KR100701199B1 - A cell derived from a mammalian cell producing a sialylated glycoprotein and CMP-sialic acid transporter and a method of producing a glycoprotein having reduced N-glycolylneuraminic acid and enhanced N-acetylylneuraminic acid content - Google Patents

Abstract

In the present invention, mammalian-derived cells expressing sialylated glycoproteins and CMP-SAT to higher levels than endogenous levels, and the content of N-glycolylneuraminic acid are reduced, and the content of N-acetylneuraminic acid is increased. Provided are methods for producing sialylated glycoproteins.

CHO, CMP-SAT, sialic acid, glycoprotein

Description

Mammalian-derived cells expressing sialylated glycoproteins and CPM-SAT to levels higher than endogenous levels and sialylation sugars having reduced content of N-glycolylneuraminic acid and increased content of N-acetylneuraminic acid using the same A cell derived from a mammalian cell producing a sialylated glycoprotein and CMP-sialic acid transporter and a method of producing a glycoprotein having reduced N-glycolylneuraminic acid and enhanced N-acetylylneuraminic acid content}

1 is a diagram schematically showing a complex oligosaccharide attached to a glycoprotein expressed in a mammal.

2 is a diagram schematically showing the structure of two substrates of sialation reaction, CMP-Neu5Ac and CMP-Neu5Gc.

3 is a diagram schematically showing a pathway in which sialation reaction occurs from Neu5Ac.

4 is a diagram schematically showing the hEPO expression vector pSEpoh.

5 is a diagram schematically showing the expression vector pTH of CMP-SAT.

Fig. 6 is a diagram showing the result of analyzing the hEPO in the medium by His-tag metal affinity chromatography method, and analyzing the SDS-PAGE (A) and Western blot (B).

7 is a culture of the CHO-EPO-SAT cell line to isolate the entire RNA therefrom, RT-PCR was performed using oligonucleotides of SAT-N (SEQ ID NO: 3) and SATH-C (SEQ ID NO: 4) as a primer It is a figure which shows the result confirmed through the electrophoresis on 1% agarose gel.

Fig. 8 is a diagram showing the result of Western blotting on CMP-SAT in culture obtained by culturing CHO-EPO-SAT cell line.

9 is a photograph showing the results of observing the expression position in the cells of CMP-SAT through a fluorescence microscope.

10 is a diagram showing the results of two-dimensional electrophoresis analysis of hEPO obtained by culturing CHO-EPO-SAT cell line according to isoelectric point and size.

FIG. 11 shows the results of sialic acid content of hEPO expressed from CHO-EPO-SAT through MAA.

In the present invention, mammalian-derived cells expressing sialylated glycoproteins and CMP-SAT to higher levels than endogenous levels, and the content of N-glycolylneuraminic acid are reduced, and the content of N-acetylneuraminic acid is increased. A method for producing sialylated glycoproteins is disclosed.

Many foreign glycoproteins of human origin are expressed in mammals other than humans. This is because vector systems for expressing foreign proteins in mammals are well established and post-translational modification systems are similar. Examples of such foreign glycoproteins include erythropoietin (EPO), transferrin, plasminogen and tyrotropin, which are mainly expressed and produced in Chinese hamster ovary (CHO) cells.

