WO2008047966A2 - Method for enhancing glycosylation of recombinant protein - Google Patents

Method for enhancing glycosylation of recombinant protein Download PDF

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Publication number
WO2008047966A2
WO2008047966A2 PCT/KR2006/004523 KR2006004523W WO2008047966A2 WO 2008047966 A2 WO2008047966 A2 WO 2008047966A2 KR 2006004523 W KR2006004523 W KR 2006004523W WO 2008047966 A2 WO2008047966 A2 WO 2008047966A2
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recombinant protein
glycosylation
protein
cho
host cell
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PCT/KR2006/004523
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French (fr)
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Tai Hyun Park
Shin Sik Choi
Ju Hyun Park
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Hanson Biotech Co., Ltd
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2511/00—Cells for large scale production

Definitions

  • the present invention relates to a method for the enhancement of the glycosylation of recombinant proteins using in vitro cultures of Chinese hamster ovarian (CHO) cells. More specifically, the present invention is directed to a method of enhancing glycosylation of recombinant proteins by adding 3OK protein to a CHO cell culture medium, a glycosylation enhancer comprising the 3OK protein, a method for enhancing glycosylation of recombinant protein by using CHO cell line transformed with 3OK gene, and a CHO cell line transformed with 3OK gene to enhance the glycosylation of the recombinant protein.
  • glycosylation of the present invention, which is a post- transitional modification (PTM), refers to the process of the addition of saccharides to proteins so as to make a carbohydrate chain.
  • PTM post- transitional modification
  • the glycosylation is classified into N-linked glycosylation to the amide nitrogen of asparagine side chains, and 0-linked glycosylation to the hydroxy oxygen of serine and theronine side chains.
  • glycosylation affects the solubility of the protein and the immunological reaction when administered into the human body and, above all, plays a key role in the duration time of the in vivo activity of the protein.
  • the negatively charged sialic acid pretects the proteins in the blood from the attack of protease and also protects proteins in the liver from degradation and prolongs the half-life of the proteins. With this increased half-life, the effectiveness of medication containing glycosylated proteins can be maintained for a longer duration upon a single dosage. Thus, glycosylation is one of the most important factors in the production of recombinant proteins.
  • the present inventors have found that, in the production of recombinant proteins, by using CHO host cells with the addition of 3OK proteins to CHO host cell culture medium, the glycosylation of the produced recombinant protein is enhanced and the sialylation is increased, and thus it can be achieved to qualitatively enhance recombinant proteins. As a result, the present invention has been completed. [Disclosure] [Technical Problem]
  • the primary object of the present invention is to provide a method for producing a recombinant protein using a Chinese Hamster Ovarian (CHO) host cell, characterized in that the process further comprises adding a 3OK protein to a CHO cell culture medium to enhance the glycosylation of recombinant proteins.
  • CHO Chinese Hamster Ovarian
  • Another object of the present invention is to provide a glycosylation enhancer comprising a 3OK protein to enhance the glycosylation of the recombinant protein by using a CHO host cell.
  • Yet another object of the present invention is to provide a method for producing a recombinant protein using a CHO host cell, characterized in that the CHO cell is transformed with a 3OK gene.
  • Yet another object of the present invention is to provide a CHO host cell line for producing a recombinant protein, characterized in that said CHO host cell line is a cell line transformed with a 3OK gene to produce a recombinant protein with enhanced glycosylation.
  • the primary object of the present invention can be achieved by providing a method for producing a recombinant protein by using a host cell, characteriszed in that the method further comprises adding a 3OK protein to a host cell culture medium to enhance the glycosylation of the recombinant proteins.
  • the present invention relates to a gene of 3OK protein which enhances the glycosylation of recombinant proteins for medical applications, produced by CHO cells. Also, the present invention relates to recombinant 3OK proteins produced by the E.coli which uses said gene.
  • CHO (Chinese hamster ovarian) cells are widely used as host cells. CHO cells are originated from the ovary of Chinese Hamster, which were approved to be used to produce recombinant proteins by the U.S. FDA. Reasons for the wide use of CHO cells are because viruses such as Polio, Herpes, Herpes, HIV, Adenovirus, etc. are not replicatable in the CHO cells and are safe for medicinal use. Also, CHO cells provide an efficient gene amplification system, and through suspension culture are able to be produced on a large scale. However, above all, the most important reason for the use of CHO cells is the similarity of glycosylation of proteins in CHO cells and human cells.
