Mixed transient transfection using transposase for high yield product production

Abstract

The invention discloses a method for producing a target protein. The method comprises the following steps: providing a host cell, a vector containing a nucleic acid sequence for coding the target protein and transposase; transfecting the host cell using the vector and the transposase to obtain a transfected cell; 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 of the culture are not passaged prior to harvesting the culture.

Description

Technical Field

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

[0002] Transient gene expression (TGE) can be used to generate small amounts of gene products over a short and finite time span. In this approach, the expression vector enters the host cell line but does not impose the highly stringent selection pressure required to stably integrate the vector DNA into the host cell line's genome. On the other hand, stable pool gene expression can be used to generate large amounts of gene products over a longer time span, where stringent selection is applied so that only cells that have incorporated the expression vector into the transcriptionally active regions of the genome survive. Both TGE and stable gene expression have their limitations. Summary of the Invention

[0003] In some embodiments, this document provides a method for producing a target protein, the method 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 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. This method leverages the advantages of transient gene expression by expressing the target protein over a short period without stringent selection and passage steps, and leverages the advantages of stable pool expression by providing high yields of the target protein. Attached Figure Description

[0004] Figure 1 A transient expression platform for glutamine synthase PIGGYBAC® according to some embodiments of the present disclosure is shown, which enables extended production of the target protein and high titers of the target protein.

[0005] Figure 2 A polyethyleneimine (PEI) transfection procedure according to some embodiments of the present disclosure is shown.

[0006] Figure 3A Product titers from three different product expression platforms are shown according to some embodiments of this disclosure.

[0007] Figure 3B The bYlok bsAb titers using standard transient expression and transient expression of glutamine synthase PIGGYBAC® are shown in some embodiments according to this disclosure.

[0008] Figure 4AIsotypes of cB72.3 products expressed using the stable pool of glutamine synthase PIGGYBAC® and transiently expressed using glutamine synthase PIGGYBAC® are shown according to some embodiments of this disclosure.

[0009] Figure 4B The aggregation of cB72.3 products expressed using a stable pool of glutamine synthase PIGGYBAC® and transient expression of glutamine synthase PIGGYBAC® is shown according to some embodiments of the present disclosure.

[0010] Figure 4C N-glycans of cB72.3 products expressed using the stable pool of glutamine synthase PIGGYBAC® and transient expression of glutamine synthase PIGGYBAC® are shown according to some embodiments of this disclosure.

[0011] Figure 5A The expression of trastuzumab using standard transient expression and transient expression of glutamine synthase PIGGYBAC® is shown in some embodiments of this disclosure.

[0012] Figure 5B The expression of AMS002 using standard transient expression and transient expression of glutamine synthase PIGGYBAC® is shown in some embodiments according to this disclosure.

[0013] Figure 5C The expression of bsAb using standard transient expression and glutamine synthase PIGGYBAC® transient expression is shown in some embodiments according to this disclosure. Detailed Implementation

[0014] In the claims and / or specification, when used in conjunction with the term “comprising,” the use of the word “a / an” may mean “one,” but is also consistent with “one or more,” “at least one,” and “one or more than one.”

[0015] Throughout this application, the term "about" is used to indicate the inherent error variation of values, including those of the methods / apparatus used to measure the values. Generally, depending on the circumstances, the term is intended to cover about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% variability.

[0016] The use of the term "or" in the claims is intended to mean "and / or" unless it is explicitly stated that it refers only to alternatives or that alternatives are mutually exclusive, although this disclosure supports the definition of referring only to alternatives and referring to "and / or".

[0017] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional unlisted elements or method steps.

[0018] As used herein, “nucleic acid,” “nucleic acid molecule,” or “oligonucleotide” refers to a polymeric compound containing covalently linked nucleotides. The term “nucleic acid” includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or MIRNA.

[0019] As used herein, “gene” refers to an assembly of nucleotides encoding a polypeptide and comprising cDNA and genomic DNA nucleic acid molecules. “Gene” also refers to a nucleic acid fragment that can act as a regulatory sequence preceding (5' non-coding sequence) and following (3' non-coding sequence). In some embodiments, a gene is integrated with multiple copies. In some embodiments, a gene is integrated at a predefined copy number.

[0020] As used in this article, "transfection" means the introduction of a foreign nucleic acid molecule containing a vector into a cell. "Transfected" cells include foreign nucleic acid molecules inside the cell, and "transformed" cells are those in which foreign nucleic acid molecules inside the cell induce phenotypic changes in the cell.

[0021] The term "transfection" encompasses a range of techniques used to introduce a target gene into a host cell line. These techniques include, for example, liposome-based transfection (in which a transfection reagent is mixed with DNA to form "liposomes"), electroporation, and the use of cationic polymers. In some embodiments, the cationic polymer used for transfection is the cationic polymer polyethyleneimine (PEI). The PEI complex binds to DNA to form positively charged particles that bind to the negatively charged cell surface before being endocytosed. After being released into the cytoplasm, the DNA can migrate to the nucleus, where gene expression can be initiated. PEI exists in branched and linear forms of varying molecular weights, with transfection efficiencies varying considerably between the different forms. In some embodiments, the PEI reagent used in this disclosure is in a linear form with a molecular weight of 40,000 MW. In some embodiments, the transfection procedure can be further optimized to improve efficiency and reduce toxicity.

[0022] In some aspects, this disclosure provides a method for producing a target protein or other type of gene product. In some embodiments, the method does not require stringent selection and complex passage, and can produce the target protein in high yield over a short period of time.

[0023] In some embodiments, the method includes: providing a host cell, a vector containing a nucleic acid sequence encoding a 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 were not passaged prior to harvesting the culture.

