Hybrid transient transfection using transposases for high-yield product production

Abstract

A method for producing a target protein, comprising: providing a host cell, a vector comprising a nucleic acid sequence encoding the target protein, and a transposase; transfecting the host cell using the vector and the transposase to obtain transfected cells; culturing the transfected cells to form a culture; and harvesting the culture to obtain the target protein from the harvested culture, wherein the cells of the culture are not passaged before the culture is harvested.

Description

[Technical Field]

[0001] This disclosure provides a method for producing a target protein by hybrid transient transfection with a transposase. By combining a transposase with transient transfection, the target protein can be produced in high yield within a short period of time. [Background technology]

[0002] Transient gene expression (TGE) is useful for generating small amounts of gene products (or more) over short, limited time spans. In this approach, the expression vector enters the host cell line, but the highly stringent selective pressures required for the stable integration of the vector DNA into the host cell line genome are not applied. On the other hand, stable pooled gene expression is useful for generating large amounts of gene products (or more) over long periods, where stringent selection is applied to ensure that only cells with the expression vector integrated into the transcriptionally active regions of the genome survive. Both TGE and stable gene expression have their limitations. [Overview of the project]

[0003] In some embodiments, a method for producing a target protein is provided herein, comprising: providing a host cell, a vector containing a nucleic acid sequence encoding the target protein, and a transposase; transfecting the host cell using the vector and transposase to obtain transfected cells; culturing the transfected cells to form a culture; and recovering the culture to obtain the target protein from the recovered culture, wherein the cells of the culture are not passaged before the culture is recovered. The method utilizes the advantages of transient gene expression by expressing the target protein in a short time without stringent selection and passage steps, and utilizes the advantages of stable pooled expression by providing the target protein in high yield. [Brief explanation of the drawing]

[0004] [Figure 1] This disclosure presents a transient expression platform for glutamine synthase PIGGYBAC® according to several embodiments, which achieves long-term production of the target protein and high titer of the target protein. [Figure 2] The present disclosure describes polyethyleneimine (PEI) transfection procedures according to several embodiments of this disclosure. [Figure 3A] The product titers from three different product expression platforms according to several embodiments of this disclosure are shown. [Figure 3B] The bYlok bsAb titers using standard transient expression and glutamine synthase PIGGYBAC® transient expression according to some embodiments of this disclosure are shown. [Figure 4A] This disclosure shows isoforms of the cB72.3 product using stable pooled expression and transient expression of glutamine synthase PIGGYBAC® according to some embodiments of this disclosure. [Figure 4B] This document shows the aggregation of cB72.3 products using stable pooled expression and transient expression of glutamine synthase PIGGYBAC® according to some embodiments of the present disclosure. [Figure 4C] The N-glycans of cB72.3 products using stable pooled expression and transient expression of glutamine synthase PIGGYBAC® according to some embodiments of this disclosure are shown. [Figure 5A] This document shows standard transient expression of trastuzumab and trastuzumab expression using glutamine synthase PIGGYBAC® transient expression according to some embodiments of this disclosure. [Figure 5B] The expression of AMS002 using standard transient expression and transient expression of glutamine synthase PIGGYBAC® according to some embodiments of this disclosure is shown. [Figure 5C]This document shows standard transient expression of bsAb and bsAb expression using glutamine synthase PIGGYBAC® transient expression according to some embodiments of this disclosure. [Modes for carrying out the invention]

[0005] The use of the words "a" or "an," when used in conjunction with the term "including" in the claims and / or herein, may mean "one," but also coincide with the meanings of "one or more," "at least one," and "one or more."

[0006] Throughout this application, the term “approximately” is used to indicate that a value includes variability due to errors inherent in the method / apparatus used to determine that value. Typically, the term “approximately” means, depending on the context, that the variability includes approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less.

[0007] The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated to refer only to substitutes or the substitutes are not mutually exclusive, but this disclosure supports the definitions of substitutes only and “and / or” as such.

[0008] As used herein and in the claims, the words “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are comprehensive or open-ended and do not exclude any additional, undescribed, elements or method steps.

[0009] As used herein, “nucleic acid,” “nucleic acid molecule,” or “oligonucleotide” means a polymer compound containing covalently linked nucleotides. The term “nucleic acid” includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid DNA or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or MIRNA.

[0010] As used herein, “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes nucleic acid molecules such as cDNA and genomic DNA. “Genes” also refers to nucleic acid fragments that can function as regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence) a coding sequence. In some embodiments, genes are incorporated in multiple copies. In some embodiments, genes are incorporated in a predetermined number of copies.

[0011] As used herein, “transfection” means introducing an exogenous nucleic acid molecule, including a vector, into a cell. A “transfected” cell contains the exogenous nucleic acid molecule within the cell, while a “transformed” cell is one in which the exogenous nucleic acid molecule within the cell induces an intracellular phenotypic change.

[0012] The term “transfection” covers a range of techniques used to introduce a target gene(s) into a host cell line. These techniques include, for example, liposome-based transfection (where the transfection reagent is mixed with DNA to form a “liposome”), electroporation, and the use of cationic polymers. In some embodiments, the cationic polymer used for transfection is the cationic polymer polyethyleneimine (PEI). The PEI polyplex, in combination with DNA, forms positively charged particles that bind to the negatively charged cell surface before endocytosis. After release into the cytoplasm, the DNA can migrate to the nucleus, where gene expression can begin. PEI exists in both branched and linear forms with different molecular masses, and transfection efficiency varies considerably between the different forms. In some embodiments, the PEI reagent used in this disclosure has a linear format of 40,000 MW. In some embodiments, the transfection procedure can be further optimized to increase efficiency and reduce toxicity.

[0013] In certain embodiments, the disclosure provides a method for producing a target protein or another type of gene product. In some embodiments, the method can produce the target protein in high yield in a short time without requiring stringent selection and complex passaging.

[0014] In some embodiments, the method comprises providing a host cell, a vector containing a nucleic acid sequence encoding a protein of interest, and a transposase; transfecting the host cell using the vector and transposase to obtain transfected cells; culturing the transfected cells to form a culture; and harvesting the culture to obtain the protein of interest from the harvested culture, wherein the cells in the culture are not passaged before the culture is harvested.

