KR101971614B1 - Cell line with increased production of target protein and producing method for target protein using the same - Google Patents

Abstract

One embodiment of the present invention relates to a cell in which the expression of a target protein is enhanced, which is a cell line in which PyLT and EBNA-1 genes are simultaneously introduced and MTX is amplified. Using the cell in a transient gene expression system not only improves the productivity of the target protein, but also has an advantage that an N-glycan pattern is similar to the protein produced from wild-type cells. Accordingly, the cell according to one embodiment of the present invention and a method for producing the target protein using the same can be used in fields such as preclinical studies, new drug screening, therapeutic protein production, and the like.

Description

TECHNICAL FIELD [0001] The present invention relates to a cell line having increased production of a target protein and a method for producing a target protein using the cell line. [0002]

One embodiment of the present invention relates to a cell line in which expression of a target protein is enhanced and a method for producing a target protein using the same.

As proteins are used as drug substances and the need to select new drug proteins increases, a technology for rapidly and efficiently producing proteins is required. Generally, a gene encoding a target protein is inserted into an expression vector and transfected into a cell line in which the expression vector is to be produced. After culturing the prepared cell line in a large amount, the desired recombinant protein is isolated and purified by an appropriate method to obtain a desired recombinant protein.

On the other hand, a recombinant protein production system based on transient gene expression (TGE) is a system capable of efficiently producing proteins. Mammalian cell-based TGE systems have undergone significant technological improvements for decades. HEK293 cells and CHO cells are known as suitable mammalian host cells for application to large scale TGE systems of the desired protein. Among these two cell types, TGE system using HEK293 cells shows high yield of transgene and excellent transformation efficiency. However, the HEK293 cell-based TGE system has disadvantages such that the protein product has a different N-sugar chain structure or is contaminated by human viruses.

Therefore, in order to develop a TGE system employing stable CHO cells, various methods for solving relatively low transfection efficiency and low protein productivity, which are shortcomings of CHO cells, have been studied.

An object of the present invention is to provide a cell in which expression of a target protein is enhanced in a transient gene expression system.

Another object of the present invention is to provide a method for producing the above cells.

It is another object of the present invention to provide a method for producing a target protein using the above cells.

In order to achieve the above object,

One embodiment of the present invention is a nucleic acid encoding at least 100 copies of a nucleic acid encoding PyLT (Polyoma virus large T-antigen) or a fragment thereof, and a nucleic acid encoding EBNA-1 (Epstein Barr virus nuclear antigen 1) Lt; RTI ID = 0.0 > 100 < / RTI > copies.

Another embodiment of the present invention relates to a method for the production of a nucleic acid encoding PyLT or EBNA-1 or a fragment encoding EBNA-1 or fragment thereof by introducing into a cell a nucleic acid encoding PyLT or a fragment thereof and a nucleic acid encoding EBNA-1 or fragment thereof, followed by a dhfr / MTX (methotrexate) And then amplifying the transformed cell line.

In another embodiment of the present invention, there is provided a method for producing an EBNA-1 comprising: 1) introducing an expression vector containing a nucleic acid encoding a target protein into a cell line containing at least 100 copies of a nucleic acid encoding PyLT and containing at least 100 copies of a nucleic acid encoding EBNA- Transient tansfection, and 2) culturing the cell line to obtain a target protein.

The cell line expressing and amplifying the PyLT and EBNA-1 gene according to an embodiment of the present invention not only has an excellent productivity of target protein in a transient gene expression system but also has a protein produced from wild type cell line without genetic manipulation and N - The oligosaccharide pattern has almost similar advantages. Therefore, the cell line according to one embodiment of the present invention and the method for producing a target protein using the same can be usefully used in the fields of preclinical studies, new drug screening, therapeutic protein production, and the like.