[0024] Figure 1 A method for producing a target protein according to some embodiments of this disclosure is illustrated. For example... Figure 1As shown, host cells are co-transfected using a vector (DNA prep) containing the target gene and a transposase. The period after transfection, during which the transfected cells are cultured and produce the target protein, is called the mixed production phase. During this phase, the transfected cells are selectively expanded and produce the target protein from the target gene. Selection can be performed under conditions less stringent than those required for stable pool selection. In this mixed production phase, the post-transfection process is not interrupted by passages, and there is no need to remove cells from the culture. This contrasts with the stable pool construction process, which typically uses transposases, where there is a precise culture selection phase to expand the transfected cells before inoculation into a dedicated production vessel for recombinant protein synthesis and before at least one subsequent passage culture step. Instead, cell harvesting and protein collection can begin immediately after the transfection procedure (i.e., from minutes to hours to less than a day). Cell passage is not required, not mandated, and not expected. This means that any protein product produced immediately after transfection may ultimately be harvested at the end of the mixed production phase. This contrasts with the stabilization pool process, where substances generated and secreted into the growth medium during the post-transfection selection phase may be lost due to subsequent passage culture steps. In this mixing process, the protein products generated after transfection are not lost from the endpoint harvest due to intermediate passage culture steps. As used herein, “passage” refers to the procedure of harvesting cells from a culture, transferring these cells to one or more culture vessels with fresh growth medium, and using these cells to begin a new culture, and is also known as passage culture. As is known in the art, cell passage allows a subset of cells to continue to proliferate and grow. In such cases, these passaged cells and their progeny belong to a different cell lineage from the original transfected cell line.

[0025] like Figure 1 As shown, in some embodiments, the vector may be a glutamine synthase PIGGYBAC® transposon, and the transposon may be a PIGGYBAC® transposon (including highly active super PIGGYBAC®). The vector PIGGYBAC® transposon carries cargo (the target gene encoding the target protein) and an inverted terminal repeat (ITR) sequence flanking the cargo. The ITR sequence is recognized by the highly active PIGGYBAC® transposon. By using the highly active glutamine synthase PIGGYBAC® transposon, this disclosure integrates the post-stabilization pool transfection "selection-amplification-production" stage into a "mixed" production stage to support high titers of multiple protein forms. The transfected vector containing the product suitably has a PIGGYBAC® ITR. The transposon may be delivered to cells as mRNA, a separate non-GS vector DNA fragment, or even contain a GS transposon along with the product gene.

[0026] In some embodiments, glutamine (e.g., about 3 mM to 10 mM, suitably 6 mM) is added to the culture medium prior to transfection, while the culture medium after transfection does not contain glutamine, in order to provide selection. In some embodiments, selection is less stringent, and about 0.5 mM to about 5 mM of glutamine may be added to the culture medium after transfection. In some embodiments, the concentration of added glutamine is about 1 mM to 4 mM. In some embodiments, the concentration of added glutamine is about 3 mM. Reduced or eliminated glutamine in the culture medium can promote the growth of productive cells containing the expression vector, which can allow for extended culture duration to achieve higher product titers.

[0027] In some embodiments, in this transient expression system based on glutamine synthase, sulfoxide (MSX) is not added to the culture medium after transfection. Conversely, in a stable pool expression system based on glutamine synthase, MSX is added to the culture medium after transfection. As a further comparison, the need for glutamine-containing culture medium (such as a medium with 6 mM glutamine) after transient transfection is generally retained because the glutamine synthase vector may not yet have been efficiently integrated into the host cell genome.

[0028] A host cell or organism expressing a foreign nucleic acid molecule or fragment is referred to as a “recombinant,” “transformed,” or “transgenic” organism. In some embodiments, the host cell is a cell suitable for expressing a foreign nucleic acid molecule or fragment. Suitably, the host cell that can be used in the various methods described herein is a mammalian cell and cell line or culture. As used herein, the term “mammalian cell” includes cells from any member of the mammalian order, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. In some embodiments, the cell is a mouse cell, human cell, Chinese hamster ovary (CHO) cell, CHOK1 cell, CHO-DXB11 cell, CHO-DG44 cell, CHOK1SV cell (including all variants, e.g., POTELLIGENT), or other variants. ® Lonza, Slough, UK) and CHOK1SV GS-KO (glutamine synthase knockout) cells (including all variants, e.g., Xceed) ® Lonza, Slough, UK) and baby hamster kidney (BHK) cells. Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK-293, HeLa cells, or HT1080 cells. In some embodiments, the target protein of this disclosure is produced from HEK-293 cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs). In some embodiments, the target protein of this disclosure is derived from CHOK1SV GS-KO. ®Host cell production. In some embodiments, the host cell is glutamine synthase Xceed. ® cell.

[0029] Mammalian cells comprise mammalian cell cultures, which can be adherent or suspension cultures. Adherent cultures refer to cells that grow on a substrate surface (e.g., a plastic plate, culture dish, or other suitable cell culture growth platform) and can be anchorage-dependent. Suspension cultures refer to cells that can be maintained in, for example, culture flasks or large suspension tanks, allowing for a larger surface area for gas and nutrient exchange. Suspension cell cultures typically utilize stirring or agitation mechanisms to provide adequate mixing. The culture media and conditions used to maintain cells in suspension are generally well known in the art. An exemplary suspension cell culture comprises human embryonic kidney (HEK293) clone cells.

[0030] In some embodiments, the host cell (such as CHOK1SV GS-KO) ® Host cells are pre-cultured for approximately 4 to 28 days to form a pre-culture prior to the transfection step. In some embodiments, host cells are pre-cultured for approximately 6 to 14 days. In some embodiments, host cells are pre-cultured for approximately 8 to 10 days. In some embodiments, host cells (such as CHOK1SVGS-KO) are pre-cultured for approximately 4 to 28 days to form a pre-culture prior to the transfection step. ® The host cells were cultured in chemically defined Chinese hamster ovary (CD CHO) medium. In some embodiments, the CD CHO medium was supplemented with glutamine prior to the transfection step. In some embodiments, the CD CHO medium was substantially free of sulfoxides.

[0031] In some embodiments, the host cells may be cultured in other media such as Duchenne Modified Eagle Medium (DMEM), Los Vickers Memorial Institute (RPMI) 1640, Minimal Essential Medium (MEM), CD CHO medium without glutamine supplementation, or a combination thereof.

[0032] In some embodiments, the pre-cultured host cells prior to transfection have at least 70% cell viability. In some embodiments, the pre-cultured host cells prior to transfection have at least 75%, 80%, 85%, 90%, or at least 95% cell viability. In some embodiments, the pre-cultured host cells prior to transfection have at least 90% cell viability. 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 from SIGMA.