In mammals, glycoproteins are expressed in which complex oligosaccharides terminated with sialic acid (SA) by post-translational modification are attached to Asn residues of Asn-X-Ser / Thr (FIG. 1). ). 1 is a diagram schematically showing a complex oligosaccharide attached to a glycoprotein expressed in a mammal. In therapeutic proteins, the terminal residues of the sugar chains are particularly important because they often regulate in vivo half-life. Glycoproteins with oligosaccharides terminated with sialic acid bind to structures with mannose (Man), N-acetylglucosamine (GlcNac) and galactose (Gal) from the bloodstream and rapidly remove receptors for hepatocytes and macrophages. It is usually present in the bloodstream for a longer time. The sialic acid content in such complex oligosaccharides expressed in mammals is often not known to be completely consistent in humans and other mammalian cells except humans. For example, in humans only N-acetylneuraminic acid (Ne5Ac) is found as sialic acid, while in other mammalian cells N-acetylneuraminic acid (Ne5) is known as Neu5Ac. Two kinds of sialic acid, N-glycolylneuraminic acid (hereinafter referred to as Neu5Gc), have been found. The content of Neu5Gc in glycoproteins expressed in CHO cells depends on the type of glycoprotein and is known to be 2 to 3% and as high as 14% (TG Warner, 1999, Glycobiology , 9, 841-850). In addition, certain CHO cells may have 100% Neu5Gc of all sialic acid of glycoproteins (TG Warner, 1999, Glycobiology , 9, 841-850). 2 is a diagram schematically showing the structure of two substrates of sialation reaction, CMP-Neu5Ac and CMP-Neu5Gc. Since sialic acid in the form of Neu5Gc is not present in the human body, it is recognized as a foreign substance, and when a glycoprotein containing Neu5Gc is administered to humans, it may induce the formation of an immune response, for example, a Hanganutziu-Deicher antibody (H Higashi et al., 1977, Biochemical and Biophysical Research Communications , 79, 388-395; JM Merrick et al., 1978, International Archives of Allergy and Applied Immunology , 57, 477-480; M. Odaka et al., 1998, Annals of neurology, 43. , 829-834).

Attempts have been made to reduce the content of N-glycolylneuraminic acid of glycoproteins expressed in mammals in the past. For example, S. Chenu et al., 2003, Biochimica et Biophysica Acta, 1622, 133-144, disclose a method for reducing the Neu5Gc content of glycoproteins by inhibiting CMP-Neu5Ac hydroxylase. This inhibits the synthetic pathway in which CMP-Neu5Gc is converted from CMP-Neu5Ac by hydroxylation reaction by CMP-Neu5Ac hydroxylase in the cytoplasm, thereby reducing the level of CMP-Neu5Gc, a substrate for sialylation, resulting in a final glycoprotein. It is a method of reducing the content of Neu5Gc in the.

Even in the prior art as described above, a method for reducing Neu5Gc in glycoprotein expressed in mammalian cells using a CMP-sialic acid transporter (CMP-SAT) has not been disclosed. . In mammalian sialylation reactions, CMP-sialic acid (CMP-SA) synthesized in the cytoplasm is transferred to the Golgi apparatus by CMP-sialic acid transporter (CMP-SAT), and CMP-SA transferred into the Golgi apparatus Sialyltransferase is used as a substrate so that sialic acid in CMP-SA is transferred to the precursor of the complex oligosaccharide to form a complex oligosaccharide. 3 is a diagram schematically showing a pathway in which sialation reaction occurs from Neu5Ac.

Therefore, there is still a need for a method capable of increasing Neu5Ac content while decreasing the content of Neu5Gc in glycoproteins expressed in mammals using CMP-SAT.

It is an object of the present invention to provide mammalian derived cells expressing sialylated glycoproteins and CMP-SAT at levels above endogenous levels.

Another object of the present invention is to provide a method for producing sialylated glycoproteins having reduced N-glycolneuraminic acid content and increased N-acetylneuraminic acid content using the cells of the present invention.

The present invention provides mammalian derived cells expressing sialylated glycoproteins and cytidine monophosphate-sialic acid transporters (hereinafter referred to as CMP-SAT) at levels above intrinsic levels.

In the cell of the present invention, any of the sialylated glycoproteins includes any sialic acid residue at the terminal of the sugar chain. One example of the sialylated glycoprotein may be selected from the group consisting of erythropoietin, plasminogen, transferrin and tyrotropin, but is not limited thereto.

The cell of the present invention is not particularly limited as long as it is a cell derived from a mammalian animal. For example, hamster kidney (BHK), mouse whole embryo (3T3), monkey kidney (COS) and chinese hamster ovary (CHO) cell lines may be included, but are not limited to these examples. The cells are preferably cells of rodents, more preferably Chinese hamster ovary (CHO) cells. The cell of the present invention is a cell expressing sialylated glycoprotein and CMP-SAT at a level higher than the endogenous level, for example, a cell in which the gene encoding the endogenous sialylated glycoprotein and CMP-SAT is amplified in natural cells. Or a cell into which a sialylated glycoprotein and a foreign nucleic acid encoding CMP-SAT are introduced.