FIGS. 1A and 1B are Western blot analyzes of the expression levels of EBNA-1 and PyLT in Null, EBNA-1-amplified cells and PyLT-amplified cells.
Fig. 1C and Fig. 1D were obtained by harvesting the culture supernatant obtained after transient transfection in a vector producing an Fc-fusion protein in Null, EBNA-1 amplified cells and PyLT-amplified cells, and cultured on a Cedex Bio HT analyzer with Fc - the result of quantifying the concentration of the fusion protein.
FIG. 2A is a Western blot analysis of the expression levels of EBNA-1 and PyLT in cells in which EBNA-1 / PyLT was simultaneously amplified according to Null and MTX concentrations.
2B and 2C, the culture supernatant obtained after transient transfection was harvested from the vector producing the Fc-fusion protein in the cell in which EBNA-1 / PyLT was amplified according to Null and MTX concentrations, Fc-fusion protein concentration and qp.
3A and 3B show the EBNA-1 / EB virus potential replication origin (oriP) sequence and the PyLT / Py virus origin replication origin (Pyori) sequence in EBNA-1 / PyLT-amplified cells according to Null and MTX concentrations Fc-fusion protein was produced by harvesting the culture supernatant obtained after transient transfection and measuring the concentration of Fc-fusion protein using a Cedex Bio HT analyzer on the third day.
Fig. 4a shows the results of quantifying the Fc-fusion protein concentration in 16 selected clones by harvesting the culture supernatant obtained after transient transfection and measuring the Fc-fusion protein concentration on the third day using a Cedex Bio HT analyzer .
FIG. 4B is a Western blot analysis of the expression levels of EBNA-1 and PyLT in 16 selected clones.
Figures 5A and 5B show linear regression plots of expression levels of q _p versus EBNA-1 and q _p versus PyLT, respectively, for 16 selected clones. Were normalized to the lowest expression levels of EBNA-1 or PyLT, EP-amp-67 or EP-amp-32, respectively (black triangles). CHO-DG44 cells (white circles) were used as controls.
Figures 6a and 6b show the survival cell concentration and viability profiles of EBX-1 / PyLT simultaneously MTX amplified clones during batch culture. Control: white circle, EP-amp-3: black circle, EP-amp-19: black triangle and EP-amp-20: black rectangle.
Figures 7a-7d show the profiles of the metabolites of EBNA-1 / PyLT co-amplification clones (glucose 7a, lactate 7b, glutamine 7c, ammonia 7d, respectively) during batch incubation. Control: white circle, EP-amp-3: black circle, EP-amp-19: black triangle and EP-amp-20: black rectangle.
Figures 8a-8c show cell growth (8a), viability (8b) and Fc-fusion protein concentration (8c) of MTX amplified clones simultaneously EBNA-1 / PyLT during TGE-based incubation. Control: white circle, EP-amp-3: black circle, EP-amp-19: black triangle and EP-amp-20: black rectangle.
FIG. 9 is a Western blot analysis of the expression levels of EBNA-1 and PyLT in TGE-cultured EP-amp-20 cells. Equivalent amounts of cellular proteins were isolated and validated using EBNA-1, PyLT and beta-actin antibodies.
10a to 10d show q _p (10a), relative mRNA expression (10b), relative amount of duplicated DNA (10c), and transfection efficiency (10d), respectively, of TGE-based cultured EBNA-1 / PyLT co- Lt; / RTI >
FIG. 11 shows the results of analysis of the N-linked glycans of the Fc-fusion non-glycoprotein obtained in the TGE-based culture.
The error bars in the figure show the standard deviation calculated from data obtained from three experiments. * P < 0.05. Also, q _p is based on data from day 0 to day 3 and is calculated from a plot of Fc-fusion protein concentration versus time-integrated value of the survival cell growth curve.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention relates to a transformed cell comprising at least 100 copies of a nucleic acid encoding PyLT or a fragment thereof and at least 100 copies of a nucleic acid encoding EBNA-1 or a fragment thereof. At this time, the cells may be cell lines exhibiting homogeneous genotypes and phenotypes while being continuously subcultured.

In transient gene expression systems based on mammalian cell lines, the productivity of recombinant proteins also decreases rapidly because the number of plasmids present in the episomal state during cell division gradually decreases. To solve this problem, mammalian cells were transfected with virus-derived proteins such as Epstein Barr virus nuclear antigen-1 (EBNA-1), polyoma virus large T antigen (PyLT), or simian virus 40 large T antigen (SV40LT) Has been attempted to improve replication and persistence of episomal plasmids.

Thus, the inventors of the present invention found that a cell line that exists in the state of simultaneous expression and amplification of PyLT and EBNA-1 protein, that is, EBNA-1 / EB virus potential replication origin ( ori P) and PyLT / Py virus origin replication origin ( Py ori) System, the plasmid replication and persistence are improved in the cell line in which the system is simultaneously implemented, and the present invention has been completed.

As used herein, the term " copy " refers to a replicate of a nucleic acid or nucleic acid fragment replicated and amplified in a gene expression system. That is, " number of gene copies " can be used in the same meaning as " number of genes ". In addition, the level of intracellular gene expression may increase in proportion to the number of gene copies.

In addition, the term " at least 100 copies or more " in the present application means that the copy number of intracellular replication target gene is larger than that of a general gene expression system. For example, the inclusion of a large number of gene copies in comparison with a general gene expression system may mean a gene expression system with improved productivity of the target protein.

The cell contains 100 or more copies of the nucleic acid encoding PyLT and may comprise 100 copies to 10,000 copies, 300 copies to 5,000 copies, 500 copies to 3,000 copies, or 800 copies to 1,200 copies. The cell line may also contain from 100 copies to 10,000 copies, from 300 copies to 5,000 copies, from 500 copies to 3,000 copies, or from 800 copies to 1,200 copies of the nucleic acid encoding EBNA-1.

For example, the PyLT may be the amino acid sequence of SEQ ID NO: 1 (GenBank: AAB59901.1). The EBNA-1 may be an amino acid sequence of SEQ ID NO: 2 (GenBank: BAB03282.1).

Specifically, the cell may be deficient in the dhfr (dihydrofolate reductase) gene. In addition, the cell may be an animal cell, but not limited thereto, a CHO cell or a CHO cell in which a dhfr gene has been deleted. For example, CHO-DG44 cell strain.

In addition, the cell may be a dfhr / MTX (methotrexate) system in which a nucleic acid encoding PyLT and EBNA-1 is amplified. The dhfr / MTX system is a system capable of transforming a vector containing a dhfr gene and a foreign gene as a selection gene into a cell line and amplifying the foreign gene using methotrone sulfate (MTX), which is an inhibitor of dhfr.

For example, a vector containing a dhfr gene and a PyLT gene, and a vector containing a dhfr gene and an EBNA-1 gene are introduced into a cell in which the dhfr gene has been deleted. Thereafter, single clones were selected from the cells simultaneously expressed with PyLT / EBNA-1, and the number of copies of intracellular PyLT and EBNA-1 gene was increased by increasing the MTX concentration in the obtained clone culture medium for a certain period of time . That is, ultimately, cells (cell lines) having an increased expression rate of PyLT and EBNA-1 protein can be obtained at the same time. As described above, it was confirmed that the cells having multiple copies of PyLT / EBNA-1 had excellent yields of the target protein in the transient gene expression system.