[0033] In some embodiments, the method provides a vector containing a nucleic acid sequence encoding a target protein. The nucleic acid sequence is also referred to herein as the target gene. In some embodiments, the vector includes an adenovirus vector, pSV vector, pCMV vector, vaccinia virus vector, retrovirus vector, or baculovirus vector. In some embodiments, the vector contains a glutamine synthase gene, and 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, allowing selective insertion of the vector into the host cell using a glutamine-free culture medium. In some embodiments, the vector contains a highly active superPIGGYBAC® inverted terminal repeat (ITR) sequence. In some embodiments, the vector contains two ITR sequences, with one of two ITR sequences flanking each side of the target gene. The ITR sequences make the vector compatible with PIGGYBAC® transposases. In some embodiments, the vector is a glutamine synthase PIGGYBAC® transposon. In some embodiments, the vector is a GSquad™ expression vector.

[0034] In some embodiments, a vector is provided that contains a complete target gene for expressing a target protein. In some embodiments, a vector is provided that contains several gene fragments of the target gene. Each fragment of the target gene is used to express a portion of a protein, and the expressed portions of the protein can be assembled to form the target protein. The assembly of the protein can be a self-assembly process. In some embodiments, a vector is provided that contains two or more target genes. The vector is used to simultaneously express two or more protein products. 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 a macromolecular assembly.

[0035] In some embodiments, two or more vectors are provided, each containing a fragment of a target gene and expressing a portion of a target protein. The expressed portion of the target protein can be assembled or self-assembled to form the target protein. In some embodiments, two or more vectors are provided, each containing the target protein. Multiple vectors can be used simultaneously with a transposase to transfect host cells, and the transfected cells can be used to simultaneously express multiple target proteins. The target proteins can be functionally related. In some embodiments, the expressed target protein is assembled or self-assembled to form a polymeric protein or a macromolecular assembly.

[0036] Exemplary 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.

[0037] In exemplary embodiments, the target gene (i.e., the target viral gene) may encode components required for assembling viral particles (including lentiviral and adeno-associated virus particles). Suitably, the methods described herein can be used to produce three AAV constructs: pHelper containing E2A, E4, and VA; pRepCap containing Rep and Cap; and pAAV containing the target gene (GOI). In other embodiments, the methods described herein can be used to produce lentiviral constructs including a lentivirus group-specific antigen (GAG) gene and a lentiviral polymerase (POL) protein; an envelope protein (typically vesicular stomatitis virus glycoprotein (VSV-G)); a Rev protein (a protein that regulates the expression of HIV viral particle proteins); and the target gene (GOI).

[0038] Suitablely, the amount of target protein or target gene produced using the methods described herein is at least 20% greater than that produced using a typical transient transfection system. More suitably, the amount of target protein or target gene produced using the methods 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% greater.

[0039] Various transposases can be used in the methods described herein. In some embodiments, the transposase includes RNase H-like transposases, HUH single-stranded DNA transposases, serine transposases, tyrosine transposases, glutamine synthase PIGGYBAC® transposase, or combinations thereof. In some embodiments, the transposase includes highly active glutamine synthase PIGGYBAC® transposase. In some embodiments, the transposase is contained in a transposase vector or plasmid such that the transposase is encoded by the cell, rather than being added directly to the cell as a functional enzyme. In other embodiments, the transposase may be delivered as mRNA.

[0040] As used herein, “transfection” means the introduction of exogenous nucleic acid molecules, including plasmids and / or vectors, into a cell. “Transfected” cells include exogenous nucleic acid molecules within the cell, and “transformed” cells are those in which exogenous nucleic acid molecules within the cell induce phenotypic changes in the cell. Transfected nucleic acid molecules may integrate into the host cell’s genomic DNA and / or may be temporarily or permanently maintained by the cell outside the chromosome. In some embodiments, “transduction” means infection of mammalian cells with a viral vector and is used interchangeably with “transfection” in this disclosure.

[0041] In some embodiments, host cells are determined to be highly viable prior to transfection. A variety of transfection techniques can be applied. In some embodiments, host cell transfection is performed using polyethyleneimine (PEI), liposome transfection, electroporation, magnetic transfection, microinjection, gene gun insertion, puncture infection, hydrostatic pressure, or sonication. In some embodiments, transfection is performed using PEI.

[0042] Figure 2 An example of transfection is shown, in which the GSquad™ vector containing the target gene and the highly active PIGGYBAC® transposase are used together to transfect Xceed glutamine synthase-KO using PEI transfection reagent. ® Host cell.

[0043] Following transfection, the transfected cells are cultured in a medium but not passaged (i.e., the cells are not removed to prepare other cultures for the production of the target protein). A variety of media can be used to culture the transfected cells, such as Duchenne Modified Eagle Medium (DMEM), Los Vickers Memorial Institute (RPMI) 1640, Minimum Essential Medium (MEM), and chemically defined Chinese hamster ovary (CD CHO) medium. 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 sulfoxide imine (MSX). This differs from stable pool expression, where MSX is used to rigorously select transfected cells with the vector stably integrated into the cell genome. In some embodiments, bubbling feeding is performed to extend production. In some embodiments, cell cultures are bubbled feeding approximately day 3 after host cell transfection. In some embodiments, cell cultures are bubbled feeding on days 2 and 5 after host cell transfection.

[0044] In some embodiments, culturing transfected cells serves as both selection / amplification and production. The absence or low concentration of glutamine in the culture medium allows for near-immediate selection of transfected cells containing the vector, which may or may not be integrated into the cell genome. Transfected cells containing the vector proliferate much faster than cells without the vector, leading to the amplification of the transfected cells. Because glutamine and MSX are not added to the culture medium, selection is less stringent than selection of a stable cell pool. As described herein, all original cells present in the culture at the time of transfection are retained in the culture, and these cells are used for production of the target protein in an early culture phase, rather than for passaged cells for subsequent protein harvesting. In some embodiments, non-stringent selection criteria other than glutamine / MSX medium can be used based on the host cells and the vector. As discussed above, the culture comprises selection / amplification and production, and is therefore also referred to as a hybrid production phase.

[0045] In some embodiments, transfected cells are cultured in a humidified CO2 orbital shaker incubator using Erlenmeyer shake flasks with vented caps. In some embodiments, transfected cells are cultured in shake flasks with sealed caps, provided that a sterile, pre-mixed air source containing 5% CO2 is available to aerate the top space of the flask or bottle during culture preparation and after opening.