In one embodiment of the invention, the cells of the invention are preferably cells into which sialylated glycoproteins and foreign nucleic acids encoding CMP-SAT are introduced. In particular, preferably, it is a CHO-EPO-SAT cell line (Accession No. KCTC 10843BP). Methods of introducing foreign nucleic acids into cells are well known in the art related to producing recombinant cells. For example, a desired sialylated glycoprotein and a nucleic acid encoding CMP-SAT may be introduced into a vector using a suitable restriction enzyme to prepare an expression vector, and the expression vector is introduced into a cell, thereby introducing a foreign nucleic acid into the cell. Can be prepared. Methods of introducing foreign nucleic acids such as expression vectors into cells, such as calcium phosphate, liposomes and electroporation, are well known in the art.

As used herein, the term "endogenous level" refers to the form and / or amount of a biological function or biochemical composition present in a naturally occurring cell or recombinant cell in a state prior to manipulating the cell according to the present invention. do. As used herein, "heterologous" refers to the form and / or amount of a biological function or biochemical composition that is not present in a naturally occurring cell or recombinant cell in a state prior to manipulating the cell according to the present invention. .

The present invention also comprises the steps of culturing the cells of the present invention to produce the sialylated glycoprotein; And

It provides a method for producing a sialylated glycoprotein having a reduced content of N-glycolylneuraminic acid and an increased content of N-acetylneuraminic acid, comprising recovering the sialylated glycoprotein from the culture. do.

Conventional methods for producing sialylated glycoproteins such as erythropoietin by culturing animal cells, such as rodent-derived CHO cells, are well known in the art. In the method of the present invention, the step of culturing the cells of the present invention and recovering the sialylated glycoprotein from the culture may be conventional methods known in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

In the following examples, foreign nucleic acids encoding hEPO and CMP-SAT, which are sialylated glycoproteins, were introduced into CHO cells, and the respective sialic acid contents of hEPO produced therefrom were analyzed.

Example 1: Cloning of hEPO Gene and Expression Vector Construction

In this example, a human erythropoietin (hereinafter referred to as hEPO) was selected as an example of sialylated glycoprotein, and a vector capable of expressing the same was prepared.

First, the gene of hEPO is based on the pMT / Bip / hEPO (HS Shin and HJ Cha, 2003, Protein Expression and Purification, 28, 331-339) plasmid, and EPO-N (SEQ ID NO: 1) and EPO-C ( The oligonucleotide of SEQ ID NO: 2) was amplified by PCR using a primer. The amplified product was inserted into HindIII and XhoI restriction sites of the pSecTag2 / Hygro A vector (Invitrogen, USA) to prepare hEPO expression vector pSEpoh (FIG. 4). 4 is a diagram schematically showing the hEPO expression vector pSEpoh. As shown in FIG. 4, in the hEPO gene, a histidine tag for isolation and a myc tag for expression confirmation are fused to the C-terminus of the hEPO gene. In addition, in the hEPO gene, an IgK secretion sequence is fused to the N-terminus of the hEPO gene for secretion of hEPO into the medium.

Example 2: Cloning and Expression Vector Construction of CMP-SAT Gene

In this example, a vector capable of expressing the CMP-SAT gene (Genbank License No. # Y12074) was prepared.

Oligonucleotides of SAT-N (SEQ ID NO: 3) and SATH-C (SEQ ID NO: 4) capable of confirming the nucleotide sequence of the CMP-SAT gene from the gene sequencing database (Genbank, # Y12074) and amplifying the gene To prepare a primer consisting of. Next, mRNA of CHO cells was used as a template, and RT-PCR was performed using the oligonucleotide as a primer to amplify the gene. The amplified product was inserted into the NheI and EcoRI restriction sites of the pIRES (Clontech, USA) vector to prepare a CMP-SAT expression vector pTH (FIG. 5). 5 is a diagram schematically showing the expression vector pTH of CMP-SAT. As shown in FIG. 5, in the CMP-SAT gene, influenza hemaclutinin (HA) tag is fused to the C-terminus of the CMP-SAT gene to confirm CMP-SAT expression.