In addition, another embodiment of the present invention relates to a transformed cell into which an expression vector comprising a nucleic acid encoding a target protein is additionally introduced into the cell.

The term " vector " as used herein means a carrier that allows a gene to be introduced into a host cell. Or the vector is understood as nucleic acid means comprising a nucleotide sequence that can be spontaneously replicated as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. (Invitrogen, USA), pcDNA3.1 (Invitrogen, USA), pCI vector (Promega, USA), and mammalian expresscion vector (Sigma) were used as the expression vector containing the nucleic acid encoding the target protein. , USA) and pCMV-Tag epitope tagging mammalian vector (Stratagene, USA).

The term " target protein " as used herein means a substance to be produced, for example, an antibody protein, an Fc-fusion protein, or a recombinant protein, but is not limited thereto.

Another embodiment of the present invention is a method of screening for a compound that encodes PyLT and EBNA-1 with a dhfr / MTX (methotrexate) system after introducing a nucleic acid encoding PyLT or a fragment thereof into a cell and a nucleic acid encoding EBNA- And a method for producing a transformed cell line.

Specifically, the cell line can be transfected with an expression vector comprising the EBNA-1 gene and an expression vector comprising the PyLT gene. More preferably, the cell line is transfected with an expression vector comprising the dhfr gene and the EBNA-1 gene, an expression vector comprising the dhfr gene and the PyLT gene, and the PyLT and EBNA-1 genes are transfected into the dhfr / MTX system And a cell line having at least 100 copies each can be prepared by amplification. In this method, the treatment concentration of MTX may be 1 nM to 500 nM, 1 nM to 1 uM, or 10 nM to 10 uM.

In another embodiment of the present invention, there is provided a method for producing an EBNA-1 comprising: 1) culturing an expression vector comprising a nucleic acid encoding a target protein in a cell line containing at least 100 copies of a nucleic acid encoding PyLT and containing at least 100 copies of a nucleic acid encoding EBNA- Transient tansfection, and 2) culturing the cell line to obtain a target protein.

The step 1) is a step of preparing a cell line containing at least 100 copies of a nucleic acid encoding PyLT and containing at least 100 copies of a nucleic acid encoding EBNA-1, wherein the characteristics of the cell line are as described in one embodiment of the present invention Gt; cell line, < / RTI > For example, the cell line may be a CHO cell strain deficient in the dhfr gene, or a CHO-DG44 cell strain. In addition, for example, a PCR product of oriP and Pyori may be subcloned into an expression vector containing a nucleic acid encoding a target protein, and the vector may be transiently transfected into the cell line.

In addition, in the step 2), it is preferable to use a medium suitable for the cell line when culturing the cells. For example, the serum-containing medium and the serum-free medium may be used alternately. In addition, purification of the desired protein can be applied without limitation as long as it is a method known to those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Preparation Example 1. Transient gene expression (TGE) method

Adherent cells grown in serum-containing medium were transiently transfected with the expression vector using the TransIT-X2 Dynamic Delivery system (Mirus Bio LLC, Madison, WI, USA). Transiently transfected cells were cultured in serum-containing adherent culture medium for 3 days.

Remove the serum-free medium to cells grown in rich medium and the exposing step centrifugation were resuspended at a density of 2 × 10 ⁶ cell / mL in 125 ㎖ Erlenmeyer flask of a culture medium (CHOgro Expression Medium, Mirus Bio LLC ) distinct chemically. Cells were transfected using TransIT-PRO Transfection Reagent (Mirus Bio LLC). The transfected cells were cultured at 110 rpm in a 5% -CO ₂ vibrated incubator at 37 ° C and stirred. For temperature changes, the temperature was reduced from 37 ° C to 32 ° C at 24 h post-transfection (hpt).

For oil-price cultivation, a HyClone Cell Boost mixture (0.25%) was added to the culture medium twice in two days and four days. Thereafter, samples were taken to determine cell concentration and viability. After centrifugation, the cell culture supernatant was aliquoted and frozen at -70 < 0 > C for further analysis.

Preparation Example 2. Cell concentration, viability, Fc-fusion protein analysis and metabolite analysis

Cell viability and viability were measured using a Vi-CELL XR analyzer (Beckman Coulter, Brea, CA, USA) as a trippin blue dye exclusion method. Secreted Fc-fusion protein concentrations and metabolites were quantified using a Cedex Bio HT analyzer (Roche Diagnostics, Basel, Switzerland).

Preparation Example 3. Western blot analysis

Total cellular protein was assessed by Western blot analysis. The same amount of cell lysate was loaded onto separate 4% to 12% Bis-Tris Plus gel (Invitrogen) under non-reducing conditions and transferred to a polyvinylidene fluoride membrane (Bio-Rad). Specifically, anti-EBV EBNA-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PyLT (Santa Cruz Biotechnology) and anti- Aldrich).

Preparation Example 4. Transfection efficiency

Transfection efficiency was measured from cell fractions expressing green fluorescent protein (GFP). Cells were harvested at 72 hpt and fluorescence intensity was determined using the Guava EasyCyte HT system (Millipore, Burlington, MA, USA) according to the manufacturer's instructions.

Preparation Example 5. Quantitative real-time PCR (qRT-PCR) analysis

To measure the amount of replicated DNA, qRT-PCR analysis and measurement of mRNA expression were performed with GoTaq qPCR Master Mix (Promega, Madison, Wis., USA). Total DNA and RNA were isolated from each individual clone using MiniBEST Universal Genomic DNA Extraction Kit and MiniBest Universal RNA Extraction Kit (TaKaRa, Tokyo, Japan). Plasmid DNA extracted from dam + Escherichia coli was digested with DpnI. Each cDNA sample from total RNA was prepared using the PrimeScript 1st strand cDNA synthesis kit (TaKaRa) for RT-PCR. All samples were normalized to expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used in qRT-PCR are listed in Table S1 below.