[0015] Figure 1 illustrates a method for producing a target protein according to several embodiments of the present disclosure. As shown in Figure 1, a vector (DNA prep) containing the target gene and transposase is used to co-transfect host cells. After transfection, the period during which the transfected cells are cultured and produce the target protein is called the hybrid production phase. During this period, the transfected cells are selectively grown and the production of the target protein from the target gene occurs. The selection may be carried out under conditions that are less stringent than those required for stable pool selection. In this hybrid production phase, there is no interruption of the post-transfection process by subculturing, and it is not necessary to remove cells from the culture. This is in contrast to the stable pool construction process in which transposases are typically used, which involves a distinct culture selection phase for growing transfected cells before at least one subsequent subculturing step and before inoculation into a production vessel for dedicated synthesis of recombinant protein. Instead, cell and protein harvesting can be initiated immediately after the end of the transfection procedure (i.e., from a few minutes to a few hours, or less than a day). There is no need, requirement, or desire to subculture the cells. This means that any protein product produced immediately after transfection can eventually be harvested at the end of the hybrid production phase. This is in contrast to a stable pooling process, where material generated during the post-transfection selection phase and secreted into the growth medium can be lost as a result of subsequent subculturing steps. In this hybrid process, protein products produced after transfection are not lost from the endpoint harvested material as a result of intermediate subculturing steps. As used herein, “subculturing” refers to the procedure of harvesting cells from a culture, transferring the cells to one or more culture vessels containing fresh growth medium, and using those cells to start a new culture, also known as subculturing. As is known in the art, subculturing cells allows a subset of cells to then continue to proliferate and grow.In such cases, these passaged cells and their progeny are thus a different cell line from the original transfected cell line.

[0016] As shown in FIG. 1, in an embodiment, the vector can be a glutamine synthetase PIGGYBAC® transposon, and the transposase can be a PIGGYBAC® transposase (including hyperactive PIGGYBAC®). The vector PIGGYBAC® transposon carries a cargo (a gene of interest encoding a protein of interest) and an inverted terminal repeat (ITR) sequence adjacent to the cargo. The ITR sequence is recognizable by the hyperactive PIGGYBAC® transposase. By using the hyperactive glutamine synthetase PIGGYBAC® transposase, the present disclosure can incorporate the stable pool “select-grow-produce” phase after transfection into a “hybrid” production phase to support high titers across various protein formats. The transfected vector containing the product preferably has PIGGYBAC® ITRs. The transposase can be delivered to the cells as mRNA, as a separate fragment of non-GS vector DNA, or even contained together with the product gene in the GS transposon.

[0017] In an embodiment, glutamine (e.g., about 3 to 10 mM, preferably 6 mM) is added to the medium before transfection, but the medium after transfection does not contain glutamine so as to have a selection function. In some embodiments, the selection is not more stringent, and about 0.5 mM to about 5 mM of glutamine can be added to the medium after transfection. In some embodiments, the added glutamine is at a concentration of about 1 mM to 4 mM. In some embodiments, the added glutamine is at a concentration of about 3 mM. The reduced or depleted glutamine medium can promote the growth of production cells containing the expression vector, which may enable the culture period to be extended to achieve a higher production titer.

[0018] In some embodiments, methionine sulfoximine (MSX) is not added to the medium after transfection in this glutamine synthetase-based transient expression system. In contrast, MSX is added to the medium after transfection in a glutamine synthetase-based stable pool expression system. As a further comparison, the requirement for a glutamine-containing medium, such as a medium with 6 mM glutamine, is usually maintained after transient transfection because very efficient integration of the glutamine synthetase vector into the host cell genome may not occur.

[0019] Host cells or organisms that express exogenous nucleic acid molecules or fragments are referred to as “recombinant,” “transformed,” or “transgenic” organisms. In some embodiments, the host cell is a cell suitable for expressing the exogenous nucleic acid molecule or fragment. Preferably, the host cells that can be utilized in the various methods described herein are mammalian cells and cell lines or cultures. As used herein, the term “mammalian cell” includes cells derived from any member of the order Mammalia, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. In some embodiments, the cells include mouse cells, human cells, Chinese hamster ovary (CHO) cells, CHOK1 cells, CHO-DXB11 cells, CHO-DG44 cells, CHOK1SV cells containing all variants (e.g., POTELLIGENT®, Lonza, Slough, UK), CHOK1SV GS-KO (glutamine synthase knockout) cells containing all variants (e.g., Xceed®, Lonza, Slough, UK), and baby hamster kidney (BHK) cells. Exemplary human cells include human embryonic kidney (HEK) cells, e.g., HEK-293, HeLa cells, or HT1080 cells. In some embodiments, the protein of interest is produced from HEK-293 cells, human white colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs). In some embodiments, the protein of interest is produced by CHOK1SV GS-KO® host cells. In some embodiments, the host cell is a glutamine synthase Xceed® cell.

[0020] Mammalian cells include mammalian cell cultures, which may be either adherent cultures or suspension cultures. Adherent cultures refer to cells that grow on a substrate surface, e.g., a plastic plate, dish, or other suitable cell culture and growth platform, and may be adhesion-dependent. Suspension cultures refer to cells that can be maintained in a culture flask or large suspension tank, for example, to allow for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize agitation or agitation mechanisms to provide proper mixing. Media and conditions for maintaining cells in suspension are generally known in the art. An exemplary suspension cell culture includes human embryonic kidney (HEK293) cloned cells.

[0021] In some embodiments, host cells, such as CHOK1SV GS-KO® host cells, are pre-cultured for about 4 to 28 days to form a pre-culture before the transfecting step. In some embodiments, host cells are pre-cultured for about 6 to 14 days. In some embodiments, host cells are pre-cultured for about 8 to 10 days. In some embodiments, host cells, such as CHOK1SV GS-KO® host cells, are cultured in chemically defined Chinese hamster ovary (CD CHO) medium. In some embodiments, the CD CHO medium is supplemented with glutamine before the transfecting step. In some embodiments, the CD CHO medium is substantially devoid of methionine sulfoxamine.

[0022] In some embodiments, host cells may be cultured in other media such as Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Laboratory (RPMI) 1640, or Minimum Essential Medium (MEM), CD CHO medium without glutamine supplementation, or a combination thereof.

[0023] In some embodiments, pre-cultured host cells before transfection have a cell viability of at least 70%. In some embodiments, pre-cultured host cells before transfection have a cell viability of at least 75%, 80%, 85%, 90%, or at least 95%. In some embodiments, pre-cultured host cells before transfection have a cell viability of at least 90%. In some embodiments, cell viability is determined by trypan blue dye exclusion. In some embodiments, trypan blue dye is a 0.4% sterile filtered solution derived from SIGMA.

[0024] In some embodiments, the method provides a vector comprising a nucleic acid sequence encoding a protein of interest. The nucleic acid sequence is also referred to herein as the gene of interest. In some embodiments, the vector includes an adenovirus vector, a pSV vector, a pCMV vector, a vaccinia vector, a retrovirus vector, or a baculovirus vector. In some embodiments, the vector has a glutamine synthase gene, while the host cell is a knockout of the glutamine synthase gene. That is, the host cell does not express endogenous glutamine synthase, and transfection of the vector into the cell provides exogenous expression of glutamine synthase. Insertion of the vector into the host cell can be selectively performed using a glutamine-free medium. In some embodiments, the vector comprises a highly active PIGGYBAC® inverted terminal repeat (ITR) sequence. In some embodiments, the vector comprises two ITR sequences, with each side of the gene of interest adjacent to one of the two ITR sequences. The ITR sequences enable a vector compatible with PIGGYBAC® transposase. In some embodiments, the vector is the glutamine synthase PIGGYBAC® transposon. In some embodiments, the vector is the GSquad® expression vector.

