactions essential for the de novo synthesis of glycine and purines and the synthesis of DNA precursors. DHFR is a common selectable marker gene, commonly used in mammalian cells, especially DHFR-deficient Chinese hamster ovary cells (CHO), such as DG44 and DXB11.

[0138] The DHFR of SEQ ID NO:6 is an example of a DHFR that can be used in the present invention. Other suitable DHFR sequences can also be used in embodiments of the present invention.

[0139] The following examples will describe the present invention in more detail, but the scope of the present invention is not limited to these examples. Those skilled in the art can make various changes or modifications according to the description of the present invention, and these changes or modifications are also included in the present invention.

[0140] [Examples]

[0141] Example 1: Vector Design

[0142] The target parameters during mammalian cell line development and the elements used to achieve these goals are shown in Table 1.

[0143] [Table 1]

[0144] Target parameters and vector elements

[0145]

[0146] By integrating the obtained sequences into template DNA, the designed expression vector models were obtained. These vectors were designed using the online vector design program GeneART Webportal. GeneArt Webportal has the sequence information of the elements visible in its portal.

[0147] [Table 2]

[0148] Examples of the expression vector model designed.

[0149]

[0150] Example 1A: In the figure above, there are two CMV promoters (SEQ ID NO: 3) in the Example 1A vector. The UCOE element is placed at the 5' end of the two promoters. Although this model is the simplest design model, it has the potential to cause homologous recombination in the genome because it contains two identical elements and promoters.

[0151] Example 1B: There are two EF1α promoters (SEQ ID NO: 2) in the Example 1B vector. Since it contains two identical elements and promoters, there is a possibility of homologous recombination.

[0152] Example 1C: Two different promoters, CMV and EF1α, are used in the Example 1C vector.

[0153] Example 1D: Two different promoters, CMV and EF1α, are used in the Example 1D vector. The CMV promoter is prone to silencing. EF1α is more resistant to silencing due to its intron region. Therefore, in the Example 1D vector model, assuming EF1α remains active, the UCOE element is only used before the CMV promoter.

[0154] Example 1E: There is one CMV promoter (SEQ ID NO: 3) in the Example 1E vector. The IRES element is inserted between two multiple cloning sites (MCS1 and MSC2). An antibody is used as the target protein. Both the heavy chain (HC) and the light chain (LC) are expressed under the control of the CMV promoter. LC is cloned into MCS1 and HC is cloned into MCS2 to ensure that the LC:HC ratio is greater than 1.

[0155] Example 1F: There is one EF1α promoter (SEQ ID NO: 2) in the Example 1F vector. The IRES element is used and both HC and LC are expressed under the control of the EF1α promoter. An antibody is used as the target protein. LC is cloned into MCS1 and HC is cloned into MCS2 to ensure that the LC:HC ratio is greater than 1.

[0156] A2UCOE

[0157] The A2UCOE sequence of SEQ ID NO: 4 was obtained from the NCBI Epigenome Browser (accession number: NC_000007.13)

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

[0158] A2UCOE of SEQ ID NO:4 was used in the examples provided herein.

[0159] IRES

[0160] The wild-type IRES of EMCV (IRESwt) of SEQ ID NO:5 is the sequence corresponding to nucleotides 260 to 848 in the EMCV-R genome (Genbank: M81861, NC_001479; Non-Patent Document 15). The IRESwt sequence of SEQ ID NO:5 was used in this example.

[0161] [Obtaining the Designed Vector Model]

[0162] After inserting the antibody HC into IRESwt using the NcoI restriction site in the 5’ region, translation can start from ATG-11 and ATG-12 (SEQ ID NO: 7). However, this does not affect the production of HC because if translation starts from ATG-11, 4 amino acids forming the “MAAT” site will be additionally added to the N-terminus of the signal sequence, which does not affect the cleavage of the signal peptide at the correct position. This was confirmed by using the Nucleofector Kit V and program U-24 on the Nucleofector I system (Lonza, Cologne, Germany). Specifically, the mAb heavy chain analysis was performed on the preproalbumin signal sequence from the MAAT region. The results showed that it was cleaved at the correct position before QV, which are the first two amino acids of the mAb heavy chain.