Example  3: CHO  In the cell hEPO  Expression and Separation Purification

In this example, the CSE-EPO cell line stably expresses hEPO after transforming the prepared pSEpoh vector into CHO cells. First, a mixture of vector DNA and LIPOFECTAMINE ^™ (Invitrogen, USA) was made and the mixture was treated with media to representative CHO-K1 cells (Cricetulus griseus (hamster, Chinese); ATCC CCL-61) in CHO cells. After 24 hours, 10 ⁴ mice per well were put in a 96-well plate, and then treated with hygromycin (GibcoBRL, USA) at a concentration of 200 mg / ml and cultured for 2 weeks. Cells surviving the hygromycin treatment were put in a 96-well plate containing 0.5 cells per well again, and the cells were incubated for 1 month after changing the hygromycin concentration to 200 mg / ml to 500 mg / ml. CHO-EPO cell lines were selected by recovering the medium of each well and confirming the expression level of hEPO.

The obtained CHO-EPO cell line was cultured in MEM-α (GibcoBRL, USA) medium to which 10% fetal bovine serum (FBS) was added for 7 days at 5% CO ₂ and 37 ° C., and then the medium was recovered. Out secreted hEPO was isolated.

Since hEPO secreted in the medium has a hexa-histidine tag to facilitate separation and purification, hEPO was separated by His-tag metal affinity chromatography using Ni-NTA agarose (Qiagen). SDS-PAGE samples (F) received when the medium was first flowed into the His-tag column, samples received when washing with 20 mM imidazole (W) and samples received after the 150 mM imidazole spill (E), respectively And analyzed by Western blot method (FIG. 6). Fig. 6 is a diagram showing the result of analyzing the hEPO in the medium by His-tag metal affinity chromatography method, and analyzing the SDS-PAGE (A) and Western blot (B). As shown in FIG. 6, hEPO having a very high purity could be purified by His-tag metal affinity chromatography.

Example 4: CMP-SAT Expression in CHO-EPO Cells

In the present Example, the pTH vector prepared in Example 2 was transformed into the CHO-EPO cells prepared in Example 3, and then a CHO-EPO-SAT cell line stably expressing hEPO and CMP-SAT was prepared.

First, the pTH vector prepared in Example 2 was treated with LIPOFECTAMINE ^™ (GibcoBRL, USA) for 24 hours in CHO-EPO cells prepared in Example 3, and then 300 mg in a 96-well plate at 10 ⁴ cells per well. After treatment with G-418 (Sigma) and 200 mg / ml of hygromycin at a concentration of 2 ml / ml, the surviving cells were transferred to a 96-well plate at 0.5 cells per well and cultured for 1 month, followed by CMP-SAT expression. CHO-EPO-SAT cell lines were selected.

CHO-EPO-SAT cell line expressing the obtained CMP-SAT was cultured in MEM-α (GibcoBRL, USA) medium to which 10% fetal calf serum (FBS) was added for 24 hours at 5% CO ₂ and 37 ° C. Afterwards, the expression of CMP-SAT was analyzed. First, RT-PCR was performed to confirm that CMP-SAT is expressed at the mRNA level. RT-PCR was performed by culturing the CHO-EPO-SAT cell line to isolate whole RNA from it, and using oligonucleotides of SAT-N (SEQ ID NO: 3) and SATH-C (SEQ ID NO: 4) as primers. 7 is a culture of the CHO-EPO-SAT cell line to isolate the entire RNA therefrom, RT-PCR was performed using oligonucleotides of SAT-N (SEQ ID NO: 3) and SATH-C (SEQ ID NO: 4) as a primer The obtained product is a view showing the result confirmed by the electrophoresis on 1% agarose gel. As shown in Figure 7, it was confirmed that the expression of hEPO and CMP-SAT at the mRNA level in the CHO-EPO-SAT cell line.