[0025] In some embodiments, a single vector is provided containing the intact gene of interest for expressing the protein of interest. In some embodiments, a single vector is provided containing several gene fragments of the gene of interest. Each fragment of the gene of interest can be used to express a portion of the protein, and the expressed portions of the protein can be organized to form the protein of interest. Protein assembly can be a self-assembly process. In some embodiments, a single vector is provided containing two or more genes of interest. The vector is used to express two or more protein products simultaneously. In some embodiments, the two or more protein products are functionally related. In some embodiments, the two or more protein products can self-assemble to form a polymeric protein or macromolecule assembly.

[0026] In some embodiments, two or more vectors are provided, each containing a fragment of the gene of interest and expressing a portion of the protein of interest. The expressed portion of the protein of interest can be organized or self-assembled to form the protein of interest. In some embodiments, two or more vectors are provided, each containing the protein of interest. Multiple vectors can be used to simultaneously transfect host cells together with a transposase, and the transfected cells can be used to simultaneously express multiple proteins of interest. The proteins of interest may be functionally related. In some embodiments, the expressed proteins of interest are organized or self-assembled to form a polymeric protein or a macromolecular assembly.

[0027] Examples of target proteins that can be produced using the methods described herein include, for example, various antibodies, including antibody fragments and single-chain antibodies, as well as other therapeutic or diagnostic proteins.

[0028] In exemplary embodiments, the gene of interest may encode components necessary for organizing viral particles (i.e., the viral gene of interest), including lentivirus and adeno-associated virus particles. Preferably, the methods described herein can provide for the production of three AAV constructs: pHelper containing E2A, E4, and VA; pRepCap containing Rep and Cap; and pAAV containing the gene of interest (GOI). In other embodiments, the methods described herein can be used to produce lentivirus constructs containing a lentivirus group-specific antigen (GAG) gene and lentiviral polymerase (POL) protein, envelope protein (typically vesicular stomatitis virus glycoprotein (VSV-G)), HIV regulators of virion protein (Rev) protein expression, and the gene of interest (GOI).

[0029] Preferably, the amount of the protein of interest or gene of interest produced using the method described herein is at least 20% greater than that of a typical transient transfection system. More preferably, the amount of the protein of interest or gene of interest produced using the method described herein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or about 50% to about 200%.

[0030] Various transposases can be used in the methods described herein. In some embodiments, the transposase includes RNase H-like transposase, HUH single-stranded DNA transposase, serine transposase, tyrosine transposase, glutamine synthase PIGGYBAC® transposase, or a combination thereof. In some embodiments, the transpose includes highly active glutamine synthase PIGGYBAC® transposase. In some embodiments, the transposase is contained in a transposase vector or plasmid, and the transposase is encoded by the cell rather than being directly added to the cell as a functional enzyme. In other embodiments, the transposase can be delivered as mRNA.

[0031] As used herein, “transfection” means the introduction of an exogenous nucleic acid molecule, including plasmids and / or vectors, into a cell. A “transfected” cell contains an exogenous nucleic acid molecule within the cell, while a “transformed” cell is one in which the exogenous nucleic acid molecule within the cell induces an intracellular phenotypic change. The transfected nucleic acid molecule may be incorporated into the genomic DNA of the host cell and / or maintained by the cell, either transiently or for extended periods outside the chromosome. In some embodiments, “transduction” means infection of a mammalian cell with a viral vector and is used interchangeably with “transfection” in this disclosure.

[0032] In some embodiments, it is determined that the host cells have a high viability before transfection. Various transfection techniques can be applied. In some embodiments, transfection of host cells is carried out using polyethyleneimine (PEI), lipofection, electroporation, magnetofection, microinjection, gene gun insertion, imparefection, hydrostatic pressure, or sonication. In some embodiments, transfection is carried out using PEI.

[0033] Figure 2 shows an example of transfection in which a GSquad® vector containing the target gene and a highly active PIGGYBAC® transposase are used together to transfect Xceed glutamine synthase KO® host cells using a PEI transfection reagent.

[0034] After transfection, the transfected cells are cultured in a culture medium, but cell passage is not performed (i.e., the cells are not removed for further preparation of a culture for the production of the target protein). Various media, such as Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Laboratory (RPMI) 1640, Minimum Essential Medium (MEM), and chemically defined Chinese hamster ovary (CD CHO) medium, can be used to culture the transfected cells. In some embodiments, the medium is CD CHO medium. In some embodiments, the vector contains the glutamine synthase gene, and the medium is not supplemented with glutamine. In some embodiments, the medium does not contain methionine sulfoxamine (MSX). This is different from stable pooled expression, where MSX is used for stringent selection of transfected cells having a vector stably integrated into the cell genome. In some embodiments, bolus feeding is performed to prolong production. In some embodiments, the cell culture is bolus fed approximately 3 days after transfection of the host cells. In some embodiments, the cell culture is supplied in boluses on days 2 and 5 after transfection of the host cells.

[0035] In some embodiments, the culture of transfected cells acts as both a selection / proliferation and production phase. The absence of glutamine or a low concentration of glutamine in the medium allows for near-immediate selection of vector-containing transfected cells, which may or may not be integrated into the cell genome. Vector-containing transfected cells proliferate much faster than vector-free cells, so that the transfected cells proliferate. Since glutamine and MSX are not added to the medium, the selection is not stringent enough to select a stable pool of cells. As described herein, all original cells present in the culture at the time of transfection are retained in the culture, and the cells are used for the production of the protein of interest in the early culture phase rather than passage the cells for subsequent protein recovery. In some embodiments, other non-stringent selection criteria other than glutamine / MSX medium may be used based on the host cells and vector. As discussed above, the culture includes both selection / proliferation and production, and is therefore also called the hybrid production phase.

[0036] In some embodiments, transfected cells are cultured in a high-humidity CO2 orbital shaker incubator using an Erlenmeyer shaking flask with a vented cap. In some embodiments, transfected cells are cultured in a shaking flask with a sealed cap, provided that a sterile, pre-mixed supply of 5% CO2 in air is available to gas the headspace of the flask or bottle during and after the culture setup.

[0037] In some embodiments, cells and the protein of interest are produced in a bioreactor. Cells may be prepared in any suitable bioreactor (also referred to herein as a reactor), including but not limited to agitated tanks, airlifts, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds and / or jet bed bioreactors. As used herein, “bioreactor” may include a fermenter or fermentation unit or any other reaction vessel, and the terms “bioreactor” and “reactor” are used synonymously with “fermenter.” The terms fermenter or fermentation may refer to both microbial cultures and mammalian cultures. For example, in some embodiments, an exemplary bioreactor unit may perform one or more or all of the following: supplying nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), fermentation or inlet and outlet flows of cell culture medium, separation of gaseous and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or washing / sterilization. An exemplary reactor unit, such as a fermentation unit, may contain multiple reactors within the unit. For example, a unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit and / or facility, and / or a facility may contain multiple units, each having one or more reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-fed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor may have a volume from about 100 mL to about 50,000 L.Non-limiting examples include 100mL, 250mL, 500mL, 750mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. Volumes include 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. In addition, preferred reactors may be multi-use, single-use, disposable, or non-disposable and may be formed from any preferred material including metal alloys such as stainless steel (e.g., 316L or any other preferred stainless steel) and Inconel, plastics, and / or glass.