[0163] Configuration of UCOE in the vector:

[0164] In Non-Patent Document 14, different configurations of the UCOE element were tested according to the positions of the heavy chain and light chain in the vector. The results showed that the 5'LC 5'HC and 3'HC 5'LC 5'HC configurations provided the highest efficiency. Based on this information, the UCOE element was inserted at the 5’ end of the promoter.

[0165] DHFR:

[0166] DHFR of SEQ ID NO:6 was used in this example.

[0167] Example 2. Transformation, Vector Transcription, and Protein Isolation

[0168] Compared with host cells that have successfully integrated the vector or vector combination according to the present invention, host cells that have not successfully integrated the vector or vector combination according to the present invention preferably die or are growth-impaired under selective culture conditions. During the screening process, host cells that have successfully integrated the vector or vector combination can be enriched as a pool from the population of transfected host cells. During the screening process, individual host cells can be isolated from the population of transfected host cells and amplified, for example, by clonal selection.

[0169] In the cell culture step, the target protein expressed by the host cell is secreted into the culture medium. By purifying this secreted protein, a large amount of the target protein can be obtained. The purification step of the present invention may include conventional purification methods well-known to those skilled in the art, for example, solubility fractionation by ammonium sulfate or polyethylene glycol, ultrafiltration, fractionation by molecular weight, fractionation by various chromatography methods (such as based on size, charge, hydrophobicity or affinity), or a combination thereof.

[0170] Once the target amino acids of the gene sequence are designed, the codons are optimized according to the preference of CHO tRNA and then submitted to Twist Bioscience for synthesis. In this example, an antibody is used as the target protein. The weights of the heavy and light chains and the absorbance value of the twist vector are determined by gel electrophoresis. The vector is cut at the selected restriction enzyme cleavage sites (incubated with the restriction enzyme at 37 °C for 1 hour and inactivated at 80 °C for 2 hours). First, the light chain is cloned and extracted from the gel. Ligation is performed. The ligation product and the control are transformed into Top 10 Escherichia coli competent cells. Different vector clones are collected and inoculated into separate petri dishes containing 10 ml of growth medium. The cultures are incubated overnight at 37 °C with shaking at 225 rpm. Mini-prep plasmid isolation is performed. After mini-prep plasmid isolation, the concentration and absorbance value of each clone are measured. The plasmid isolated from the transformed colonies is digested with an appropriate restriction endonuclease. The insert and the digestion product are examined by agarose gel electrophoresis. PCR using specific primers is used as a secondary control. Finally, diagnostic digestion is performed with an appropriate restriction endonuclease. For large-scale production, the selected clone is cultured overnight at 37 °C with shaking at 225 rpm in 100 ml of medium. Thereafter, the plasmid is isolated by maxi-prep and its concentration is determined.

[0171] Example 3: Transfection and Random Integration

[0172] Vectors carrying nucleic acid sequences encoding monoclonal antibody heavy and light chains were transferred into E. coli cells by bacterial transformation. The transformed bacterial cells were cultured and propagated in selective agar media. This process was carried out separately for each vector. The vectors expressing monoclonal antibodies were linearized by appropriate restriction enzyme digestion. The appropriate restriction enzyme cleavage site was located in the ampicillin resistance gene that was no longer needed after transformation.

[0173] Example 4: Selection of Cells Expressing the Target Antibody

[0174] HT Selection and MTX Gene Amplification

[0175] HT selection and MTX gene amplification were carried out by methods known in the art. MTX can be used to increase the gene copy number of DHFR, which usually leads to co-amplification of the transgene of the target recombinant protein and increases the overall protein yield. MTX amplification can be carried out in a single round or multiple rounds by gradually increasing the concentration of MTX added to the selection medium.

[0176] CHO cells deficient in DHFR were transfected with recombinant DNA (containing a gene closely linked to the nucleotide sequence encoding DHFR). The MTX selection system was used to select CHO cells that produce the target protein. MTX is a drug similar to folic acid, which binds to DHFR, thereby inhibiting the production of tetrahydrofolic acid, which is necessary for de novo synthesis of purines and pyrimidines. During gene amplification, CHO cells were cultured at gradually increasing MTX levels. CHO cells with increased DHFR gene copy numbers and bound to the target gene were selected. CHO cells with insufficient DHFR levels lacked nucleoside precursors (hypoxanthine and thymidine) and died. Once selected, the transfected cell line from a CHO DHFR-negative host did not require the addition of MTX to the medium.