Next, Western blot was performed to confirm the expression of CMP-SAT at the protein level. After culturing the cultured cells were placed in a buffer for SDS-PAGE (0.5M Tris-HCl (pH 6.8), 10% glycerol, 5% SDS, 5% beta-mercaptoethanol, 0.25% bromophenol blue) After dilution, the mixture was boiled at 100 ° C. for 5 minutes for denaturation. The sample was electrophoresed on a 12% SDS-poly acrylamide gel and then transferred to a nitrocellulose membrane under a voltage of 15 V and the CMP- using monoclonal influenza hemaclotinin (HA) affinity ligand antibody (Sigma, USA). Color reactions specific to SAT were performed (FIG. 8). Fig. 8 is a diagram showing the result of Western blotting on CMP-SAT in culture obtained by culturing CHO-EPO-SAT cell line. As shown in FIG. 8, it was confirmed that CMP-SAT was expressed at the protein level in the CHO-EPO-SAT cell line.

Next, it was confirmed by immunofluorescence analysis whether CMP-SAT is expressed in Golgi apparatus in cells. First, cells were cultured on a cover glass and then immobilized with 4% paraformaldehyde. Immobilized cells were endowed with 1% Triton X-100 and blocked with 3% bovine serum albumin (BSA). Next, the HA antibody and the secondary antibody (SIGMA, USA) to which the FITC is attached were treated, and then observed through a fluorescence microscope. 9 is a photograph showing the results of observing the expression position in the cells of CMP-SAT through a fluorescence microscope. As shown in Figure 9, it was confirmed that the CMP-SAT is expressed in the Golgi apparatus in the cell.

The CHO-EPO-SAT cell line prepared in this example was deposited on September 6, 2005 to the Korean Collection for Type Collection, an international depository institution under the Butafest Treaty (Accession No. KCTC 10843BP) .Z

Example 5 Two-Dimensional Electrophoresis Analysis of Separated Purified hEPO

In the present embodiment, the CHO-EPO-SAT cell line prepared in Example 4 was cultured, hEPO and CMP-SAT were simultaneously expressed, and the hEPO obtained from the culture was separated and purified by His-tag metal affinity chromatography. Two-dimensional electrophoresis analysis was performed to analyze sugar residue patterns of hEPO.

First, hEPO isolated by His-tag metal affinity chromatography method as in Example 3 was separated according to isoelectric point in 7 cm IMMOBILINE ™ DRYSTIP GEL (pH 3.0-5.6) (Amersham Biosciences, Sweden). The primary separation gels according to isoelectric points were secondaryly separated according to size by electrophoresis on SDS-PAGE gels. After SDS-PAGE electrophoresis, the gel was Coomassie blue staining to detect spots (FIG. 10). 10 is a diagram showing the results of two-dimensional electrophoresis analysis of hEPO obtained by culturing CHO-EPO-SAT cell line according to isoelectric point and size. As control, hEPO expressed in CHO-EPO cell line was used. As shown in FIG. 10, it can be seen that spots having lower isoelectric points appear darker in hEPO (B) expressed in the CHO-EPO-SAT cell line than in the hEPO (A) expressed in the CHO-EPO cell line.

The pI (isoelectric point) value of glycoproteins is known to be related to the degree of terminal sialylation of glycoproteins, and it is reported that the more sialylation, the lower isoelectric point value (W. Schlags et al. 2002, Proteomics , 2, 679-682). Thus, as shown in FIG. 10, the lower isoelectric point of hEPO expressed in cells co-expressed with CMP-SAT is an indirect result showing that its sialic acid content is higher.

Example 6: MAA (Maarckia amurensis agglutinin) binding experiment of purified hEPO