[0038] As discussed above, protein production is initiated in the early stages of cell culture. In some embodiments, transfection may be performed on day 1, and cell harvesting may be initiated on day 2. There is no subculturing of cells after transfection, and there is no need to remove cells from the culture except for harvesting. In some embodiments, harvesting continues from day 2 until at least day 14. In some embodiments, harvesting continues from day 2 until approximately day 8–12.

[0039] In some embodiments, harvesting is performed once at the end of the culturing process, and harvesting is performed on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 and can be continued for additional days to additional weeks.

[0040] The hybrid production phase utilizes transient expression and stable pooled expression. Compared to transient expression, production can be extended to approximately 13 days after transfection to obtain higher titers. Compared to stable pooled expression, highly stringent selection and passage processes are avoided, cells (i.e., less than 10%, less than 5%, or less than 1% of the original cell culture) are not removed from the cell culture, and the production period is much shorter.

[0041] In additional embodiments, the Disclosure provides a method for producing a protein of interest. In some embodiments, the method comprises providing a host cell, a vector containing a nucleic acid sequence encoding the protein of interest, and a transposase; transfecting the host cell using the vector and transposase to obtain transfected cells; culturing the transfected cells to form a culture; and recovering the culture to obtain the protein of interest from the recovered culture, wherein the cells in the culture are of the same cell lineage as the transfected cells. In embodiments, stringent selection and cell passage are not required, so the cells in the culture are of the same cell lineage as the transfected cells. Thus, “cells of the same cell lineage” refers to the original transfected cells that have not been isolated for separate cell culture and proliferation before protein recovery.

[0042] Embodiment In a first embodiment, a method for producing a target protein is provided herein, comprising: providing a host cell, a vector comprising a nucleic acid sequence encoding the target protein, and a transposase; transfecting the host cell using the vector and the transposase to obtain transfected cells; culturing the transfected cells to form a culture; and recovering the culture to obtain the target protein from the recovered culture, wherein the cells of the culture are not subcultured before the culture is recovered.

[0043] Embodiment 2 comprises the method of Embodiment 1, wherein the host cell includes a mammalian cell line.

[0044] Embodiment 3 comprises the method of Embodiment 2, wherein the mammalian cell line includes a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, a mouse myeloma cell line, a human embryonic kidney (HEK) cell line, or a HeLa cell line.

[0045] Embodiment 4 comprises the method of Embodiment 1, wherein the host cells include a CHOK1SV glutamine synthase knockout cell line.

[0046] Embodiment 5 includes any of the methods of Embodiments 1 to 4, wherein the host cells are pre-cultured for about 4 to about 28 days to form a pre-culture before transfection.

[0047] Embodiment 6 comprises the method of Embodiment 5, wherein the host cells are pre-cultured in chemically defined Chinese hamster ovary (CD CHO) medium supplemented with 6 mM glutamine before transfection.

[0048] Embodiment 7 comprises the method of Embodiment 5 or 6, wherein the pre-culture has a cell viability of at least 90%.

[0049] Embodiment 8 includes any of the methods of Embodiments 1 to 7, wherein the vector includes an adenovirus vector, a pSV vector, a pCMV vector, a vaccinia vector, a retrovirus vector, or a baculovirus vector.

[0050] Embodiment 9 comprises any of the methods of Embodiments 1 to 8, wherein the vector includes a glutamine synthase gene.

[0051] Embodiment 10 comprises the method of Embodiment 9, wherein the vector contains a highly active ultra-PIGGYBAC® (glutamine synthase PIGGYBAC®) inverted terminal repeat (ITR) sequence.

[0052] Embodiment 11 includes any of the methods of Embodiments 1 to 10, wherein the vector comprises one to about five nucleic acid sequences encoding the protein of the objective, or the vector comprises one to about five different vectors comprising the nucleic acid sequences.

[0053] Embodiment 12 includes any one of Embodiments 1 to 11, wherein the transposase includes an RNase H-like transposase, a HUH single-stranded DNA transposase, a serine transposase, a tyrosine transposase, a glutamine synthase PIGGYBAC® transposase, or a combination thereof.

[0054] Embodiment 13 includes the method of Embodiment 12, wherein the transposase comprises glutamine synthase PIGGYBAC® transposase.

[0055] Embodiment 14 includes any of the methods of Embodiments 1 to 13, wherein the transposase is contained in a transposase vector.

[0056] Embodiment 15 includes any method of Embodiments 1 to 14, wherein the transfecting of the host cells is carried out using polyethyleneimine (PEI), lipofection, electroporation, magnetofection, microinjection, gene gun insertion, imparefection, hydrostatic pressure, or sonication.

[0057] Embodiment 16 includes the method of Embodiment 15, wherein the transfection of the host cells is carried out using PEI.

[0058] Embodiment 17 comprises any method of Embodiments 1 to 16, wherein the transfected cells are cultured in a medium, the medium comprising Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, Minimum Essential Medium (MEM), chemically defined Chinese hamster ovary (CD CHO) medium without glutamine supplementation, or a combination thereof.

[0059] Embodiment 18 includes the method of Embodiment 17, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

[0060] Embodiment 19 includes the method of Embodiment 18, wherein the culture lacks methionine sulfoxamine (MSX).

[0061] Embodiment 20 includes any of the methods of Embodiments 1 to 19, wherein the culture is supplied in bolus form on the 2nd and 5th days after the host cells have been transfected.

[0062] Embodiment 21 includes any of the methods of Embodiments 1 to 20, wherein transfection is performed on day 1 of the method and recovery is initiated on day 2 of the method.

[0063] Embodiment 22 includes the method of Embodiment 21, wherein the recovery continues from the second day to at least the 14th day of the method.

[0064] Embodiment 23 provides a method for producing a target protein, comprising: providing a host cell, a vector comprising a nucleic acid sequence encoding the target protein, and a transposase; transfecting the host cell using the vector and the transposase to obtain transfected cells; culturing the transfected cells to form a culture; and recovering the culture to obtain the target protein from the recovered culture, wherein the cells of the culture are of the same cell lineage as the transfected cells.

[0065] Embodiment 24 includes the method of Embodiment 23, wherein the host cell includes a mammalian cell line.

[0066] Embodiment 25 includes the method of Embodiment 24, wherein the mammalian cell line includes a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, a mouse myeloma cell line, a human embryonic kidney (HEK) cell line, or a HeLa cell line.

[0067] Embodiment 26 comprises the method of Embodiment 23, wherein the host cells include a CHOK1SV glutamine synthase knockout cell line.

[0068] Embodiment 27 includes any of the methods of Embodiments 23 to 26, wherein the host cells are pre-cultured for about 4 to 28 days to form a pre-culture before transfection.