Preparation Example 6. N-glycan analysis of Fc-fusion non-glycoprotein

Fc-fusion non-glycoproteins were purified by protein A chromatography using MabSelect SuRe (GE Healthcare, Little Chalfont, UK) and a chromatography system (AKTA explorer 100 Air, GE Healthcare). N-glycans of the purified Fc-fusion non-glycoprotein were released by treatment with N-glycosidase F (Roche Diagnostics) and labeling with the fluorescent reagent 2-aminobenzoic acid (2-AA, Sigma-Aldrich) . The mixture was separated on a TSK gel Amide-80 column (4.6 x 250 mm, TOSOH, Tokyo, Japan) connected to an HPLC system (1260 Infinity, Agilent Technologies, Santa Clara, Calif. The 2-AA derivatized N-glycans were each monitored with a fluorescence detector having an excitation and emission wavelength of 360 nm and 425 nm, respectively.

Example 1. Fabrication of EBNA-1 or PyLT amplification vector

An internal ribosome entry site (IRES) dhfr gene, an EBNA-1 gene and a PyLT gene were obtained from each of the pOptiVEC vectors (Invitrogen, Carlsbad, CA, USA), pCEP4 vector (Invitrogen) and pBsd- PyLT vector . The PCR products of each gene were subcloned into pcDNA3.1 / Zeo (+) vector (Invitrogen) to obtain pcDNA-EBNA-1 / dhfr and pcDNA-PyLT / dhfr vectors. The expression of EBNA-1 / dhfr and PyLT / dhfr was induced by the CMV promoter and the single cistron was activated by IRES.

Example 2. Development of rCHO cells amplified with EBNA-1 and / or PyLT

CHO-DG44 cells were cultured in Iscove's modified Dulbecco's medium supplemented with 5% dialyzed fetal bovine serum (dFBS, Gibco, Waltham, MA, USA), 4 mM glutamine and hypoxanthine-thymidine (HT; Gibco) ; HyClone, Little Chalfont, UK). PyLT amplification pool and EBNA-1 / PyLT co-amplification pool were established by transfecting CHO-DG44 cells with pcDNA-EBNA-1 / dhfr, pcDNA-PyLT / dhfr and combinations thereof. The concentration of MTX (Sigma-Aldrich, St. Louis, MO, USA) was gradually increased from 1 to 500 nM in the resulting stable pool and applied to dhfr-mediated gene amplification.

To screen for EBNA-1 / PyLT co-amplification clones, single clones were selected by limiting dilution from the EBNA-1 / PyLT co-amplification pool at 100 nM MTX. Representative clones were screened by western blot analysis of intracellular EBNA-1 and PyLT. Clones were adapted to serum-free suspension culture in a 125 mL Erlenmeyer flask (Corning, Corning, NY, USA) at 110 rpm humidified 5% -CO ₂ vibrotic incubator (INFORS HT Ecotron, Basel, Switzerland). SFM4CHO (HyClone) supplemented with 4 mM glutamine (HyClone) and 100 nM MTX was used as the suspension culture medium.

Example 3. Preparation of Fc-Fusion Protein Expression Vector

Fc-fusion non-glycoprotein gene was prepared from pDR-OriP-Fc1 and subcloned into pcDNA3.1 / Zeo (+) vector to obtain pSF. The oriP and Pyori sequences were prepared from pCEP4 and pWP-Ang-Pyori vectors, respectively. oriP, Pyori and oriP / Pyori were subcloned into pSF vector to obtain pSF-oriP, pSF-Pyori and pSF-oriP-Pyori. All plasmids were isolated using EndoFree Plasmid Maxi Kit (Qiagen, Hilden, Germany). In this study, plasmids of A260 / A280> 1.8 were used.

Experimental Example 1. Confirmation of protein expression of EBNA-1 or PyLT-amplified rCHO cells

EBNA-1 transfected cells (EBNA-1 amplification pool) and PyLT transfected cells (PyLT amplification pool) were pooled and maintained in a selection medium containing MTX. Western blot analysis was performed to confirm EBNA-1 or PyLT amplification in this pool.

As shown in FIGS. 1A and 1B, it was confirmed that the expression level of EBNA-1 or PyLT in the gene amplification pool was sequentially increased according to the stepwise increase (1 nM to 500 nM) of the MTX concentration. Thus, the problem of relatively low EBNA-1 or PyLT expression in CHO cells is expected to be overcome by the dhfr / MTX gene amplification system.

To evaluate the effect of EBNA-1 or PyLT amplification on Fc-fusion protein production, EBNA-1 and PyLT amplification pools were incubated with two different Fc- Transfected with a fusion protein expression vector. Transient transfection was performed with two expression vectors, respectively.

Fig. 1c and Fig. 1d show Fc-fusion protein production according to various MTX concentrations in EBNA-1 amplified pool and PyLT amplified pool caused by transient transfection in serum-containing adhesion cultures. As MTX concentration increased, Fc-fusion protein production gradually increased from the PyLT amplification pool to 50 nM MTX and stagnated, while reaching a peak at 50 nM MTX in the EBNA-1 amplification pool and decreased. Generally, Fc-fusion protein production in EBNA-1 and PyLT amplified pools established at various MTX concentrations was higher than Null. Thus, EBNA-1 or PyLT amplification has been shown to increase transient transfection Fc-fusion protein production in serum-containing adherent cultures.