[0177] DHFR-deficient strains require supplementation with glycine, hypoxanthine, and thymidine. These strains were used to demonstrate that exogenous DHFR genes can be stably transfected and selected into originally DHFR-deficient cells using glycine / hypoxanthine / thymidine-deficient (GHT-minus) medium. This selection method has become the standard method for establishing stable transfection in CHO cell lines used for the production of therapeutic proteins. The gene expressing the target protein and the DHFR gene were incorporated into a mammalian expression vector or placed separately into different vectors. Then the plasmid or multiple plasmids were transfected into CHO cells, and the cells were cultured in GHT-minus medium to provide a selective environment. In this way, copies of the DHFR gene and the target gene were integrated into the genomes of all surviving cells. DHFR of SEQ ID NO:8 was used in this example.

[0178] Selection of Glutamine Synthetase (GS) and Amplification of Methionine Sulfoximine (MSX)

[0179] GS-deficient CHO cells can be transfected with recombinant DNA containing the target gene linked to the GS gene. Then, the MSX selection system is used to select CHO cells that produce the target protein. MSX is a drug similar to glutamic acid that binds to GS, thereby inhibiting the production of glutamine, which is essential for cell growth. During gene amplification, CHO cells are cultured in a high concentration of MSX. CHO cells with an increased copy number of the GS gene and containing the target gene are selected. CHO cells with insufficient GS levels will die. Once selected, the transfected cell line derived from a CHO GS-negative host does not require MSX in the medium.

[0180] Example 5: Limiting Dilution Cloning

[0181] CHO production cell lines are usually clonal populations derived from a single cell to ensure consistent quality of the produced protein. This is achieved by physically separating individual cells from a heterogeneous population of stably transfected cells into separate culture dishes and allowing these cells to grow into clonal cell populations. The challenge of single-cell growth is that mammalian cells grow slowly or even cannot survive when cultured at a low cell density in a protein-free medium, resulting in low efficiency of the single-cell cloning process.

[0182] This article describes the process of single-cell cloning and amplification. This process is used to select monoclonal clones from a pool of CHO DG44 cells that stably produce the target protein.

[0183] Cells are seeded into 96-well plates. Since the initial cell density is too high, the cells are first diluted with a dilution factor of 1:100. Prepare the amount required for 100 wells × 30 plates. Add 50 μl of the 1:100 cell suspension to the control wells, with a total volume of 200 μl. After 4 hours, imaging is performed. This is an important step in the CSI imaging experiment because the focus is determined at this step. To obtain a good focus, multiple cells rather than a single cell are needed; therefore, control wells are set up in 96-well plates containing approximately 500 cells at the initial seeding. Once the focus is selected, it will be used for each well in 30 96-well plates. On days 7 and 14, imaging is also performed using the same focus. It is very important to select a good and universal focus value suitable for all wells. Two to three days after seeding, a predetermined volume (usually 60 μl) of the medium is collected from the selected wells for productivity testing. The productivity test in this step is used to quickly screen the selected clones. If the protein concentration is higher than 10 μg / ml, the medium is diluted with a sample diluent (usually 1:1).

[0184] Example 6: Process Development and Titer

[0185] CHO cells have become the preferred expression system for large-scale production of complex biopharmaceuticals. However, due to the lack of a profound understanding of intracellular processes, the industrial strategy for upstream process development is based on empirical results.

[0186] Cell culture media and cultivation methods play important roles in determining optimal growth and productivity in any culture system. To ensure optimal performance across various CHO cell sources, media and feeds were obtained from a library containing over 70 chemically defined media and over 40 chemically defined feeds, based on historical performance and nutrient diversity evaluated by multivariate data analysis. According to these criteria, we identified four basal media and four new feeds that were able to support excellent growth and productivity of various clones.

[0187] Cell adaptation to serum-free conditions facilitates the easy adaptation of cells to suspension growth. By CaCl 2 Transform competent Top 10 E. coli. Pick out two colonies and inoculate them into 10 mL of LB broth. Perform restriction digestion with two enzymes and then agarose gel electrophoresis. Isolate plasmids by this method.