[0069] Embodiment 28 comprises the method of Embodiment 27, wherein the host cells are pre-cultured in chemically defined Chinese hamster ovary (CD CHO) medium supplemented with 6 mM glutamine before transfection.

[0070] Embodiment 29 includes the method of Embodiment 27 or 28, wherein the pre-culture has a cell viability of at least 90%.

[0071] Embodiment 30 includes any of the methods of Embodiments 23 to 29, wherein the vector includes an adenovirus vector, a pSV vector, a pCMV vector, a vaccinia vector, a retrovirus vector, or a baculovirus vector.

[0072] Embodiment 31 includes any of the methods of Embodiments 23 to 30, wherein the vector contains a glutamine synthase gene.

[0073] Embodiment 32 comprises the method of Embodiment 31, wherein the vector contains a highly active ultra-PIGGYBAC® (glutamine synthase PIGGYBAC®) inverted terminal repeat (ITR) sequence.

[0074] Embodiment 33 includes any method of Embodiments 23 to 32, wherein the vector comprises one to about five nucleic acid sequences encoding the protein of the objective, or the vector comprises one to about five different vectors comprising the nucleic acid sequences.

[0075] Embodiment 34 includes any one of Embodiments 23 to 33, wherein the transposase includes an RNase H-like transposase, a HUH single-stranded DNA transposase, a serine transposase, a tyrosine transposase, a glutamine synthase PIGGYBAC® transposase, or a combination thereof.

[0076] Embodiment 35 includes the method of Embodiment 34, wherein the transposase comprises the glutamine synthase PIGGYBAC® transposase.

[0077] Embodiment 36 includes any of the methods of Embodiments 23 to 35, wherein the transposase is contained in a transposase vector.

[0078] Embodiment 37 includes any method of Embodiments 23 to 36, wherein the transfection of the host cells is carried out using polyethyleneimine (PEI), lipofection, electroporation, magnetofection, microinjection, gene gun insertion, imparefection, hydrostatic pressure, or sonication.

[0079] Embodiment 38 includes the method of Embodiment 37, wherein the transfection of the host cells is carried out using PEI.

[0080] Embodiment 39 comprises any of the methods of Embodiments 23 to 38, wherein the transfected cells are cultured in a medium, the medium comprising Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, Minimum Essential Medium (MEM), chemically defined Chinese hamster ovary (CD CHO) medium without glutamine supplementation, or a combination thereof.

[0081] Embodiment 40 includes the method of Embodiment 39, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

[0082] Embodiment 41 includes the method of Embodiment 40, wherein the culture lacks methionine sulfoxamine (MSX).

[0083] Embodiment 42 includes any of the methods of Embodiments 23 to 41, wherein the culture is supplied in bolus form on the second and fifth days after the host cells have been transfected.

[0084] Embodiment 43 includes any of the methods of Embodiments 23 to 42, wherein the transfection is performed on day 1 of the method and the recovery is initiated on day 2 of the method.

[0085] Embodiment 44 includes the method of Embodiment 43, wherein the recovery continues from the second day to at least the 14th day of the method.

[0086] Embodiment 45 is a method for producing a target viral gene, comprising: providing a host cell, a vector containing a nucleic acid sequence encoding the target viral gene, and a transposase; transfecting the host cell using the vector and the transposase to obtain transfected cells; culturing the transfected cells to form a culture; and recovering the culture to obtain the target viral gene from the recovered culture, wherein the cells of the culture are not passaged before the culture is recovered.

[0087] Embodiment 46 includes the method of claim 45, wherein the target viral gene produces AAV virus particles or lentiviral particles. [Examples]

[0088] Example 1: Transient PEI outbreak In this example, PEI baseline transient expression is performed according to a specific embodiment of the present disclosure. The procedure times are shown in Table 1 below. [Table 1]

[0089] I. Preparation before the day of transfection (-Day 1) 1. The CHOK1SV GS-KO® host cell line is revived from cryopreservation and routinely cultured until the day before transfection. The CHOK1SV GS-KO® host cells are preferably cultured for 4 to 28 days prior to transfection. The culture can have a viability of more than 90% prior to transfection (measured by trypan blue exclusion). The culture is grown in medium CD CHO / 6mM L-glutamine until transfection. CD CHO may be, for example, Thermo Fisher Scientific, catalog no. 10743-029.

[0090] 2. Prepare the vector construct for transfection. The uncut vector DNA is suspended in sterile Tris-EDTA (TE) buffer at 1000 μg / mL. Furthermore, for products with two, three, or four genes, a single dual-gene vector (DGV), triple-gene vector (TGV), or quadruple-gene vector (QGV) can be used, or multiple part vectors can be co-transfected for transient expression.

[0091] 3. Perform subculturing of the host cell line the day before transfection: 3.1 Preheat the appropriate growth medium (CD CHO + 6 mM glutamine) to 35.5-37.0°C. 3.2 Aseptically remove the cell suspension sample from the counted CHOK1SV GS-KO(registered trademark) cells using a sterile serological pipette. 3.3 Count cells using a hemocytometer or automated cell counter, and assess cell viability using trypan blue dye or other methods. Use the cell count and viability data to determine cell concentration and culture viability. 3.4 Add 1.0 × 10 to each 20 mL of the required transient transfect culture. 6 Inoculate 30 mL of fresh culture at a target concentration of 100 viable cells / mL. The recommended range of culture volumes is shown in Table 2. 3.5 If using a ventilated cap, reattach the cap and place the new culture in a high-humidity CO2 orbital shaker incubator set to 35.5-37.0°C, 140±5 rpm, >85% relative humidity, and 5% CO2 in the air. 3.6 If using a sealing cap, reattach the cap and release headspace gas using a sterile, pre-mixed feed of 5% CO2 air before placing the new culture into an orbital shaker incubator set to 35.5–37.0°C and 140±5 rpm.

[0092] Table 2 shows the culture volume specific to transient transfection in Example 1. [Table 2]

[0093] II. Transfection (Day 0) Transfection day (1 day after subculturing, considered day 0 in this procedure):

[0094] 1. Calculate the total amount of culture medium, PEI, sodium acetate, cells, and glutamine synthase vector DNA required for transfection. 1.1 For a single standard transfection in a 20 mL scale, a total of 20 × 10 6 The following are required: 1 viable cell, 40 μg of GS vector DNA encoding the product gene, 100 μL of PEI (Transporter 5, Polysciences catalog number 26008-5), and 67 μL of sodium acetate (3M, pH 5.2, Lonza Bioscience catalog number 51203). 1.2 Transfection can be scaled up or down, but the relative ratio of reagents per mL of culture is preferably maintained as shown in Table 3. [Table 3]

[0095] It should be noted that the process described above is for the transfection of a single glutamine synthase vector carrying up to four product genes. However, if two vectors (e.g., for co-expression of multiple part vectors) are co-transfected, the amount of plasmid DNA is shared between the two vectors. For example, for a 20 mL transfection, 20 μg of each vector is transfected. Too little DNA means that transfection will be inefficient, while too much DNA can be harmful to cells.