[0046] In some embodiments, cells and target proteins are produced in a bioreactor. Cells can be prepared in any suitable bioreactor (also referred to herein as a reactor), including but not limited to stirred tanks, airlift bioreactors, fibrous, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or sputtered bed bioreactors. As used herein, “bioreactor” can include fermenters or fermentation units or any other reaction vessel, and the terms “bioreactor” and “reactor” are used interchangeably with “fermenter.” The term fermenter or fermentation refers to both microbial cultures and mammalian cultures. For example, in some aspects, an example bioreactor unit may perform one or more or all of the following: feeding of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flow of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Example reactor units, such as fermentation units, may contain multiple reactors within a 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. A facility may contain multiple units having one or more reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-feed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor volume may range from about 100 mL to about 50,000 L. Non-limiting examples include volumes of 100 mL, 250 mL, 500 mL, and 750 L. mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 15 L, 20 L, 25 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, 500 L, 550 L, 600 L, 650 L, 700 L 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, suitable reactors can be reusable, single-use, disposable or non-disposable, and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics and / or glass.

[0047] As discussed above, protein production begins in the early stages of cell culture. In some embodiments, transfection occurs on day 1, and cell harvesting can begin on day 2. No passage is performed after transfection, and cells are not removed 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 to day 12.

[0048] In some embodiments, harvesting is carried out once at the end of the cultivation process, and the harvesting time is 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 may continue for additional days to additional weeks.

[0049] The hybrid production phase leverages the advantages of both transient and stable pool expression. Compared to transient expression, production time can be extended to approximately 13 days post-transfection to achieve high titers. Compared to stable pool expression, it avoids the highly rigorous selection and passage processes, eliminates the need to remove cells from the cell culture (i.e., less than 10%, 5%, or 1% of the original cell culture), and significantly shortens the production cycle.

[0050] In an additional aspect, this disclosure provides a method for producing a target protein. In some embodiments, the method includes: 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 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 in the culture have the same cell lineage as the transfected cells. In some embodiments, the cells in the culture have the same cell lineage as the transfected cells because rigorous selection and cell passage are not required. Therefore, cells of "same cell lineage" refer to the original transfected cells that were not removed for individual cell culture and proliferation prior to protein harvesting.

[0051] Example

[0052] In a first embodiment, this document provides a method for producing a target protein, the method 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 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 have not been passaged prior to harvesting the culture.

[0053] Example 2 includes the method of Example 1, wherein the host cell includes a mammalian cell line.

[0054] Example 3 includes the method of Example 2, wherein the mammalian cell lines include Chinese hamster ovary (CHO) cell line, young hamster kidney (BHK) cell line, mouse myeloma cell line, human embryonic kidney cell line (HEK) cell line or HeLa cell line.

[0055] Example 4 includes the method of Example 1, wherein the host cell comprises the CHOK1SV glutamine synthase knockout cell line.

[0056] Example 5 includes the method of any one of Examples 1 to 4, wherein the host cells are precultured for about 4 days to about 28 days to form a preculture prior to transfection.

[0057] Example 6 includes the method of Example 5, wherein the host cells are pre-cultured in chemically defined Chinese hamster ovary (CD CHO) medium supplemented with 6 mM glutamine prior to transfection.

[0058] Example 7 includes the method of Example 5 or 6, wherein the preculture has at least 90% cell viability.

[0059] Example 8 includes the method of any one of Examples 1 to 7, wherein the vector includes an adenovirus vector, a pSV vector, a pCMV vector, a vaccinia virus vector, a retrovirus vector, or a baculovirus vector.

[0060] Example 9 includes the method of any one of Examples 1 to 8, wherein the vector contains a glutamine synthase gene.

[0061] Example 10 includes the method of Example 9, wherein the vector contains a highly active super PIGGYBAC® (glutamine synthase PIGGYBAC®) inverted terminal repeat (ITR) sequence.

[0062] Example 11 includes the method of any one of Examples 1 to 10, wherein the vector comprises one to five nucleic acid sequences encoding a target protein, or the vector comprises one to five different vectors containing nucleic acid sequences.

[0063] Example 12 includes the method of any one of Examples 1 to 11, wherein the transposase includes RNase H-like transposase, HUH single-stranded DNA transposase, serine transposase, tyrosine transposase, glutamine synthase PIGGYBAC® transposase, or a combination thereof.

[0064] Example 13 includes the method of Example 12, wherein the transposase includes glutamine synthase PIGGYBAC® transposase.

[0065] Example 14 includes the method of any one of Examples 1 to 13, wherein the transposase is contained in a transposase vector.

[0066] Example 15 includes the method of any one of Examples 1 to 14, wherein transfection of host cells is performed using polyethyleneimine (PEI), liposome transfection, electroporation, magnetic transfection, microinjection, gene gun insertion, puncture infection, hydrostatic pressure, or sonication.

[0067] Example 16 includes the method of Example 15, wherein PEI is used to transfect host cells.

[0068] Example 17 includes the method of any one of Examples 1 to 16, wherein the transfected cells are cultured in a culture medium comprising DuPont Modified Eagle Medium (DMEM), Los Vickers Memorial Institute (RPMI) 1640, Minimum Essential Medium (MEM), chemically defined Chinese hamster ovary (CD CHO) medium without glutamine supplementation, or a combination thereof.

[0069] Example 18 includes the method of Example 17, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

[0070] Example 19 includes the method of Example 18, wherein the culture is free of sulfoxide imine (MSX).

[0071] Example 20 includes the method of any one of Examples 1 to 19, wherein the culture is fed in a bolus on day 2 and day 5 after transfection of the host cells.

[0072] Example 21 includes the method of any one of Examples 1 to 20, wherein transfection occurs on day 1 of the method and harvest begins on day 2.

[0073] Example 22 includes the method of Example 21, wherein harvesting continues from day 2 until at least day 14 of the method.

[0074] Example 23 provides a method for producing a target protein, the method 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 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 have the same cell lineage as the transfected cells.

[0075] Example 24 includes the method of Example 23, wherein the host cell includes a mammalian cell line.

[0076] Example 25 includes the method of Example 24, wherein the mammalian cell lines include Chinese hamster ovary (CHO) cell lines, young hamster kidney (BHK) cell lines, mouse myeloma cell lines, human embryonic kidney (HEK) cell lines, or HeLa cell lines.