  • the 3OK protein of the present invention is selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23.
  • the specific amino acid sequences of said 3OK proteins are provided in the annexed Sequence List 1 to 5.
  • EPO erythropoietin
  • erythropoietin refers to the glycoprotein hormone produced by the kidney. EPO enhances the division and differentiation of erythrocyte precursors. EPO is administered to a patient with anemia and a patient undergoing a surgical operation with excessive bleeding. Genetic code for EPO was first identified in the 1980' s, and production of the recombinant protein using CHO cells began soon after. The molecular weight of EPO is approximately 30KDa, however only 18,398Da of the weight is attributed to amino acid composition while the remainder of the weight is from the carbohydrate chain.
  • FSH follicle stimulating hormone
  • Another object of the present invention can be achieved by providing a glycosylation enhancer comprising 3OK proteins added to the culture medium of a host cell which produces recombinant proteins.
  • the 3OK protein is one selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23 and the specific amino acid sequences of said 3OK proteins are provided in the annexed Sequence List 1 to 5.
  • Another object of the present invention can be achieved by providing a method for producing a recombinant protein using a CHO host cell, characterized in that the CHO cell is transformed with a 3OK gene.
  • Said 3OK gene used for cell transformation is one selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23, whereupon the base sequences are provided in the annexed Sequence Lists 6 to 10.
  • Another object of the present invention can be achieved by providing a CHO host cell for producing a recombinant protein transformed with a 3OK gene.
  • Said 3OK gene used for cell transformation is one selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23, whereupon the base sequences are provided in the annexed Sequence Lists 6 to 10. [Advantageous Effects]
  • the present invention provides many advantageous features over the conventional methods.
  • First, the method enhances the degree of glycosylation of the recombinant proteins.
  • Second, said technique is able to increase in vivo retention time by increasing the sialylation. Consequently, this invention allows for the qualitative improvement of bio-medical drugs.
  • the present invention is widely applicable for the production of bio ⁇ medical drugs which market share is presently about 10% of the pharmaceutical drug market with 20% yearly growth. [Description of Drawings]
  • Fig. 1 is an image showing the results of RT-PCR from the RNA of 30Kc6 expressed CHO cell.
  • Fig. 2 is an image showing an SDS-PAGE result of 30Kc6 produced by an E.coli host.
  • Fig. 3, Fig.4, Fig. 5 are images showing the results of 2-Dimensional electrophoresis Western blot test of EPO and FSH produced by the 30Kc6 expressed CHO cell.
  • Fig.6 is an image showing the results of 2-Dimensional electrophoresis Western blot test of FSH produced by the 30Kc6 expressed CHO cell.
  • Fig.7 is a picture showing the results of MAA lectin assay of EPO produced by the 30Kc6 expressed CHO cell.
  • Fig.8 is a reaction scheme of the sialylation.
  • Fig.9 shows the results of lectin assay of the influence of silkworm hemolymph and 3OK protein.
  • Fig.10 shows the mass spectrometer analysis results of the reaction product of sialylation with 30Kcl9.
  • the object of the present invention can be achieved by providing a method for producing a recombinant protein by using a host cell, characteriszed in that the method further comprises adding a 3OK protein to a host cell culture medium to enhance the glycosylation of the recombinant proteins.
  • Another object of the present invention can be achieved by providing a method for producing recombinant proteins using a CHO cell as the host cell which is transformed with the 3OK gene.
  • a 30Kc6 gene was inserted between the Baml and Not I site of the pIRES-Hyg vector.
  • Said plasmid was used to transfect a CHO host cell for producing FSH, with Lipofectamine 2000 (Invirtrogen) reagent, and the CHO host cell was cultured in 200(micro[upsilon]gram)/ml concentration of hygromicine, whereupon a large number of 30Kc6 expressing CHO cell clones for FSH production were sorted.
  • a 30Kc6 gene was inserted in a pcDNA3 vector, transfected using Lipofectamine plus (Invitrogen) reagent, whereupon the clones were sorted in the same aforementioned fashion using G418 (Invitrogen).
  • RNA was extracted from the 30K6 expressed CHO cell, and cDNA synthesis was carried out using reverse transcriptase and anti-sense primer, whereupon said cDNA underwent PCR using the RT-PCR method.