[0096] Prepare an appropriate volume of CD CHO containing 2.6 mM L-glutamine aseptically and preheat to 35.5–37.0°C.

[0097] 3. Using a sterile serological pipette, prepare 1.0 × 10 the day before. 6 A cell suspension sample is aseptically removed from a CHOK1SV GS-KO(registered trademark) culture inoculated with 100 viable cells / mL.

[0098] 4. Count the cells using a hemocytometer or automated cell counter and assess cell viability using trypan blue dye or other methods. Use the cell count and viability data to determine cell concentration, culture viability, and total number of viable cells. Note that the culture should be at least 90% viable before transfection. Low viability reduces transfection efficiency.

[0099] 5. Calculate the volume of culture needed to obtain the number of cells required for transfection (20 x 10 per 20 mL transfection). 6 (Individual viable cells). Note that cells may be lost during the centrifugation and washing steps. Therefore, in the next step, it is recommended to centrifuge up to twice as many cells as needed, and to centrifuge the cells at 200 × g. 5.1 Aseptically remove the calculated volume of culture (from the previous step) and centrifuge at 200 × g for 5 minutes. 5.2 Remove the supernatant, remove the pellet by gently tapping the bottom of the tube, and disperse the cell pellet in a sufficient volume of preheated CD CHO + 6 mM L-glutamine to a concentration of 1.0 × 10⁻⁶. 6 Obtain 100 viable cells / mL. 5.3 Determine the viable cell concentration and use CD CHO + 6 mM L-glutamine to obtain 1.0 × 10⁻⁶ cells. 6 Adjust to the final concentration of 10

[0100] The cell suspension is incubated at ambient temperature, while transfection is performed for up to 30 minutes. Furthermore, it is crucial that the prepared cell suspension is maintained at ambient temperature for up to 30 minutes, and not at high temperatures. At high cell concentrations, cells maintained at 37°C would quickly become oxygen-depleted, leading to rapid cell death.

[0101] 6. For a single standard transfection, transfer 20 mL of cell suspension from the previous step to a 125 mL Erlenmeyer flask.

[0102] 7. Add the following to the flask in the order listed, shaking vigorously after each addition. 7.1 40 μL (= 40 μg) of sterile circular plasmid DNA at a concentration of 1 mg / mL, 7.2 100 μL of PEI, and 7.3 67 μL of sodium acetate.

[0103] 8. Return the cells to a high-humidity CO2 orbital shaker incubator set to 35.5-37.0°C, 140±5 rpm, >85% relative humidity, and 5% CO2 in the air for 4 hours.

[0104] 9. After 4 hours, transfer the cells to a shaking incubator set to 32°C, 5% CO2, 85% humidity, and 140 rpm, or lower the temperature of the existing incubator to 32°C. If sealing caps are used, reattach the caps and release headspace gases using a sterile, pre-mixed feed of 5% CO2 air before placing the new culture into an orbital shaker incubator set to 32°C and 140±5 rpm.

[0105] 10. Incubate the transfected cells in a shaking incubator for 8–10 days. The cultures may be regularly monitored for growth and viability, with the goal of harvesting them before the average viability falls below 90%.

[0106] 11. Collect the culture using a suitable method, remove the sample from the culture vessel, purify the culture supernatant, and store it at an appropriate temperature for further assays. Note that the shift to 32°C can be performed at any point between 4 and 24 hours after transfection, allowing for better fit with the laboratory schedule. However, the temperature shift should not be performed at least 4 hours prior.

[0107] Example 2: Transient expression of GS PIGGYBAC(registered trademark) PEI / transposase In this example, a highly active GS PIGGYBAC® transposase enzyme is used for transient gene expression to maximize the final product yield. Example 2 has similarities to the stable pool construction process but also has important adaptations to the transient gene expression workflow. The timeline for Example 2 is shown in Table 4 below. [Table 4]

[0108] Firstly, while highly stringent selection for vector integration into transcriptional active sites within the host cell genome using MSX is not applied, the cultures are not supplemented with L-glutamine. Secondly, the application of bolus supplies on days 2 and 5 post-transfection helps extend the culture and maximize yield towards the end of the culture. Thirdly, the GS PIGGYBAC® transposase enzyme is co-transfected into cells as circular plasmid DNA, not as mRNA. Since transiently transfected cultures are not intended for long-term culture or to generate clonal cell lines, inadvertent integration of the transposase gene into the genome is not considered a critical factor. Fourthly, PEI is used for transfection, not electroporation.

[0109] I. Preparation before the day of transfection (-2 days) 1. The CHOK1SV GS-KO® host cell line is revived from cryopreservation and routinely cultured until two days before transfection. In this embodiment, the CHOK1SV GS-KO® cells are cultured for 4 to 28 days prior to transfection. The culture has a viability of over 90% prior to transfection (measured by trypan blue exclusion). The culture is grown in CD CHO medium supplemented with 6 mM L-glutamine (CD CHO / 6 mM glutamine) until prior to transfection.

[0110] 2. Prepare the vector construct for transfection.

[0111] After preparation, the uncut GS vector DNA is suspended in sterile Tris-EDTA (TE) buffer at 1000 μg / mL. The GS vector contains the GS PIGGYBAC® ITR sequence to enable this method. In this example, the latest GSquad® vector platform is used.

[0112] 3. Two days before transfection, perform subculturing of the host cell line: 3.1 Preheat the appropriate growth medium (CD CHO / 6mM glutamine) to 35.5-37.0°C. 3.2 Aseptically remove the cell suspension sample from the counted CHOK1SV GS-KO(registered trademark) cells using a sterile serological pipette. 3.3 Count cells using a hemocytometer or automated cell counter, and assess cell viability using trypan blue dye or other methods. Use the cell count and viability data to determine cell concentration and culture viability. 3.4 Add 0.2 × 10 to each 20 mL of the required transient transfect culture. 6 Inoculate 30 mL of fresh culture at a target concentration of 100 viable cells / mL. Larger transfection volumes can be achieved, and the recommended range of culture volumes is shown in Table 5 below. [Table 5] 3.5 If using a ventilated cap, reattach the cap and place the new culture in a high-humidity CO2 orbital shaker incubator set to 35.5-37.0°C, 140±5 rpm, >85% relative humidity, and 5% CO2 in the air. 3.6 If using a sealing cap, reattach the cap and release headspace gas using a sterile, pre-mixed feed of 5% CO2 air before placing the new culture into an orbital shaker incubator set to 35.5–37.0°C and 140±5 rpm.

[0113] II. Transfection (Day 0) Transfection day (2 days after subculturing, considered day 0 in this procedure):

[0114] 1. Calculate the total amount of culture medium, PEI, cells, and DNA (both GS PIGGYBAC® compatible vector containing the product gene, and also the transposase-coding plasmid SPB-DNA) required for transfection.