EXPERIMENTAL EXAMPLE 2 Development of rCHO Cells Simultaneously with EBNA-1 / PyLT

EBNA-1 / PyLT co-amplification pools were pcDNA-EBNA-1 / dhfr and pcDNA-PyLT / dhfr Vector. ≪ / RTI > The EBNA-1 / PyLT co-amplification pool was assessed by transient transfection with an Fc-fusion protein expression vector with oriP and Pyori sequences (pSF-oriP-Pyori) according to a stepwise increase in MTX concentration (1 nM to 500 nM) .

Western blot analysis showed that EBNA-1 and PyLT expression levels were successfully co-amplified by sequential increases in MTX concentration (Fig. 2a). In Fc- fusion protein production and protein production _(p q) is the Null respectively 52.1 ± 4.2 mg / L and 20.6 ± 2.1 pg / cell / day in the EBNA-1 / PyLT simultaneous amplification of the pool at 100 nM MTX than the culture 5.2 and 6.1 times higher (see Figures 2b and 2c).

In addition, four different Fc-fusion protein production vectors (pSF, pSF-fusion protein) were used to measure the synergy of EBNA-1 or PyLT amplification for Fc-fusion protein production in the EBNA-1 / PyLT co- oriP, pSF-Pyori and pSF-oriP-Pyori) were used for transient transfection. 100 nM MTX and treated with Null and EBNA-1 / PyLT in the simultaneous amplified pool. The results are shown in FIGS. 3A and 3B, respectively.

3A, the various expression vectors have different sizes, but did not significantly affect Fc-fusion protein production in Null. However, referring to FIG. 3B, it was highly dependent on the vector components in the EBNA-1 / PyLT co-amplification pool of 100 nM MTX. At this time, the yield of Fc-fusion protein was higher in pSF-oriP and pSF-oriP-Pyori than in pSF-Pyori. Taken together, the amplification of EBNA-1 or PyLT in the simultaneous amplification pool of EBNA-1 / PyLT at 100 nM MTX significantly increased protein production.

Experimental Example 3: Expression of EBNA-1 / PyLT q _p  Confirm the relationship between

To confirm the correlation between q _p and EBNA-1 or PyLT expression levels, 16 clones obtained from the EBNA-1 / PyLT amplification pool of 100 nM MTX were analyzed by Western blot analysis for EBNA-1 or PyLT expression levels After screening, a Quantity One intensity measurement was performed (see FIGS. 4A and 4B). Sixteen selected clones were cultured in 6-well plates using serum-containing adherent medium. In addition, q _p was evaluated by transiently transfection with the Fc-fusion protein production vector pSF-oriP-Pyori. Q _p was determined from a plot of Fc-fusion protein concentration versus time-integrated value of survival cell growth curve.

Figures 5A and 5B also show linear regression plots of q _p versus EBNA-1 and q _p versus PyLT expression levels, respectively, in 16 clones after transient transfection. Relative expression of EBNA-1 or PyLT for each clone was normalized relative to that of EP-amp-67 or EP-amp-32 clones, respectively, with the lowest level of EBNA-1 or PyLT expression (see FIG. 4b). As a control, CHO-DG44 cells were also transiently transfected. q _p was proportional to the level of EBNA-1 expression (R2 = 0.8447). In contrast, q _p and PyLT expression levels were not linearly correlated. Thus, under simultaneous amplification of EBNA-1 and PyLT in CHO cells, q _p was linearly correlated with EBNA-1 expression but not correlated with PyLT expression.

Experimental Example 4. Confirmation of effect of TGE system on rCHO cells by simultaneous amplification of EBNA-1 / PyLT

To investigate the effect of co-amplification of EBNA-1 and PyLT on cell growth and metabolite utilization in non-TGE systems, EBNA-1 / PyLT co-amplified clones were transiently serotyped without transfection. Three clones (EP-amp-3, EP-amp-3) with different q _p values were identified based on q _p in cell growth and transient transfection assays in serum containing adhesive cultures -19 and EP-amp-20) were selected.

As a result, it was confirmed that the simultaneous amplification of EBNA-1 and PyLT did not significantly affect the cell growth, viability profile or metabolite utilization of glucose, glutamine, lactate or ammonia (FIGS. 6A, 6B and 7A 7D).

In order to investigate the effect of simultaneous amplification of EBNA-1 and PyLT on Fc fusion protein production in the TGE system, EBNA-1 / PyLT co-amplified clones were transiently transfected with pSF-oriP-Pyori and suspended in serum-free medium Lt; / RTI > A TGE-based culture of CHO-DG44 was used as a control.

Typical cell growth (FIG. 8A), viability (FIG. 8A) and Fc-fusion protein production profile (FIG. 8C) during TGE-based incubation of EBNA-1 / PyLT co-amplified clones are shown in the respective figures. EP-amp3, EP-amp- 19 and EP-amp-20 clone was reached the maximum viable cell density of 3.1 to 3.6 × 10 ⁶ cell / mL, which is slightly higher than the control group. In addition, the highest Fc-fusion protein production was achieved at EP-amp-20 on day 6 (186 +/- 5.7 mg / L) and the survival rate was higher than 60%. Fc-fusion protein production of EP-amp-20 was 27.4 times higher than that of the control.