  • Fig. 1 shows the result product of RT-PCR of DNA electrophoresis.
  • Fig. 1 is an image showing the results after RT-PCR used to confirm the successful expression of the 30Kc6 gene in the EPO producing CHO cells.
  • the size of the 30Kc6 gene is approximately 770bp, and as shown in the image, lane 1 is the result of an empty vector with no 30Kc6 insertion and showed no band, while with proper 30Kc6 gene insertion and 30Kc6 expression, lane 2 shows a band at an accurate size.
  • the PCR product was cloned with pBluescript SK+ vector, and using T3 and T7 primer, the resulting read 30Kc6 gene DNA sequence of the silkworm hemolymph appeared clearly.
  • Example 2 3OK proteins produced by E.coli Of the five 3OK proteins of the aforementioned 3OK protein family group, the 30Kcl9 gene was inserted into E.coli expressed vector pET22b(+), and by using said vector, E.coli BL2KDE3) was transformed and consequently a 3OK protein producing recombinant strain was produced. His6-tag was then added to the carboxylic end of the produced 30Kcl9 so they could be easily purified. After culturing said strain in a LB culture medium, ImM of isopropyl-(beta)-D-thiogalactopranoside: IPTG was added and the prepared 30Kcl9 proteins were finally purified by HisTrap HP column (ImL, Amersham). The purified 30Kcl9 was exchanged in buffer solution with PBS and stored and used for experimentation.
  • Fig. 2 shows the SDS-PAGE results of the 30Kcl9 produced by the gene recombination method in E.coli. As shown in the image, 28KDa of the 30Kcl9 protein was produced and a high level of purity could be determined. After freeze drying and storage of said produced recombinant 30Kcl9, it was melted and filtrated in a culture when used for experimentation.
  • Example 3 CHO cell culture in a serum free medium and 2-Dementional electrophoresis Western blot analysis
  • the medium was collected 3 days after replacement, and in the case of FSH, medium was collected 4 days after replacement. Furthermore, suspension culture was carried out for the CHO cell which produced FSH. After innoculating the CHO cells, which were being grown by the adhesion culture, in a spinner flask containing 5OmL Ex-cell 301 medium, said medium was collected after 4 days of culture.
  • 0.2mg/mL 30Kcl9 protein was added at an early growth stage with serum and a protein production stage with serum free culture medium and experiments were carried out. The collected culture medium was concentrated 10 fold using CentriPlus (Millipore) and used for analysis.
  • the degree of glycosylation was analyzed using the basic 2D electrophoresis Western blot method. Complete glycosylation of the glycoprotein was determined by the attachement of sialic acid at the terminal end of the carbohydrate chain. Incomplete glycosylation was indicated by a comparatively decreased value of the pi (isoelectric point).
  • IEF isoelectro focusing
  • rehydration solution 8.0M urea, 2% CHAPS, 0.3% DTT, 0.5% IPG buffer, small quantity of bromophenol blue
  • IPG buffer 0.5% IPG buffer
  • electrophoresis was performed on a 12% polyacrylamide gel. The separated proteins by the electrophoresis were transferred to PVDF membrane and then underwent first round, second round antibody processing using the Western blot test.
  • Fig. 3 - Fig. 5 show the results of 2-Demensional electrophoresis Western blot test of EPO and FSH produced by 30Kc6 expressed Chinese Hamster Ovarian cell .
  • Fig. 3 is an image showing the results of 2-Dimensional electrophoresis Western blot test of the influence of glyosylation on the EPO produced by 30Kc6 expressed CHO cell.
  • the CHO cell underwent adhesion culture in a DMEM/F12 culture supplemented with 10% fetal bovine serum, and was then re-cultivated in replacement serum-free culture medium Ex-cell 301.
  • EPO which was produced in control group cell which did not express the 30Kc6 gene displayed 6 pi spots of various values (above) while EPO produced in a cell with proper expression of the 30Kc6 gene showed by a singular spot with lower pi value (below).
  • Fig. 4 is an image showing the results of 2-Dimensional electrophoresis Western blot test of the influence of glyosylation on the FHS produced by 30Kc6 expressed CHO cell.