[0188] The suspension culture adaptation of CHO cells was achieved by using chemically defined media (such as PFCHO, Excell advanced media) used in production. The suspension culture adaptation of CHO cells was tested by directly inoculating the cells into serum-free media in shake flasks (for about 1 month). The suspension culture adaptation of CHO cells was examined sequentially by including increasing amounts of CD media at each step. CHO DG44 cells were cryopreserved at P20 under 5%, 2.5%, 1% FBS adaptation conditions.

[0189] The CHO DG44 cell line for monoclonal antibody production was cultured in 3L-15L-50L stirred-tank glass bioreactors in an independent fed-batch process. Samples were taken daily to measure cell density, cell viability, and metabolite concentrations (glucose, lactate, glutamate, glutamine, ammonium, amino acids, and monoclonal antibody).

[0190] After transfection, MTX gene amplification was performed on the stable pool for production. The cells were thawed, passaged, and transferred to 250 mL Erlenmeyer flasks to obtain sufficient cells for storage and multiple MTX amplifications. The purpose of this step was to select cells with a greater copy number of expression cassette containing the sequence encoding the target protein (here an antibody). The cells were seeded in media containing 50, 100, 200, and 300 nM MTX and passaged every 3 - 4 days until the cells were confluent and had a viability of over 80%. Enzyme - linked immunosorbent assay, glycan analysis, resin slurry protein - A, SDS - PAGE, CEX VCD, and viability assays were performed and the results were analyzed. Biosimilarity analysis was conducted. Batch culture was used to produce sufficient product for analysis. A vial was thawed from two selected pools for limiting dilution cloning (LDC) and passaged again to increase the viability to over 95%. LDC was completed by diluting the cells at a ratio of 1:100 (1:10 and 1:10) and seeding 0.5 cells / well. The cloning media was filter - sterilized using a 0.22 μm Corning bottle - top filter. The cloning media should be filtered because the particles in FBS are not visible to the naked eye but are visible under a microscope, and these particles can interfere with single - cell imaging in 96 - well plates.

[0191] Different concentrations of FBC were established for LDC - Clone evaluation. Elisa, glycan analysis, resin slurry protein A, SDS - PAGE, CEX VCD, and viability experiments were performed and the results were analyzed.

[0192] Overall, the selected media and feeds showed excellent growth and productivity in all tested clones. The data also indicated that the performance of the selected media and feeds was comparable or superior to that of the competitor's products.

[0193] Example 7: Genetic Stability of Clones

[0194] Cell line and process stability are very important for biopharmaceutical production. A core concept of cloning is that the genetic characteristics of a specific clone remain unchanged or nearly unchanged in the offspring of the cloned cells, thus benefiting from the identified advantageous characteristics in the clone. Initially, the emerging population maintained a close relationship with the CHO MS of a member of the previous CHO quasispecies family (cloned cells); it might have been selected for its excellent specific productivity, but its growth rate might not have been satisfactory. To construct the master cell bank, conduct seed train cultures, scale up the culture, and for subsequent production phases, the time required to expand this early cell population was very long. During this period, a variety of different culture conditions were applied, using different medium components - until the cells entered a stirred bioreactor where pH value, dissolved oxygen, osmotic pressure, etc. could be fully controlled - all of which were strictly controlled following the principles of quality by design. During these stages, the cells of the new CHO quasispecies family were evolving and might have been different from the original CHO MS. cGMP-regulated stability studies start from the master cell bank and last for several months. Although it can give us some understanding of these change trends, it definitely cannot prevent this change trend.

[0195] Considering the process and method differences between P0 and P21, an acceptable 22% titer difference was observed. Clonal stability cannot be assumed without quality data. This is a key factor in maintaining quality consistency in terms of regulation.

[0196] Culturing cells for 60 generations is very important for genetic stability. The 0th generation is called the research cell bank (RCB) generation, the first 12 generations are called the master cell bank (MCB) generations, and the subsequent 12 generations are called the working cell bank (WCB) generations.

[0197] Comparative Example

[0198] The platform developed within the scope of the present invention using a variety of different transcriptional genetic elements and their combinations was compared with a reference innovative molecule and a molecule obtained using the Catalent cell line.

[0199] [Table 3]

[0200] A purification difference study was conducted on the biosimilar production processes of the Catalent cell line and the Example 1E cell line.