[0115] A single transfection on a 20 mL scale results in a total of 20 × 10 6 This requires 1 viable cell, 40 μg of GS vector DNA, 4 μg of SPB-DNA, and 100 μL of PEI (Transporter 5). Transfection can be scaled up or down, but the relative ratio of reagents per mL of culture should be preferably maintained. Table 6 below shows the reagent ratios during transfection. [Table 6]

[0116] Prepare an appropriate volume of CD CHO containing 2.10 mL / L of HT supplementation aseptically and preheat to 35.5–37.0°C.

[0117] 3. Using a sterile serological pipette, aseptically remove a sample of the cell suspension from a CHOK1SV GS-KO® culture inoculated 2 days previously at 0.2 × 10 6 viable cells / mL.

[0118] 4. Count the cells using a hemocytometer or an automated cell counter and assess cell viability using trypan blue dye or other methods. Use the cell count and viability data to determine the cell concentration, culture viability, and total number of viable cells. Note that the culture should be at least 90% viable. Low viability will reduce transfection efficiency.

[0119] 5. Calculate the volume of culture required to obtain the number of cells needed for transfection (20 × 10 6 viable cells per 20 mL of transfection). Note that cells can be lost during the centrifugation and washing steps. Therefore, in the next step, it is recommended to centrifuge up to twice as many cells as required and centrifuge the cells at 200 × g. 5.1 Aseptically remove the volume of culture calculated (from the previous step) and centrifuge at 200 × g for 5 minutes. 5.2 Remove the supernatant, remove the pellet by gently tapping the bottom of the tube, and disperse the cell pellet in a sufficient volume of pre-warmed CD CHO + 10 mL / L HT supplement to obtain a concentration of 1.0 × 10 6 viable cells / mL. 5.3 Determine the viable cell concentration and adjust to a final concentration of 1.0 × 10 6 viable cells / mL using CD CHO + 10 mL / L HT supplement (sodium hypoxanthine (10 mM) and thymidine (1.6 mM), Thermo Fisher catalog number 11067030).

[0120] 6. For a single standard transfection, transfer 20 mL of the cell suspension from the previous step to a 125 mL Erlenmeyer flask.

[0121] 7. Add the following to the flask in the order listed, shaking vigorously after each addition. 7.1 Sterile circular plasmid DNA encoding the product gene at a concentration of 1 mg / mL, 40 μL (= 40 μg) 7.2 4 μg of SPB-DNA circular plasmid DNA, 7.3 100 μL of PEI.

[0122] 8. Return the cells to a high-humidity CO2 orbital shaker incubator set to 35.5–37.0°C, 140±5 rpm, >85% relative humidity, and 5% CO2 in air. If sealing caps are used, reattach the caps and release headspace gases using a sterile, pre-mixed feed of 5% CO2 in air before placing the new culture into the orbital shaker incubator set to 35.5–37.0°C and 140±5 rpm.

[0123] 9. On days 2 and 5 post-transfection, add 10% v / v (2 mL for a total transfection volume of 20 mL) of CHO CD EfficientFeed® B liquid nutritional supplement (Thermo Fisher catalog number A 1024001).

[0124] 10. Cultures may be periodically monitored for growth and viability using a hemocytometer or automated cell counter. Note that cultures should be harvested before viability falls below 90%.

[0125] 11. After 14 days, collect the culture using a suitable method, remove the sample from the culture vessel, purify the culture supernatant, and store it at a suitable temperature for further assays.

[0126] Note that the expression vector and transposase are used together in Example 2 to transfect the host cell line using the PEI transfection reagent.

[0127] Example 3: Transient expression of optimized transposase In this example, protein expression by transposase-optimized transient expression is performed as described in Example 2, and the results are compared with those of a conventional platform (similar to that described in Example 1, but using electroporation for transfection) and a third-party system.

[0128] Figure 3A shows a comparison of GS PIGGYBAC® transposase technology with conventional electroporation processes and third-party systems in transient expression. Target proteins include cB72.3(IgG1), tolsutuzumab, AMS002, asymmetric Mab, and classical bsAb. As shown in Figure 3A, using GS PIGGYBAC®, transient titers are increased up to 30-fold compared to those obtained with conventional processes.

[0129] Figure 3B shows a comparison of the GS PIGGYBAC® transposase technology with the conventional electroporation expression process for expressing bYlok® bsAb in transient expression. The average recovery titer of the GS PIGGYBAC® transposase technology in transient expression is approximately 336 mg / L.

[0130] In conclusion, the optimized transient transfection process built around GS PIGGYBAC® achieves a substantial increase in titer compared to conventional electroporation processes and outperforms third-party systems under most conditions.

[0131] Example 4: Transposase Optimization Transient Expression Product Quality In this example, protein expression by transposase-optimized transient expression is performed as described in Example 2, and the product quality and attributes are analyzed. In particular, the GS PIGGYBAC® transposase technology is used for transient expression as described in Example 2. The same Xceed GS-KO® host cell line is used for both optimized transient expression and stable expression, and the target protein is cB72.3(IgG1). The products from the two processes are compared.

[0132] Figure 4A shows isoforms of the cB72.3 product, Figure 4B shows aggregation of the cB72.3 product, and Figure 4C shows the N-glycan of the cB72.3 product. The quality of the product from optimized transient expression corresponds to the quality of the product from stable expression.

[0133] Example 5: Determination between transient transfection processes using + / -GS PIGGYBAC® This example provides a method for determining an appropriate transient process based on a balance of time, required supply source, and required recovery titer. The + / -GS PIGGYBAC® transient transfection process is carried out as described in Example 2 and Example 1, respectively.

[0134] Figure 5A shows trastuzumab expression using the -GS PIGGYBAC® transient transfection process (standard transient) and the +GS PIGGYBAC® transient transfection process (GS PIGGYBAC® transient). Figure 5B shows AMS002 expression. Figure 5C shows bsAb-bYlok #1 expression.

[0135] The results described above are summarized in Table 7 below. [Table 7]

[0136] In conclusion, GS PIGGYBAC® transient expression achieves the highest titer in extended culture, while the standard process prioritizes speed / simplicity for rapid turnaround.

[0137] In summary, transient expression does not require separate selection and proliferation steps after transfection, and the transfected cells are used directly for protein production. It is fast and low-cost, but the product provided by transient expression has a low titer. Stable pooled expression requires selection and proliferation steps after transfection and before production. The selection criteria are stringent, and cell passaging is required. The product titer is high, but stable pooled expression is slow and high-cost. The transposase-optimized transient expression of this disclosure balances speed, cost, and product yield, with selection / proliferation and production occurring simultaneously, low-stringent selection applied, and no cell passaging required. The entire transfected cell culture is moved directly and immediately to the product production phase without the cells being removed by any intermediate passaging steps. As a result, transposase-optimized transient expression achieves a high yield of product within a shorter but extended time than stable pooled expression.

[0138] While certain embodiments are illustrated and described herein, it should be understood that the claims are not limited to any particular form or configuration of the elements described and shown. Exemplary embodiments are disclosed herein, and certain terms are used, but these terms are used only in a general and descriptive sense and are not intended to be limiting. In light of the above teachings, modifications and variations of embodiments are possible. Therefore, it should be understood that embodiments may be carried out in ways other than those specifically described.