  • the CHO cell underwent adhesion culture in a IMDM culture supplemented with 10% fetal bovine serum, and was then re-cultured in replacement serum-free culture SF CHO. Although it does not appear as a completely singular spot like EPO, proper expression of the 30Kc6 gene could be determined by a much stronger and more defined spot with lower pi value.
  • Fig. 5 shows the results of FSH producing CHO cells after suspension culture.
  • an equal number of FSHs produced by CHO cells which expressed the 30Kc6 gene and by CHO cells which did not express the 30Kc6 gene were analyzed by 2-D electrophoresis Western blot test.
  • the resulting spot indicated the increased uniformity of the FSH, lowered pi, and namely the high degree of sailic acid bonded.
  • the definitive spot with lower pi indicates that regardless of the type of protein and method of cell culture, glycosylation is enhanced when expressing 30Kc6 genes which is depicted by a stronger and more definitive spot with lower pi.
  • Fig. 6 is an image showing the 2-D electrophoresis Western blot test results which indicate the influence of the addition of recombinant 30Kcl9 produced by E.coli on the glycosylation of FSH produced in CHO cells.
  • the CHO cell underwent adhesion culture in a IMDM culture medium supplemented with 10% FBS, re-cultured in replacement serum- free culture SF CHO, and then cultured again four days later.
  • 30Kcl9 was added both in serum-included and serum-free cultures for the experiment.
  • Fig. 6 is an image showing the 2-D electrophoresis Western blot test results which indicate the influence of the addition of recombinant 30Kcl9 produced by E.coli on the glycosylation of FSH produced in CHO cells.
  • the most important factor of glycosylation is the degree of bonding of the sialic acid.
  • lectin assay was carried out.
  • the culture solution underwent SDS-PAGE electrophoresis and was then transferred onto PVDF membrane, whereupon MAA (Maackia amurensis agglutinin),which among sugars specifically binds to sialic acid, was used for assay.
  • MAA Meackia amurensis agglutinin
  • Fig. 7 is an image showing the MAA lectin assay of EPO produced by 30Kc6 expressed CHO cell. As shown in Fig. 7, in the case that the 30Kc6 gene is expressed, the lectin band appears larger, which indicates that the expression of 30Kc6 has increased the sialylation of the recombinant protein.
  • 3OK proteins were added during the reaction of producing fetuin.
  • the fetuin can be produced by adding CMP-sialic acid, a sialylation precursor, and recombinant sialyltransferase to asialofetuin produced by the asialylation of fetuin which is a typical glycoprotein.
  • the degree of influence of the addition of the 3OK proteins to the reaction was analyzed. For this analysis, lectin assay and mass spectrometry were carried out. Sialylation of asialofetuin used by sialyltransferase appears in Fig. 8.
  • sialylation pre-cursor If sialyltransferase is added to the sialylation pre-cursor, CMP-sialic acid, and asialofetuin during sialylation then sialylation advances and the complete fetuin glycoprotein is produced.
  • Fig. 9 is an image showing MAA lectin assay of the influence that silk worm hemolymph and recombinant 30Kcl9 has on the aforementioned reaction. If sialic acid is binded with asialofetuin and fetuin is made, the MAA lectin indicates the binding and a band appears (Fig. 9).
  • the basic sialic acid reaction without any additives shows almost no band (lane 1), however with the addition of silkworm hemolymph, sialic acid binds with fetuin, and appeares as a band (lane 3).
  • lane 3 After boiling silkworm hemolymph and removing proteins, almost no band was visible after adding the boiled silkworm hemolymph(lane 2), which indicates that silk worm hemolymph proteins promote sialylation.
  • 0.2 mg/ml of 30Kcl9 recombinant protein produced by E.coli was added to the reaction.
  • Fig. 9 shows that a band appeared (lane 4).
  • sialylation is increased by the addition of silkworm hemolymph and recombinant 30Kcl9 protein as indicated by the distinctive band.
  • Fig. 10 shows the results of mass spectrometry analysis after adding 30Kcl9 to the reactions shown in Fig. 8.
  • Fig. 10 shows the results of mass spectrometry analysis after adding 30Kcl9 to the reactions shown in Fig. 8.
  • a 3OK protein a large new peak in molecular weight was observed (red circle), which was due to the fetuin bound with sialic acid. Namely, it was observed that sialylation was promoted by the 3OK protein.