[0201]

[0202]

[0203] Initially, the transfected cells were purchased from the commercial company Catalent, and then vector design and transfection were carried out. Using the biosimilar antibodies obtained from these transfected cells, tests on glycan profiles, CEX charge profiles, and cIEF charge profiles were conducted. However, as shown in the figure below, the biosimilars produced by the Catalent cell line were insufficient in both glycan profiles and yields and were not suitable for use. Subsequently, CHO cells developed by Dr. Chasin's laboratory (www.biology.columbia.edu / freeform / chasin-lab) were provided. Cloning, vector design, transfection, and transformation were carried out with the provided cells as described above.

[0204] [Table 4]

[0205] Comparison of the original molecule, the biosimilar produced by the Catalent cell line, and the biosimilar produced by Example-1E.

[0206]

[0207]

[0208] Two-step purification refers to the use of two different filters with nominal pore sizes of large (0.8 - 0.4 microns) and small (0.3 - 0.1 microns) respectively. One-step clarification refers to the use of only one filter. In terms of cost and time, there is no difference in production between using the Catalent cell line and using the Example-1E cell line. The production capacities of the tested cell lines (Catalent cell line and Example-1E) are similar, except for the layout of the filters used (i.e., the installation design of the filters).

[0209] One-step purification is easier.

[0210] In the biosimilar produced by the Catalent cell line, the downstream process is a three-column chromatography mAb platform process with a recovery rate of less than 20%. The biosimilar produced by the Example-1E cell line can adopt a two-column chromatography mAb platform process. All quality attributes have been achieved. At the same time, one chromatography step is omitted, reducing the process cost and time, and having a high recovery rate (>90%).

[0211] In the biosimilarity quality standards, glycan and charge variant characteristics have the greatest impact on the activity and stability of biosimilar products.

[0212] When evaluating the glycan profiles, no obvious differences were found between the two clones. The glycan profile results of the product are similar to those of the original product.

[0213] In terms of charge variant characteristics, which are another quality criterion, a cloning platform has been developed to make the product similar to the original product without optimization. It is impossible to obtain a biosimilar profile using the Catalent vector, and there are basic variants that do not exist in the original product. Neither upstream nor downstream process optimization can eliminate these basic variants that are not present in the original product. By designing a new cloning platform, the charge profile of the biosimilar was obtained. According to the cIEF results, the basic variants of the biosimilar are higher than those of the original product. Carboxypeptidase B enzymatic cleavage demonstrated that a large number of basic variants come from C-terminal lysine. It has been widely reported in the literature that C-terminal lysine is cleaved by circulating extracellular carboxypeptidases after the product is injected into the body, so it does not pose an obstacle to the activity and biosimilarity of the product (Non-Patent Document 13 and Non-Patent Document 16).

[0214] Process optimization was carried out on the product developed using the Catalent vector in an attempt to improve the charge distribution, which plays a crucial role in the activity and stability of biosimilar products. However, it is impossible to obtain the charge curve of the biosimilar product using the original product. In the charge variant curve obtained using the Catalent vector, considering the risk that different variants not present in the original product may cause problems at the clinical stage, process optimization research was continued.

[0215] On the other hand, in the product developed using the vector platform of the present invention, the charge variant distribution in the biosimilar profile can be obtained without process optimization, the downstream process efficiency is increased by about 5 times, the cost is greatly reduced, and time is saved.

[0216] [Citation List]

[0217] [Patent Documents]

[0218] Patent Document 1: U.S. Patent No. 4,656,134 to Ringold. Ringold's Patent No. 4,656,134.

[0219] Patent Document 2: U.S.Pat.Lin's Patent No. 5,955,422.

[0220] Patent Document 3: U.S.Pat. No. 5,888,774, granted to Delcuve.

[0221] Patent Document 4: U.S. Patent No. 5,605,690 to Jacobs and Smith.

[0222] [Non-Patent Documents]

[0223] Non-Patent Document 1: Coco-Martin JM., and Harmsen MM. Review of Therapeutic Protein Expression in Mammalian Cells. BioProcess Int. 6: S28 - S33.

[0224] Non-Patent Document 2: Kwaks TH, Otte AP. Enhancing the Expression of Therapeutic Proteins in Mammalian Cells Using Epigenetics. Trends Biotechnol. Doi: 10.1016 / j.tibtech.2006.01.007. Epub 2006 Feb 7. PMID: 16460822.

[0225] Non-Patent Document 3: Esmer Duruel HE et al. (2021). Hucre Kulturlerine Genel Selcuk Universitesi Fen Fakultesi Fen Dergisi. 47. 136 - 149.