[0077] Example 26 includes the method of Example 23, wherein the host cell comprises the CHOK1SV glutamine synthase knockout cell line.

[0078] Example 27 includes the method of any one of Examples 23 to 26, wherein the host cells are precultured for about 4 days to about 28 days to form a preculture prior to transfection.

[0079] Example 28 includes the method of Example 27, wherein the host cells are pre-cultured in chemically defined Chinese hamster ovary (CD CHO) medium supplemented with 6 mM glutamine prior to transfection.

[0080] Example 29 includes the method of Example 27 or 28, wherein the preculture has at least 90% cell viability.

[0081] Example 30 includes the method of any one of Examples 23 to 29, wherein the vector includes an adenovirus vector, a pSV vector, a pCMV vector, a vaccinia virus vector, a retrovirus vector, or a baculovirus vector.

[0082] Example 31 includes the method of any one of Examples 23 to 30, wherein the vector contains a glutamine synthase gene.

[0083] Example 32 includes the method of Example 31, wherein the vector contains a highly active super PIGGYBAC® (glutamine synthase PIGGYBAC®) inverted terminal repeat (ITR) sequence.

[0084] Example 33 includes the method of any one of Examples 23 to 32, wherein the vector comprises one to five nucleic acid sequences encoding a target protein, or the vector comprises one to five different vectors containing nucleic acid sequences.

[0085] Example 34 includes the method of any one of Examples 23 to 33, wherein the transposase includes RNase H-like transposase, HUH single-stranded DNA transposase, serine transposase, tyrosine transposase, glutamine synthase PIGGYBAC® transposase, or a combination thereof.

[0086] Example 35 includes the method of Example 34, wherein the transposase includes glutamine synthase PIGGYBAC® transposase.

[0087] Example 36 includes the method of any one of Examples 23 to 35, wherein the transposase is contained in a transposase vector.

[0088] Example 37 includes the method of any one of Examples 23 to 36, wherein transfection of host cells is performed using polyethyleneimine (PEI), liposome transfection, electroporation, magnetic transfection, microinjection, gene gun insertion, puncture infection, hydrostatic pressure, or sonication.

[0089] Example 38 includes the method of Example 37, wherein PEI is used to transfect host cells.

[0090] Example 39 includes the method of any one of Examples 23 to 38, wherein the transfected cells are cultured in a culture medium comprising DuPont Modified Eagle Medium (DMEM), Los Vickers Memorial Institute (RPMI) 1640, Minimum Essential Medium (MEM), chemically defined Chinese hamster ovary (CD CHO) medium without glutamine supplementation, or a combination thereof.

[0091] Example 40 includes the method of Example 39, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

[0092] Example 41 includes the method of Example 40, wherein the culture is free of sulfoxide imine (MSX).

[0093] Example 42 includes the method of any one of Examples 23 to 41, wherein the culture is fed in bolus feed on day 2 and day 5 after transfection of host cells.

[0094] Example 43 includes the method of any one of Examples 23 to 42, wherein transfection occurs on day 1 of the method and harvest begins on day 2.

[0095] Example 44 includes the method of Example 43, wherein harvesting continues from day 2 until at least day 14 of the method.

[0096] Example 45 is a method for producing a target viral gene, the method comprising: providing a host cell, a vector containing a nucleic acid sequence encoding a 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 harvesting the culture to obtain the target viral gene from the harvested culture, wherein the cells of the culture have not been passaged prior to harvesting the culture.

[0097] Example 46 includes the method of Example 45, wherein the target viral gene produces AAV viral particles or lentiviral particles.

[0098] Example

[0099] Example 1: PEI transient expression

[0100] In this example, a PEI baseline transient expression is performed according to certain embodiments of this disclosure. The timing of the procedure is shown in Table 1 below:

[0101]

[0102] I. Preparations before the day of transfection (Day - 1)

[0103] 1. Change CHOK1SV GS-KO ® The host cell line was thawed from cryopreservation and cultured routinely until the day before transfection. CHOK1SV GS-KO ® Host cells should be cultured appropriately for at least 4 days but no more than 28 days prior to transfection. The culture should have greater than 90% viability prior to transfection (as measured by trypan blue exclusion). Cultures should be grown pre-transfection using CD CHO / 6 mM L-glutamine medium. CD CHO can be, for example, Thermo Fisher Scientific, catalog number 10743-029.

[0104] 2. Prepare vector constructs for transfection. Suspend uncut vector DNA at a concentration of 1000 µg / mL in sterile Tris-EDTA (TE) buffer. Alternatively, for products containing 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 vectors can be co-transfected for transient expression.

[0105] 3. Passage the host cell line the day before transfection:

[0106] 3.1 Preheat the appropriate growth medium (CD CHO + 6 mM glutamine) to 35.5°C to 37.0°C.

[0107] 3.2 Use sterile serum pipettes to transfer serum from CHOK1SV GS-KO ® The cells were aseptically removed as a sample of the cell suspension for counting.

[0108] 3.3 Count cells using a hemocytometer or automated cell counter, and assess cell viability using trypan blue dye or other methods. Use cell count and viability data to determine cell concentration and culture viability.

[0109] 3.4 For every 20 mL of transient transfection culture required, use 1.0 × 10⁻⁶ mg / L. 6 The target concentration of 1 live cells / mL is inoculated into 30 mL of fresh culture. Recommended culture volume ranges are given in Table 2.

[0110] 3.5 If a vent cover is used, replace the cover and place the new culture in a humidified CO2 track shaker incubator set at 35.5°C to 37.0°C, 140 ± 5 rpm, relative humidity >85%, and containing 5% CO2 air.

[0111] 3.6 If a sealed lid is used, vent the top space using a sterile, pre-mixed air supply containing 5% CO2, then replace the lid and place the new culture in an orbital shaker incubator set to 35.5°C to 37.0°C and 140 ± 5 rpm.

[0112] Table 2 shows the culture volumes for transient transfection in Example 1.

[0113]

[0114] II. Transfection (Day 0)

[0115] The day of transfection (the day after passage culture, considered day 0 in this procedure):

[0116] 1. Calculate the total amount of culture medium, PEI, sodium acetate, cells, and glutamine synthase vector DNA required for transfection.