  • the method of the present invention for producing recombinant proteins will allow the advancement and increased value of the qualitative activity of biomedical drugs. Therefore, the present invention is widely applicable for the production of bio-medical drugs which market share is presently 10% of the pharmaceutical drug market with 20% yearly growth. [Sequence List Text]
  • Sequence List 6 to 10 of base sequence of the 3OK protein gene specifically 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23, respectively.

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Description

[DESCRIPTION]
[Invention Title] METHOD FOR ENHANCING GLYCOSYLATION OF RECOMBINANT PROTEIN
[Technical Field]
The present invention relates to a method for the enhancement of the glycosylation of recombinant proteins using in vitro cultures of Chinese hamster ovarian (CHO) cells. More specifically, the present invention is directed to a method of enhancing glycosylation of recombinant proteins by adding 3OK protein to a CHO cell culture medium, a glycosylation enhancer comprising the 3OK protein, a method for enhancing glycosylation of recombinant protein by using CHO cell line transformed with 3OK gene, and a CHO cell line transformed with 3OK gene to enhance the glycosylation of the recombinant protein.
[Background Art]
The term "glycosylation" of the present invention, which is a post- transitional modification (PTM), refers to the process of the addition of saccharides to proteins so as to make a carbohydrate chain. The glycosylation is classified into N-linked glycosylation to the amide nitrogen of asparagine side chains, and 0-linked glycosylation to the hydroxy oxygen of serine and theronine side chains.
Glycosylation affects the solubility of the protein and the immunological reaction when administered into the human body and, above all, plays a key role in the duration time of the in vivo activity of the protein. At the end of the carbohydrate chain, the negatively charged sialic acid pretects the proteins in the blood from the attack of protease and also protects proteins in the liver from degradation and prolongs the half-life of the proteins. With this increased half-life, the effectiveness of medication containing glycosylated proteins can be maintained for a longer duration upon a single dosage. Thus, glycosylation is one of the most important factors in the production of recombinant proteins.
In situtations where natural glycosylation of EPO and FSH does not occur normally in the body, it is known that while in vitro activity may still occur there is a sudden decrease in in vivo activity upon administration of medication.
Up to this point, the technique relating to the glycosylation of recombinant proteins in Korea is disclosed under patent number 10-1999- 0065494. This technique relates to the method of increasing the in vivo activity of the glycoprotein by maximizing the sialic acid content of the lycoprotein. The sialic acid content of the glycoprotein is maximized by minimizing the production of sialidase through transformation of the sialidase gen of the cell line which produces the glycoprotein, and by re- binding by means of a specific enzyme, sialic acid to sialic acid-free glycoprotein of the glycoprotein produced by the cell line.
This technique allows the production of many types of glycoproteins for general use, however, the efficiency of production is not high and the degree of glycosylation enhancement is relatively low. For this reason, commercialization of this technique is not practical.
Representative of all the methods which enhance the in vivo activity of recombinant proteins by enhancing glycosylation of the recombinant proteins is a technique which produces new polypeptides which have higher in vivo activity by modifying the amino acid composition of the target protein. Reformed protein which in vivo activity has been enhanced by modifying the existing amino acid sequence were widely developed as disclosed in US Patent No. 6,989,365 which describes a method for the fusion of albumin which is known as having a long duration time for in vivo activity, and EPO, US Patent Nos. 5,457,089 and 4,835,260 which describe a method for increasing the glycosylation by the fusion of peptides with a glycosylation site at their C-terminal . These methods are applicable to the production of various glycoproteins such as EPO, FSH, etc.
However, because the functional sites for each target protein are different in these techniques, the probability of obtaining the modified proteins with high in vitro activity without losing functionality is very low. Consequently, these techniques cannot be universally applied to different types of proteins, and it is difficult to find the sites to be modified in accordance with the characteristics of each target protein, and to apply these techniques to the sites. Although much research has been done to find a method that enhances glycosylation by using CHO cells in the production of glycoproteins, there has been no real ground breaking developments up to this point.
The present inventors have found that, in the production of recombinant proteins, by using CHO host cells with the addition of 3OK proteins to CHO host cell culture medium, the glycosylation of the produced recombinant protein is enhanced and the sialylation is increased, and thus it can be achieved to qualitatively enhance recombinant proteins. As a result, the present invention has been completed. [Disclosure] [Technical Problem]
Therefore, the primary object of the present invention is to provide a method for producing a recombinant protein using a Chinese Hamster Ovarian (CHO) host cell, characterized in that the process further comprises adding a 3OK protein to a CHO cell culture medium to enhance the glycosylation of recombinant proteins.