In this example, the sialic acid content of hEPO expressed from CHO-EPO-SAT cell line was investigated. First, MAA is a lectin that binds to sialic acid attached to the end of a sugar residue of a glycoprotein, which can be used to analyze the degree of sialation of the glycoprotein. The hEPO isolated by His-tagged metal affinity chromatography method, as in Example 3, was transferred to a nitrocellulose membrane at 15 V after SDS-PAGE electrophoresis, followed by digoxy in a DIG glycan differentiation kit (Roche, Germany). After treating MAA with digoxigenin, a secondary antibody against digoxygenin with alkaline phosphatase was treated. The color reaction was carried out using BCIP / NBT (Bio-Rad, USA), which is a substrate of alkaline phosphatase. After the color reaction, the degree of MAA binding was analyzed (FIG. 11). As a control, hEPO expressed from the CHO-EPO cell line was used. FIG. 11 shows the results of sialic acid content of hEPO expressed from CHO-EPO-SAT cell line through MAA. FIG. As shown in FIG. 11, hEPO isolated from CHO-EPO-SAT cells co-expressing CMP-SAT showed a higher degree of MAA binding. This indicates that more sialic acid is attached to the sugar residue terminus of hEPO in which CMP-SAT is expressed simultaneously in the cell.

Example  7: Purified separately hEPO of Neu5Ac  And Neu5Gc  Content analysis

In this example, the sialic acid content of hEPO expressed from CHO-EPO-SAT cell line was investigated.

As in Example 3, the content analysis of sialic acid content of hEPO separated by His-tag metal affinity chromatography method was analyzed by requesting the Korea Institute of Basic Science. Analytical method hydrolyzed sugars by treating 0.1N HCl with sugars of hEPO sugar residues, and then analyzed the contents of Neu5Ac and Neu5Gc by chromatographic analysis using CarboPAK PA1 column (4.5 × 250 mm, Dionex, USA). . Table 1 shows the results of chromatograph analysis of the sialic acid content of hEPO expressed from the CHO-EPO-SAT cell line using a CarboPAK PA1 column (4.5 × 250 mm, Dionex, USA).

Table 1.

As shown in Table 1, when CMP-SAT was simultaneously expressed in cells, the content of Neu5Gc in sialic acid in hEPO was reduced by about 43% and Neu5Ac was increased by about 21%. This resulted in efficient transfer of CMP-Neu5Ac, a substrate of sialylation reaction, to Golgi apparatus by CMP-SAT co-expressed in cells, and CMP-Neu5Ac by CMP-Neu5Ac hydroxylase in the cytoplasm. It is believed that this is due to a decrease in the rate of conversion to, and an increase in sialylation efficiency due to an increase in the substrate of sialyltransferase in the Golgi apparatus.

Through the embodiments of the present invention as described above, in the case of expressing the glycoprotein while simultaneously expressing CMP-SAT in a CHO cell line, which is a mammal, particularly a rodent-derived cell, it can cause an immune response in humans of sialic acid in the glycoprotein. It was found that the content of Neu5Gc was decreased and the content of Neu5Ac was increased.

According to the cells of the present invention, sialylated glycoproteins and CMP-SAT can be expressed at levels higher than endogenous levels.

According to the method for producing sialylated glycoprotein having a reduced content of N-glycolylneuraminic acid and an increased content of N-acetylneuraminic acid, the N-acetylneuraminic acid in the sialic acid content of the glycoprotein Is increased and the content of N-glycolylneuraminic acid can easily produce a reduced glycoprotein.

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Claims (9)

Transformed CHO cells into which foreign nucleic acids encoding erythropoietin and cytidine monophosphate-sialic acid trasporter (CMP-SAT) have been introduced, with reduced content of N-glycolylneuraminic acid and N-acetylneuraminic acid Transformed CHO cells, characterized in that to produce the erythropoietin with an increased content of. delete delete delete The cell of claim 1, which is a CHO-EPO-SAT cell (Accession No. KCTC 10843BP). Culturing mammalian-derived cells into which sialylated glycoproteins and foreign nucleic acid encoding cytidine monophosphate-sialic acid trasporter (CMP-SAT) are introduced to produce the sialylated glycoproteins; And Recovering the sialylated glycoprotein from the culture, wherein the content of N-glycolylneuraminic acid is reduced and the content of N-acetylneuraminic acid is increased. The method of claim 6, wherein the sialylated glycoprotein is selected from the group consisting of erythropoietin, plasminogen, transferrin and tyrotropin. The method of claim 6, wherein the cells are selected from the group consisting of CHO, BHK, 3T3, and COS. The method of claim 6, wherein the cells are CHO-EPO-SAT cells (Accession No. KCTC 10843BP).

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