[0117] 1.1 For a single standard transfection of 20 mL, a total of 20 × 10⁻⁶ mL is required. 6 One live 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).

[0118] 1.2 Transfection can be increased or decreased proportionally, but the relative proportion of reagents per milliliter of culture should be maintained appropriately, as shown in Table 3.

[0119] Table 3: Reagent ratios during transfection

[0120]

[0121] Please note that the above procedure applies to transfecting a single glutamine synthase vector, which carries at most four product genes. However, if two vectors are co-transfected (e.g., for co-expression of multipart vectors), the amount of plasmid DNA is shared between the two vectors. For example, for a 20 mL transfection, transfect 20 µg of each vector. Too little DNA means low transfection efficiency, while too much DNA may be toxic to cells.

[0122] 2. Prepare an appropriate volume of CD CHO aseptically using 6 mM L-glutamine and preheat to 35.5°C to 37.0°C.

[0123] 3. Using a sterile serum pipette, dispense 1.0 × 10⁶ ml of serum from the previous day. 6 A sample of the cell suspension was aseptically extracted from CHOK1SV GS-KO® cultures inoculated at a concentration of live cells / mL.

[0124] 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 the total number of live cells. Note that, appropriately, the culture should be at least 90% viable before transfection. Low viability will reduce transfection efficiency.

[0125] 5. Calculate the required culture volume to obtain the desired number of cells for transfection (20 × 10⁶ cells per 20 mL transfection volume). 6 (Number of live cells). Please note that cells may be lost during the centrifugation and washing steps. Therefore, it is recommended to centrifuge up to twice the number of cells needed in the next step, at a rate of 200 × g.

[0126] 5.1 Aseptically remove the calculated culture volume (from the previous step) and centrifuge at 200 × g for 5 minutes.

[0127] 5.2 Remove the supernatant, remove the precipitate by gently tapping the bottom of the tube, and disperse the cell precipitate in a sufficient volume of pre-warmed CD CHO + 6 mM L-glutamine to obtain a concentration of 1.0 × 10⁻⁶. 6 live cells / mL.

[0128] 5.3 Determine the viable cell concentration and adjust to a final concentration of 1.0 × 10⁻⁶ using CD CHO + 6 mM L-glutamine. 6 live cells / mL.

[0129] Incubate the cell suspension at ambient temperature for a maximum of 30 minutes while transfecting. Furthermore, it is important that the prepared cell suspension be maintained at ambient temperature for a maximum of 30 minutes and not kept at high temperatures. At high cell concentrations, cells maintained at 37°C will rapidly become hypoxic, leading to rapid cell death.

[0130] 6. For a single standard transfection, transfer 20 mL of cell suspension from the previous step to a 125 mL Lerlenmeyer flask.

[0131] 7. Add the following substances to the flask in the prescribed order, shaking vigorously after each addition:

[0132] 7.140 µL (= 40 µg) of sterile circular plasmid DNA at a concentration of 1 mg / mL;

[0133] 7.2100 µL PEI; and

[0134] 7.367 µL sodium acetate.

[0135] 8. Place the cells back into a humidified CO2-equipped orbital shaker incubator set at 35.5℃ to 37.0℃, 140 ± 5 rpm, relative humidity >85%, and containing 5% CO2 air, and incubate for 4 hours.

[0136] 9. After 4 hours, transfer the cells to a shaking incubator at 32°C, 5% CO2, 85% humidity, and 140 rpm, or lower the temperature of the existing incubator to 32°C. If using a sealed lid, purge the top space with sterile, pre-mixed sterile air containing 5% CO2, then replace the lid and place the new culture in a track shaker incubator set to 32°C and 140 ± 5 rpm.

[0137] 10. Incubate the transfected cells in a shaking incubator for 8 to 10 days. Monitor the culture regularly for growth and viability, aiming to harvest before the average viability drops below 90%.

[0138] 11. Harvest the culture using appropriate methods, remove the sample from the culture container, clarify the culture supernatant, and store it at an appropriate temperature for further analysis. Note that the temperature can be switched to 32°C at any time between 4 and 24 hours after transfection to better suit laboratory schedules. However, the temperature switch should not be performed at least 4 hours prior.

[0139] Example 2: GS PIGGYBAC® PEI transient / transposase transient expression

[0140] In this example, highly active GS PIGGYBAC® transposase was used in transient gene expression to maximize the final product yield. Example 2 is similar to the stable pool construction process, but also features key adjustments to the transient gene expression workflow. The timeline for Example 2 is shown in Table 4 below:

[0141]

[0142] First, although L-glutamine was not supplemented in the culture, MSX was not used for highly selective integration of the vector into the host cell genome at transcriptional active sites. Second, bolus feeding on days 2 and 5 post-transfection helped prolong the culture and maximize yield at the end of the culture. Third, GS PIGGYBAC® transposase was co-transfected into the cells as circular plasmid DNA rather than mRNA. Since transiently transfected cultures are not intended for long-term culture or to generate clonal cell lines, accidental integration of the transposase gene into the genome is not considered a significant factor. Fourth, PEI was used for transfection, not electroporation.

[0143] I. Preparations before the day of transfection (day - 2)

[0144] 1. Thaw the CHOK1SV GS-KO® host cell line from cryopreservation and culture it routinely until two days prior to transfection. In this example, CHOK1SV GS-KO® cells were cultured for at least 4 days but no more than 28 days prior to transfection. The culture had greater than 90% viability prior to transfection (as measured by trypan blue exclusion assay). Cells were grown using CD CHO medium supplemented with 6 mM L-glutamine (CD CHO / 6 mM glutamine) until transfection.

[0145] 2. Prepare vector constructs for transfection.

[0146] After preparation, the uncut GS vector DNA was suspended in sterile Tris-EDTA (TE) buffer at a concentration of 1000 µg / mL. The GS vector contains the GS PIGGYBAC® ITR sequence for this method. In this example, the latest GSquad™ vector platform was used.

[0147] 3. Two days before transfection, perform passage culture of the host cell line:

[0148] 3.1 Preheat the appropriate growth medium (CD CHO / 6 mM glutamine) to 35.5°C to 37.0°C.

[0149] 3.2 Use a sterile serum pipette to aseptically extract a sample of the cell suspension from the CHOK1SV GS-KO® cells for counting.