Also, referring to FIG. 9, it can be seen that simultaneous expression of EBNA-1 and PyLT in EP-amp-20 was continuously detected until day 7 even though MTX was not present in the TGE-based culture. These results indicate that CHO cells co-amplified with EBNA-1 / PyLT can be used as a therapeutic protein host for TGE.

EXPERIMENTAL EXAMPLE 5. Identification of Molecular Characteristic of rCHO Cells Simultaneously Amplified with EBNA-1 / PyLT

In order to confirm the changes in molecular characteristics of EBNA-1 / PyLT co-amplified cells, q _p , mRNA expression level, amount of replicated DNA and transfection efficiency in EBNA-1 / PyLT co- Were measured. The expression of mRNA on day 2 and the amount of replicated DNA was normalized to the control group on day 2 in the EBNA-1 / PyLT co-amplified clone.

Figure 10a shows q _p calculated using data obtained from days 0 to 3 after TGE. As expected based on observed Fc-fusion protein production, TGE culture with EP-amp-20 showed the highest q _p of 20.6 ± 2.5 pg / cell / day, 22.9-fold greater than the control. Figure 10b shows the relative mRNA expression levels of EBNA-1 / PyLT co-amplified clones on day 2. As shown in Figure 10b, it can be seen that the relative mRNA expression level of the 20-EP-amp significantly increased, which is consistent with q _p. Figure 10c shows the relative amount of DNA duplicated in the EBNA-1 / PyLT co-amplification clone on day 2. The relative amount of replicated DNA of EP-amp-20 was slightly higher than that of the control. The amount of DNA replicated in the control group gradually decreased after transient transfection, but the amount of DNA replicated in EP-amp-20 peaked at day 2 (data not shown).

Figure 10d shows the transfection efficiency of a transiently transfected control with an EBNA-1 / PyLT co-amplified clone and a GFP-positive vector. The proportion of GFP positive cells in all EBNA-1 / PyLT co-amplified clones was similar to that of the control. Taken together, these results demonstrate that q _p increase induced by EBNA-1 / PyLT co-amplification contributes to mRNA expression and increased quantities of replicated DNA, but not to high transfection efficiency.

Experimental Example 6. Formation of N-linked glycans in rCHO cells by simultaneous amplification of EBNA-1 / PyLT

In order to determine the effect of simultaneous amplification of EBNA-1 and PyLT on product quality, untreated CHO cells (CHO-DG44, CHO-K1 and ExpiCHO-S) and viable CHO cells linked glycans released from the Fc-fusion protein were purified from the Fc-fusion protein, -AMP-3, EP-amp-19 and EP-amp-20. This was analyzed using a hydrophilic interaction HPLC column.

Figure 11 shows the ratio of the five major N-glycans calculated by integrating the area of each group. More than 98% of the total glycosylation pattern of G0, G0F, (1,6) G1F, (1,3) G1F and G2F constitute the five major glycosylation peaks. The ratio of G0, G0F, (1, 6) G1F, (1,3) G1F and G2F in CHO-DG44 was 2.5% ± 0.5%, 59.4% ± 0.6%, 24.0% ± 0.7% % ± 0.2% and 5.4% ± 0.5%. Glycan profiles of EBNA-1 / PyLT co-amplified clones are very similar to untreated CHO cells (CHO-DG44, CHO-K1, ExpiCHO-S). Therefore, it was confirmed that the simultaneous amplification of EBNA-1 / PyLT did not significantly affect the formation of N-glycan of the protein produced in the TGE system.