[0139] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is indicated as being specifically and individually referred to herein.

Claims

1. A method for producing a target protein, To provide a host cell, a vector containing a nucleic acid sequence encoding the target protein, and a transposase, Transfecting the host cells using the vector and the transposase to obtain transfected cells, The transfected cells are cultured to form a culture, This includes recovering the culture and obtaining the target protein from the recovered culture, A method in which the cells of the culture are not subcultured before the culture is harvested.

2. The method according to claim 1, wherein the host cell includes a mammalian cell line.

3. The method according to claim 2, wherein the mammalian cell line includes a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, a mouse myeloma cell line, a human embryonic kidney (HEK) cell line, or a HeLa cell line.

4. The method according to claim 1, wherein the host cell comprises a CHOK1SV GS knockout cell line.

5. The method according to any one of claims 1 to 4, wherein the host cells are pre-cultured for about 4 to about 28 days to form a pre-culture before transfection.

6. The method according to claim 5, wherein the host cells are pre-cultured in chemically defined Chinese hamster ovary (CD CHO) medium supplemented with 6 mM glutamine before transfection.

7. The method according to claim 5 or 6, wherein the preliminary culture has a cell viability of at least 90%.

8. The method according to any one of claims 1 to 7, wherein the vector comprises an adenovirus vector, a pSV vector, a pCMV vector, a vaccinia vector, a retrovirus vector, or a baculovirus vector.

9. The method according to any one of claims 1 to 8, wherein the vector comprises a glutamine synthase gene.

10. The method according to claim 9, wherein the vector comprises a highly active hyper-PIGGYBAC® inverted terminal repeat (ITR) sequence.

11. The method according to any one of claims 1 to 10, wherein the vector comprises one to about five nucleic acid sequences encoding the target protein, or the vector comprises one to about five different vectors comprising the nucleic acid sequences.

12. The method according to any one of claims 1 to 11, wherein the transposase includes RNase H-like transposase, HUH single-stranded DNA transposase, serine transposase, tyrosine transposase, PIGGYBAC® transposase, or a combination thereof.

13. The method according to claim 12, wherein the transposase comprises PIGGYBAC® transposase.

14. The method according to any one of claims 1 to 13, wherein the transposase is contained in a transposase vector or delivered as mRNA.

15. The method according to any one of claims 1 to 14, wherein the transfection of the host cells is carried out using polyethyleneimine (PEI), lipofection, electroporation, magnetofection, microinjection, gene gun insertion, imparefection, hydrostatic pressure, or sonication.

16. The method according to claim 15, wherein the transfection of the host cells is carried out using PEI.

17. The method according to any one of claims 1 to 16, wherein the transfected cells are cultured in a medium, the medium comprising Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, Minimum Essential Medium (MEM), chemically defined Chinese hamster ovary (CD CHO) medium without glutamine supplementation, or a combination thereof.

18. The method according to claim 17, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

19. The method according to claim 18, wherein the culture lacks methionine sulfoxamine (MSX).

20. The method according to any one of claims 1 to 19, wherein the culture is supplied as a bolus on the second and fifth day after transfecting the host cells.

21. The method according to any one of claims 1 to 20, wherein transfection is performed on the first day of the method, and recovery is started on the second day.

22. The method according to claim 21, wherein the recovery is continued from the second day to at least the 14th day of the method.

23. A method for producing a target protein, To provide a host cell, a vector containing a nucleic acid sequence encoding the target protein, and a transposase, Transfecting the host cells using the vector and the transposase to obtain transfected cells, The transfected cells are cultured to form a culture, This includes recovering the culture and obtaining the target protein from the recovered culture, A method wherein the cells of the culture are of the same cell lineage as the transfected cells.

24. The method according to claim 23, wherein the host cell includes a mammalian cell line.

25. The method according to claim 24, wherein the mammalian cell line includes a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, a mouse myeloma cell line, a human embryonic kidney (HEK) cell line, or a HeLa cell line.

26. The method according to claim 23, wherein the host cell comprises a CHOK1SV GS knockout cell line.

27. The method according to any one of claims 23 to 26, wherein the host cells are pre-cultured for about 4 to about 28 days to form a pre-culture before transfection.

28. The method according to claim 27, wherein the host cells are pre-cultured in chemically defined Chinese hamster ovary (CD CHO) medium supplemented with 6 mM glutamine before transfection.

29. The method according to claim 27 or 28, wherein the pre-culture has a cell viability of at least 90%.

30. The method according to any one of claims 23 to 29, wherein the vector comprises an adenovirus vector, a pSV vector, a pCMV vector, a vaccinia vector, a retrovirus vector, or a baculovirus vector.

31. The method according to any one of claims 23 to 30, wherein the vector comprises a glutamine synthase gene.

32. The method according to claim 31, wherein the vector comprises a highly active ultra-PIGGYBAC® inverted terminal repeat (ITR) sequence.

33. The method according to any one of claims 23 to 32, wherein the vector comprises one to about five nucleic acid sequences encoding the target protein, or the vector comprises one to about five different vectors comprising the nucleic acid sequences.

34. The method according to any one of claims 23 to 33, wherein the transposase includes RNase H-like transposase, HUH single-stranded DNA transposase, serine transposase, tyrosine transposase, PIGGYBAC® transposase, or a combination thereof.

35. The method according to claim 34, wherein the transposase comprises PIGGYBAC® transposase.

36. The method according to any one of claims 23 to 35, wherein the transposase is contained in a transposase vector.

37. The method according to any one of claims 23 to 36, wherein the transfection of the host cells is carried out using polyethyleneimine (PEI), lipofection, electroporation, magnetofection, microinjection, gene gun insertion, imparefection, hydrostatic pressure, or sonication.

38. The method according to claim 37, wherein the transfection of the host cells is carried out using PEI.

39. The method according to any one of claims 23 to 38, wherein the transfected cells are cultured in a medium, the medium comprising Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, Minimum Essential Medium (MEM), chemically defined Chinese hamster ovary (CD CHO) medium without glutamine supplementation, or a combination thereof.

40. The method according to claim 39, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

41. The method according to claim 40, wherein the culture lacks methionine sulfoxamine (MSX).

42. The method according to any one of claims 23 to 41, wherein the culture is supplied as a bolus on the second and fifth day after transfecting the host cells.

43. The method according to any one of claims 23 to 42, wherein the transfecting is performed on the first day of the method, and the recovery is started on the second day.

44. The method according to claim 43, wherein the recovery is continued from the second day to at least the 14th day of the method.

45. A method for producing the target viral gene, To provide a host cell, a vector containing a nucleic acid sequence encoding the target viral gene, and a transposase, Transfecting the host cells using the vector and the transposase to obtain transfected cells, The transfected cells are cultured to form a culture, This includes recovering the culture and obtaining the target viral gene from the recovered culture, A method in which the cells of the culture are not subcultured before the culture is harvested.

46. The method according to claim 45, wherein the target viral gene produces AAV virus particles or lentiviral particles.

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