[0150] 3.3 Count cells using a hemocytometer or automated cell counter, and assess cell viability using trypan blue dye or other methods. Use cell count and viability data to determine cell concentration and culture viability.

[0151] 3.4 For every 20 mL of transient transfection culture required, use 0.2 × 10⁻⁶ mg / L. 6 The target concentration of 1 live cells / mL is inoculated into 30 mL of fresh culture. Larger transfection volumes can be achieved; recommended culture volume ranges are given in Table 5 below:

[0152]

[0153] 3.5 If a vent cover is used, replace the cover and place the new culture in a humidified CO2 track shaker incubator set at 35.5°C to 37.0°C, 140 ± 5 rpm, relative humidity >85%, and containing 5% CO2 air.

[0154] 3.6 If a sealed lid is used, vent the top space using a sterile, pre-mixed air supply containing 5% CO2, then replace the lid and place the new culture in an orbital shaker incubator set to 35.5°C to 37.0°C and 140 ± 5 rpm.

[0155] II. Transfection (Day 0)

[0156] The day of transfection (two days after passage culture, considered day 0 in this procedure):

[0157] 1. Calculate the total amount of culture medium, PEI, cells, and DNA (both the GS PIGGYBAC® compatible vector containing the product gene and the plasmid SPB-DNA encoding the transposase) required for transfection.

[0158] A single transfection of 20 mL requires a total of 20 × 10 6 One live cell, 40 µg GS vector DNA, 4 µg SPB-DNA, and 100 µL PEI (Transporter 5). Transfection can be increased or decreased proportionally, but the relative proportions of reagents per mL of culture should be appropriately maintained. Table 6 below shows the reagent proportions for transfection:

[0159]

[0160] 2. Aseptically prepare an appropriate volume of CD CHO using 10 mL / L HT supplement and preheat to 35.5°C to 37.0°C.

[0161] 3. Using a sterile serum pipette, administer 0.2 × 10⁻⁶ serum from the first two days. 6 A sample of the cell suspension was aseptically extracted from CHOK1SV GS-KO® cultures inoculated at a concentration of live cells / mL.

[0162] 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 the total number of live cells. Note that the culture should be at least 90% viable. Low viability will reduce transfection efficiency.

[0163] 5. Calculate the required culture volume to obtain the desired number of cells for transfection (20 × 10⁶ cells per 20 mL transfection volume). 6 (Number of live cells). Please note that cells may be lost during the centrifugation and washing steps. Therefore, it is recommended to centrifuge up to twice the number of cells needed in the next step, at a rate of 200 × g.

[0164] 5.1 Aseptically remove the calculated culture volume (from the previous step) and centrifuge at 200 × g for 5 minutes.

[0165] 5.2 Remove the supernatant, remove the precipitate by gently tapping the bottom of the tube, and disperse the cell precipitate in a sufficient volume of pre-warmed CD CHO + 10 mL / L HT supplement to obtain a concentration of 1.0 × 10⁻⁶. 6 live cells / mL.

[0166] 5.3 Determine the viable cell concentration and adjust to 1.0 × 10⁻⁶ using CD CHO + 10 mL / L HT supplement (sodium hypoxanthine (10 mM) and thymidine (1.6 mM), Thermo Fisher catalog number 11067030). 6 The final concentration is 1 live cells / mL.

[0167] 6. For a single standard transfection, transfer 20 mL of cell suspension from the previous step to a 125 mL Lerlenmeyer flask.

[0168] 7. Add the following substances to the flask in the prescribed order, shaking vigorously after each addition:

[0169] 7.140 µL (= 40 µg) of sterile circular plasmid DNA encoding the product gene at a concentration of 1 mg / mL;

[0170] 7.24 µg SPB-DNA circular plasmid DNA;

[0171] 7.3100 µL PEI.

[0172] 8. Place the cells back into a humidified CO2-filled orbital shaker incubator set to 35.5°C to 37.0°C, 140 ± 5 rpm, relative humidity >85%, and containing 5% CO2 air. If using a sealed lid, purge the top space with a sterile, pre-mixed supply of 5% CO2 air, then replace the lid and place the new culture in the orbital shaker incubator set to 35.5°C to 37.0°C and 140 ± 5 rpm.

[0173] 9. On days 2 and 5 post-transfection, add 10% v / v (= 2 mL, total transfection volume 20 mL) of CHO CDEfficientFeed™ B liquid nutrient supplement (Thermo Fisher catalog number A 1024001).

[0174] 10. Cultures can be periodically monitored for growth and viability using a hemocytometer or automated cell counter. Note that cultures should be harvested before viability drops below 90%.

[0175] 11. After 14 days, harvest the culture using appropriate methods, remove the sample from the culture container, clarify the culture supernatant and store it at an appropriate temperature for further analysis.

[0176] Please note that in Example 2, the expression vector and transposase were used together to transfect the host cell line using the PEI transfection reagent.

[0177] Example 3: Optimizing the titer of transient transposase expression

[0178] In this embodiment, protein expression optimized by transposase transient expression was performed as described in Example 2, and the results were compared with those of a conventional platform (similar to the platform described in Example 1, but using electroporation for transfection) and a third-party system.

[0179] Figure 3A This diagram compares the transient expression of GS PIGGYBAC® transposase technology with conventional electroporation processes and third-party systems. Target proteins included cB72.3 (IgG1), trastuzumab, AMS002, asymmetric Mab, and classic bsAb. Figure 3AAs shown, the instantaneous titer is increased by up to 30 times compared to the conventional process using GS PIGGYBAC®.

[0180] Figure 3B This demonstrates the advantages of GS PIGGYBAC® transposase technology in transient expression compared to its use for expressing bYlok. ® Comparison of conventional electroporation expression processes for bsAb. The average harvest titer for transient expression using the GS PIGGYBAC® transposase technology is approximately 336 mg / L.

[0181] In summary, the optimized transient transfection process built around GS PIGGYBAC® achieves a significant increase in titer compared to conventional electroporation processes and outperforms third-party systems under most conditions.

[0182] Example 4: Optimizing the quality of products from transient transposase expression

[0183] In this example, protein expression optimized by transposase transient expression was performed as described in Example 2, and product quality and properties were analyzed. Specifically, GS PIGGYBAC® transposase technology was used for transient expression, as described in Example 2. The same Xceed GS-KO... ® The host cell line was used to optimize both transient and stable expression, and the target protein was cB72.3 (IgG1). The products from the two processes were compared.