<110> Korea Research Institute of Bioscience and Biotechnology <120> CELL LINE WITH INCREASED PRODUCTION OF TARGET PROTEIN AND          PRODUCING METHOD FOR TARGET PROTEIN USING THE SAME <130> FPD201803-0021C <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 785 <212> PRT <213> Artificial Sequence <220> <223> PyLT <400> 1 Met Asp Arg Val Leu Ser Arg Ala Asp Lys Glu Arg Leu Leu Glu Leu   1 5 10 15 Leu Lys Leu Pro Arg Gln Leu Trp Gly Asp Phe Gly Arg Met Gln Gln              20 25 30 Ala Tyr Lys Gln Gln Ser Leu Leu Leu His Pro Asp Lys Gly Gly Ser          35 40 45 His Ala Leu Met Gln Glu Leu Asn Ser Leu Trp Gly Thr Phe Lys Thr      50 55 60 Glu Val Tyr Asn Leu Arg Met Met Asn Leu Gly Gly Thr Gly Phe Gln Gly  65 70 75 80 Ser Pro Pro Arg Thr Ala Glu Arg Gly Thr Glu Glu Ser Gly His Ser                  85 90 95 Pro Leu His Asp Asp Tyr Trp Ser Phe Ser Tyr Gly Ser Lys Tyr Phe             100 105 110 Thr Arg Glu Trp Asn Asp Phe Phe Arg Lys Trp Asp Pro Ser Tyr Gln         115 120 125 Ser Pro Pro Lys Thr Ala Glu Ser Ser Glu Gln Pro Asp Leu Phe Cys     130 135 140 Tyr Glu Glu Pro Leu Leu Ser Pro Asn Pro Ser Ser Pro Thr Asp Thr 145 150 155 160 Pro Ala His Thr Ala Gly Arg Arg Arg Asn Pro Cys Val Ala Glu Pro                 165 170 175 Asp Asp Ser Ile Ser Pro Asp Pro Pro Arg Thr Pro Val Ser Arg Lys             180 185 190 Arg Pro Arg Pro Ala Gly Ala Thr Gly Gly Gly Gly Gly Gly Gly Val His         195 200 205 Ala Asn Gly Gly Ser Val Phe Gly His Pro Thr Gly Gly Thr Ser Thr     210 215 220 Pro Ala His Pro Pro Tyr His Ser Gln Gly Gly Ser Glu Ser Met 225 230 235 240 Gly Gly Ser Asp Ser Ser Gly Phe Ala Glu Gly Ser Phe Arg Ser Asp                 245 250 255 Pro Arg Cys Glu Ser Glu Asn Glu Ser Tyr Ser Gln Ser Cys Ser Gln             260 265 270 Ser Ser Phe Asn Ala Thr Pro Pro Lys Lys Ala Arg Glu Asp Pro Ala         275 280 285 Pro Ser Asp Phe Pro Ser Ser Leu Thr Gly Tyr Leu Ser His Ala Ile     290 295 300 Tyr Ser Asn Lys Thr Phe Pro Ala Phe Leu Val Tyr Ser Thr Lys Glu 305 310 315 320 Lys Cys Lys Gln Leu Tyr Asp Thr Ile Gly Lys Phe Arg Pro Glu Phe                 325 330 335 Lys Cys Leu Val His Tyr Glu Glu Gly Gly Met Leu Phe Phe Leu Thr             340 345 350 Met Thr Lys His Arg Val Ser Ala Val Lys Asn Tyr Cys Ser Lys Leu         355 360 365 Cys Arg Ser Phe Leu Met Cys Lys Ala Val Thr Lys Pro Met Glu Cys     370 375 380 Tyr Gln Val Val Thr Ala Ala Pro Phe Gln Leu Ile Thr Glu Asn Lys 385 390 395 400 Pro Gly Leu His Gln Phe Glu Phe Thr Asp Glu Pro Glu Glu Gln Lys                 405 410 415 Ala Val Asp Trp Ile Met Val Ala Asp Phe Ala Leu Glu Asn Asn Leu             420 425 430 Asp Asp Pro Leu Leu Ile Met Gly Tyr Tyr Leu Asp Phe Ala Lys Glu         435 440 445 Val Pro Ser Cys Ile Lys Cys Ser Lys Glu Glu Thr Arg Leu Gln Ile     450 455 460 His Trp Lys Asn His Arg Lys His Ala Glu Asn Ala Asp Leu Phe Leu 465 470 475 480 Asn Cys Lys Ala Gln Lys Thr Ile Cys Gln Gln Ala Ala Ala Ser Leu                 485 490 495 Ala Ser Arg Arg Leu Lys Leu Val Glu Cys Thr Arg Ser Gln Leu Leu             500 505 510 Lys Glu Arg Leu Gln Gln Ser Leu Leu Arg Leu Lys Glu Leu Gly Ser         515 520 525 Ser Asp Ala Leu Leu Tyr Leu Ala Gly Val Ala Trp Tyr Gln Cys Leu     530 535 540 Leu Glu Asp Phe Pro Gln Thr Leu Phe Lys Met Leu Lys Leu Leu Thr 545 550 555 560 Glu Asn Val Pro Lys Arg Arg Asn Ile Leu Phe Arg Gly Pro Val Asn                 565 570 575 Ser Gly Lys Thr Gly Leu Ala Ala Leu Ile Ser Leu Leu Gly Gly             580 585 590 Lys Ser Leu Asn Ile Asn Cys Pro Ala Asp Lys Leu Ala Phe Glu Leu         595 600 605 Gly Val Ala Gln Asp Gln Phe Val Val Cys Phe Glu Asp Val Lys Gly     610 615 620 Gln Ile Ala Leu Asn Lys Gln Leu Gln Pro Gly Met Gly Val Ala Asn 625 630 635 640 Leu Asp Asn Leu Arg Thr Thr Trp Asn Gly Ser Val Lys Val Asn Leu                 645 650 655 Glu Lys Lys His Ser Asn Lys Arg Ser Gln Leu Phe Pro Pro Cys Val             660 665 670 Cys Thr Met Asn Glu Tyr Leu Leu Pro Gln Thr Val Trp Ala Arg Phe         675 680 685 His Met Val Leu Asp Phe Thr Cys Lys Pro His Leu Ala Gln Ser Leu     690 695 700 Glu Lys Cys Glu Phe Leu Gln Arg Glu Arg Ile Ile Gln Ser Gly Asp 705 710 715 720 Thr Leu Ala Leu Leu Leu Ile Trp Asn Phe Thr Ser Asp Val Phe Asp                 725 730 735 Pro Asp Ile Gln Gly Leu Val Lys Glu Val Arg Asp Gln Phe Ala Ser             740 745 750 Glu Cys Ser Tyr Ser Leu Phe Cys Asp Ile Leu Cys Asn Val Gln Glu         755 760 765 Gly Asp Asp Pro Leu Lys Asp Ile Cys Asp Ile Ala Glu Tyr Thr Val     770 775 780 Tyr 785 <210> 2 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> EBNA-1 <400> 2 Met Ala Asp Glu Gly Leu Pro Arg Gly Asn Gly Leu Gly Ala Arg   1 5 10 15 Gly Asp Pro Gly Gln Gly Pro Arg Gly Pro Ala Gln Pro Asp Ser Thr              20 25 30 Ser Gly Ser Gly Gly Gly Gly Thr Arg Gly Arg Gly Gly Ser Arg Gly          35 40 45 His Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Gly Gly Gln Gly      50 55 60 Gly Thr Val Ala Ser Gly Gly Ser Gly Ser Gly Pro Arg Leu Gly Asp  65 70 75 80 Asp Arg Arg Pro Asp Gly Gln Arg Pro Ser Lys Arg Arg Ser Ser Cys Ile                  85 90 95 Gly Cys Arg Gly Gly Ala Gly Gly Gly Ser Gly Gly Gly Ala Gly Gly             100 105 110 Ser Gly Ala Gly Gly Ser Gly Ala Gly Gly Ser Gly Ala Gly Gly Ala         115 120 125 Gly Gly Ser Gly Ala Gly Asp Ser Gly Ala Gly Gly Ala Gly Gly Ser     130 135 140 Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly 145 150 155 160 Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly                 165 170 175 Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly             180 185 190 Gly Ser Gly Ala Gly Gly Ser Gly Ala Val Gly Ala Gly Gly Ser Gly         195 200 205 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Gly     210 215 220 Ala Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ser Gly Gly Gly Gly Gly 225 230 235 240 Gly Arg Gly Arg Gly Gly Ser Arg Gly Arg Gly Gly Ser Arg Gly Arg                 245 250 255 Gly Gly Ser Arg Gly Arg Gly Gly Ser Arg Gly Arg Gly Arg Gly Arg             260 265 270 Gly Gly Ser Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg         275 280 285 Gly Gln Gly Pro Arg Gly Gly Gly Lys Arg Pro Arg Ser Ser Pro Gly     290 295 300 Ser Ser Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Gly Arg Ala 305 310 315 320 Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly                 325 330 335 Tyr Gly Phe Pro Gly Asp Arg Pro Leu Thr Val Thr Val Pro Gly Ser             340 345 350 Ala Leu Gly Arg Tyr Arg Gly Thr Asp Gly Thr Asp Gly Gly Asp Glu         355 360 365 Pro Pro Gly Ala Met Glu Gln Gly Pro Glu Glu Asp Pro Gly Glu Gly     370 375 380 Pro Ser Arg Gln His Thr Thr Ser Gly Gly Arg Gly Gly Gly Lys Lys 385 390 395 400 Gly Gly Trp Phe Gly Arg His Arg Gly Glu Gly Arg Gly Asn Lys Lys                 405 410 415 Phe Gln Ser Ile Gly Asp Ser Ile Ser Ala Leu Leu Gly Arg Cys Glu             420 425 430 Ala Pro Arg Thr Ser Pro Glu Gly Glu Trp Cys Cys Ala Leu Phe Ile         435 440 445 Tyr Ser Tyr Ser Lys Thr Cys Cys Tyr Asn Leu Arg Arg Gly Leu Ala     450 455 460 Leu Cys Ile Pro Glu Gly Arg Ala Thr Gly Leu Gly Arg Leu Pro Tyr 465 470 475 480 Gly Val Ser Pro Gly Ser Gly Pro Gln Pro Gly Pro Leu Arg Glu Ser                 485 490 495 Ser Trp Ser Tyr Phe Leu Phe Phe Leu Gln Cys His Leu Phe Ala Glu             500 505 510 Cys Ala Lys Asp Ala Ile Leu Asp Tyr Ile Arg Thr Arg Pro Pro Pro         515 520 525 Thr Cys Asp Thr Arg Val Thr Val Cys Ser Phe Asp Asp Ser Val Met     530 535 540 Leu Pro Ile Trp Phe Pro Pro Ala Pro Gln Gly Ala Ala Ala Ala Gly 545 550 555 560 Glu Gly Ala Gly Gly Asp Asp Gly Asp Glu Gly Gly Glu Gly Gly Asp                 565 570 575 Gly Asn Glu Gly Asp Glu Gly             580 585