[0226] Non-Patent Document 4: Wurm FM. Production of Recombinant Protein Therapeutics in Cultured Mammalian Cells. Nat Biotechnol. Doi: 10.1038 / nbt1026. PMID: 15529164. Non-Patent Document 5: Wurm F, Wurm M. Cloning of CHO cells, productivity and genetic stability - a discussion. Processes 2017, 5(4): 20. doi: 10.3390 / pr5020020 Non-Patent Document 6: Schimke RT et al. Amplification of dihydrofolate reductase genes in methotrexate - resistant cultured mouse cells. Cold Spring Harb Symp Quant Biol. PMID: 277312.

[0227] Non-Patent Document 7: Lee F et al. Glucocorticoid regulation of dihydrofolate reductase cDNA expression in mouse mammary tumor virus - derived plasmids. Nature. Nov 19, 1981; 294(5838): 228 - 32. DOI: 10.1038 / 294228A0. PMID: 6272123.

[0228] 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; 159(4): 601 - 21. PMID: 6292436.

[0229] Non - Patent Document 9: Mielke C, Maass K, Tümmler M, Bode J. Anatomy of highly - expressing chromosomal sites targeted by retroviral vectors. Biochemistry. DOI: 10.1021 / BI952393Y. PMID: 8652565.

[0230] Non - Patent Document 10: Klehr and Bode. Mol. Genet. (Life Sci. Adv.) 1988; 7: 47 - 52.

[0231] 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.

[0232] Non - Patent Document 12: Jang HP et al. MAbs. 2014; 6(5): 1145 - 54. Doi: 10.4161 / mabs.29883. Epub 2014 Oct 30. PMID: 25517300; PMCID: PMC4622420.

[0233] Non - Patent Document 14: Saunders F. et el. Comparison of chromatin - function - modifying elements - UCOE, MAR, STAR and cHS4 elements in industrial antibody production platforms. PLoS One. 2015 Apr 7; 10(4): e0120096. Doi: 10.1371 / journal.pone.0120096. PMID: 25849659; PMCID: PMC4388700.

[0234] Non-Patent Document 15: Koh EY et al. Library of internal ribosome entry site (IRES) mutants for modulating the levels of multi-gene expression in mammalian cells. PLoS One. Dec 9, 2013; 8(12): e82100. Doi: 10.1371 / journal.pone.0082100. PMID: 24349195; PMCID: PMC3857217.

[0235] Non-Patent Document 16: Cai B et al. C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo. Biotechnol Bioeng. Doi: 10.1002 / bit.22933. PMID: 20830675.

[0236] The content of all patent documents and non-patent documents or references specifically cited in this specification can be incorporated into this specification as part of this specification.

Claims

1. An expression vector comprising the following elements: A nucleotide sequence encoding one or more target proteins or peptides; One or more terminator sequences; One or more promoters; a chromatin opening element (UCOE), a nucleotide sequence encoding an internal ribosome entry site (IRES), or a combination thereof; And a nucleotide sequence encoding dihydrofolate reductase (DHFR) as a selection marker; Wherein the elements are operably linked.

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

3. The expression vector according to claim 1, wherein the expression vector is a dual-promoter vector.

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

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

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

8. The expression vector according to any one of claims 1-7, wherein DHFR is human DHFR, and wherein the nucleotide sequence encoding human DHFR is operably linked to a murine DHFR promoter.

9. The expression vector according to any one of claims 6-7, wherein The first promoter and the second promoter are each selected from: CMV promoter and EF1-α promoter; UCOE comprises the sequence of SEQ ID NO:4; The nucleotide sequence encoding IRES comprises the sequence of SEQ ID NO:6; and DHFR comprises the sequence of SEQ ID NO:

8.

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

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

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

1.

13. A cell or cell population, wherein the cell or cell population is transfected with the expression vector according to any one of claims 1-12.

14. The cell or cell population according to claim 13, wherein the cell or cell population is a mammalian cell.

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

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

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

18. The method according to claim 17, wherein the cell or cell population is a clone stably expressing the target protein or peptide.

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

20. The method according to any one of claims 17 or 19, wherein the target protein or peptide is a monoclonal antibody, an antibody heavy chain or an antibody light chain, or a combination thereof.

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