[0184] Figure 4A The isotype of cB72.3 product is shown. Figure 4B The aggregation of cB72.3 products was shown, and Figure 4C The N-glycan of the cB72.3 product is shown. The quality of the product from optimized transient expression is comparable to that from stable expression.

[0185] Example 5: Determining the process between + / - GS PIGGYBAC® transient transfection.

[0186] This example provides a method for determining a suitable transient transfection process based on a balance of time, required resources, and desired harvest titer. The + / - GS PIGGYBAC® transient transfection process is performed as described in Example 2 and Example 1, respectively.

[0187] Figure 5A The expression of trastuzumab using the -GS PIGGYBAC® transient transfection procedure (standard transient) and the +GS PIGGYBAC® transient transfection procedure (GS PIGGYBAC® transient) is demonstrated. Figure 5B The expression for AMS002 is shown. Figure 5CThe expression for bsAb-bYlok #1 is shown.

[0188] The results are summarized in Table 7 below:

[0189]

[0190] In summary, GS PIGGYBAC® transient expression achieves the highest titers with extended incubation, while standard procedures prioritize speed / simplicity for rapid turnaround.

[0191] In summary, transient expression requires no separate selection and amplification steps after transfection, and transfected cells can be used directly for protein production. While fast and inexpensive, transient expression provides lower product titers. Stable-pool expression requires selection and amplification steps after transfection and before production. Selection criteria are stringent, and cell passage is required. While product titers are high, stable-pool expression is slow and expensive. The transposase-optimized transient expression of this disclosure balances speed, cost, and product yield, where selection / amplification and production occur simultaneously, employing less stringent selection and eliminating the need for cell passage. Transfected cell cultures proceed directly and immediately to the product production stage, without the need for cell removal through any intermediate passage culture steps. Therefore, transposase-optimized transient expression achieves high product yields within a longer timeframe than transient expression (but shorter than stable-pool expression).

[0192] It should be understood that although certain embodiments have been shown and described herein, the claims are not limited to the specific form or arrangement of the described and illustrated portions. Illustrative embodiments have been disclosed in this specification, and although specific terminology has been used, it is for general and descriptive purposes only and not for limiting purposes. In view of the above teachings, modifications and variations to the described embodiments are possible. Therefore, it should be understood that the described embodiments can be practiced in ways different from those specifically described.

[0193] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. A method for producing a target protein, the method comprising: Provide a host cell, a vector containing a nucleic acid sequence encoding the target protein, and a transposase; The host cells were transfected using the vector and the transposase to obtain transfected cells; Culture the transfected cells to form a culture; and The culture is harvested to obtain the target protein from the harvested culture. The cells in the culture were not passaged before the culture was harvested.

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

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

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

5. The method according to any one of claims 1 to 4, wherein the host cells are precultured for about 4 days to about 28 days to form a preculture prior to the transfection.

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

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

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 virus 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 of claim 9, wherein the vector comprises a highly active super PIGGYBAC® inverted terminal repeat (ITR) sequence.

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

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

13. The method of 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 transfection of the host cells is performed using polyethyleneimine (PEI), liposome transfection, electroporation, magnetic transfection, microinjection, gene gun insertion, puncture infection, hydrostatic pressure, or sonication.

16. The method of claim 15, wherein the transfection of the host cells is performed using PEI.

17. The method according to any one of claims 1 to 16, wherein the transfected cells are cultured in a culture medium comprising DuPont Modified Eagle Medium (DMEM), Los Vickers 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 of claim 17, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

19. The method of claim 18, wherein the culture is free of sulfoxide imine (MSX).

20. The method according to any one of claims 1 to 19, wherein the culture is fed in bolus feed on days 2 and 5 after transfection of the host cells.

21. The method according to any one of claims 1 to 20, wherein transfection occurs on day 1 of the method and harvest begins on day 2.

22. The method of claim 21, wherein harvesting continues from the second day of the method to at least the fourteenth day.

23. A method for producing a target protein, the method comprising: Provide a host cell, a vector containing a nucleic acid sequence encoding the target protein, and a transposase; The host cells were transfected using the vector and the transposase to obtain transfected cells; The transfected cells were cultured to form a culture. as well as The culture is harvested to obtain the target protein from the harvested culture. The cells in the culture have the same cell lineage as the transfected cells.

24. The method of claim 23, wherein the host cell comprises a mammalian cell line.

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

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

27. The method according to any one of claims 23 to 26, wherein the host cells are precultured for about 4 days to about 28 days to form a preculture prior to the transfection.

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

29. The method of claim 27 or claim 28, wherein the preculture has at least 90% cell viability.

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 virus 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 of claim 31, wherein the vector comprises a highly active super PIGGYBAC® inverted terminal repeat (ITR) sequence.

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

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

35. The method of 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 performed using polyethyleneimine (PEI), liposome transfection, electroporation, magnetic transfection, microinjection, gene gun insertion, puncture infection, hydrostatic pressure, or sonication.

38. The method of claim 37, wherein the transfection of the host cells is performed using PEI.

39. The method according to any one of claims 23 to 38, wherein the transfected cells are cultured in a culture medium comprising DuPont Modified Eagle Medium (DMEM), Los Vickers 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 of claim 39, wherein the culture medium comprises CD CHO medium without glutamine supplementation.

41. The method of claim 40, wherein the culture is free of sulfoxide imine (MSX).

42. The method according to any one of claims 23 to 41, wherein the culture is fed in bolus feed on days 2 and 5 after transfection of the host cells.

43. The method according to any one of claims 23 to 42, wherein the transfection occurs on day 1 of the method and the harvest begins on day 2.

44. The method of claim 43, wherein the harvest continues from the second day of the method to at least the fourteenth day.

45. A method for producing a target viral gene, the method comprising: Provide a host cell, a vector containing a nucleic acid sequence encoding the gene of the target virus, and a transposase; The host cells were transfected using the vector and the transposase to obtain transfected cells; Culture the transfected cells to form a culture; and The culture is harvested to obtain the target viral gene from the harvested culture. The cells in the culture were not passaged before the culture was harvested.

46. ​​The method of claim 45, wherein the target viral gene produces AAV viral particles or lentiviral particles.

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