Claims (12)

As transformed cells containing 100 copies or more of a nucleic acid encoding PyLT (Polyoma virus Large T-antigen) and containing 100 or more nucleotides encoding EBNA-1 (Epstein Barr virus nuclear antigen 1)
Wherein said cells are deficient in the dihydrofolate reductase (dhfr) gene and amplified with a nucleic acid encoding PyLT and EBNA-1 in a dhfr / MTX (methotrexate) system.
The method according to claim 1,
Wherein the PyLT comprises the amino acid sequence shown in SEQ ID NO: 1.
The method according to claim 1,
Wherein said EBNA-1 comprises the amino acid sequence of SEQ ID NO: 2.
delete The method according to claim 1,
Wherein the cell is an animal cell.
6. The method of claim 5,
Wherein the animal cell is a Chinese hamster ovary (CHO) cell.
delete The method according to claim 1,
A transformed cell in which an expression vector comprising a nucleic acid encoding a target protein is additionally introduced.
amplifying PyLT and EBNA-1-encoding nucleic acid into a dhfr / MTX (methotrexate) system after introducing a nucleic acid encoding PyLT and a nucleic acid encoding EBNA-1 into cells lacking the dihydrofolate reductase (dhfr) / RTI &gt;
A method for producing a transformed cell line.
1) Transient transfection of an expression vector containing a nucleic acid encoding a target protein into a cell line containing 100 copies or more of PyLT-encoding nucleic acid and containing 100 or more nucleotides encoding EBNA-1 ; And
2) culturing the cell line to obtain a target protein,
The cell line was deficient in the dihydrofolate reductase (dhfr) gene and was amplified with a nucleic acid encoding PyLT and EBNA-1 in a dhfr / MTX (methotrexate) system.
A method for producing a target protein.
11. The method of claim 10,
Wherein the cell line is a CHO cell line deficient in the dhfr gene.
A method for producing a target protein.
11. The method of claim 10,
Wherein the target protein is any one selected from the group consisting of an antibody protein, an Fc-fusion protein and a recombinant protein.
A method for producing a target protein.

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