Another object of the present invention is to provide a glycosylation enhancer comprising a 3OK protein to enhance the glycosylation of the recombinant protein by using a CHO host cell.
Yet another object of the present invention is to provide a method for producing a recombinant protein using a CHO host cell, characterized in that the CHO cell is transformed with a 3OK gene.
Yet another object of the present invention is to provide a CHO host cell line for producing a recombinant protein, characterized in that said CHO host cell line is a cell line transformed with a 3OK gene to produce a recombinant protein with enhanced glycosylation. [Technical Solution] The primary object of the present invention can be achieved by providing a method for producing a recombinant protein by using a host cell, characteriszed in that the method further comprises adding a 3OK protein to a host cell culture medium to enhance the glycosylation of the recombinant proteins.
The present invention relates to a gene of 3OK protein which enhances the glycosylation of recombinant proteins for medical applications, produced by CHO cells. Also, the present invention relates to recombinant 3OK proteins produced by the E.coli which uses said gene.
CHO (Chinese hamster ovarian) cells are widely used as host cells. CHO cells are originated from the ovary of Chinese Hamster, which were approved to be used to produce recombinant proteins by the U.S. FDA. Reasons for the wide use of CHO cells are because viruses such as Polio, Herpes, Herpes, HIV, Adenovirus, etc. are not replicatable in the CHO cells and are safe for medicinal use. Also, CHO cells provide an efficient gene amplification system, and through suspension culture are able to be produced on a large scale. However, above all, the most important reason for the use of CHO cells is the similarity of glycosylation of proteins in CHO cells and human cells.
The 3OK protein of the present invention is selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23. The specific amino acid sequences of said 3OK proteins are provided in the annexed Sequence List 1 to 5.
The term "erythropoietin (EPO)" of the present invention refers to the glycoprotein hormone produced by the kidney. EPO enhances the division and differentiation of erythrocyte precursors. EPO is administered to a patient with anemia and a patient undergoing a surgical operation with excessive bleeding. Genetic code for EPO was first identified in the 1980' s, and production of the recombinant protein using CHO cells began soon after. The molecular weight of EPO is approximately 30KDa, however only 18,398Da of the weight is attributed to amino acid composition while the remainder of the weight is from the carbohydrate chain.
The term "follicle stimulating hormone (FSH)" of the present invention refers to a glycoprotein sex hormone synthesized and secreted by the anterior pituitary gland. FSH is also used for in vitro fertilization. FSH is a glycoprotein of which 40% molecular weight is attributed to the carbohydrate chain, and consists of two sub-units, alpha and beta, each of which has two N-linked glycosylation sites, respectively.
Another object of the present invention can be achieved by providing a glycosylation enhancer comprising 3OK proteins added to the culture medium of a host cell which produces recombinant proteins.
The 3OK protein is one selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23 and the specific amino acid sequences of said 3OK proteins are provided in the annexed Sequence List 1 to 5.
Another object of the present invention can be achieved by providing a method for producing a recombinant protein using a CHO host cell, characterized in that the CHO cell is transformed with a 3OK gene.
Said 3OK gene used for cell transformation is one selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23, whereupon the base sequences are provided in the annexed Sequence Lists 6 to 10.
Another object of the present invention can be achieved by providing a CHO host cell for producing a recombinant protein transformed with a 3OK gene.
Said 3OK gene used for cell transformation is one selected from the group consisting of 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23, whereupon the base sequences are provided in the annexed Sequence Lists 6 to 10. [Advantageous Effects]
The present invention provides many advantageous features over the conventional methods. First, the method enhances the degree of glycosylation of the recombinant proteins. Second, said technique is able to increase in vivo retention time by increasing the sialylation. Consequently, this invention allows for the qualitative improvement of bio-medical drugs. The present invention is widely applicable for the production of bio¬ medical drugs which market share is presently about 10% of the pharmaceutical drug market with 20% yearly growth. [Description of Drawings]
Fig. 1 is an image showing the results of RT-PCR from the RNA of 30Kc6 expressed CHO cell.
Fig. 2 is an image showing an SDS-PAGE result of 30Kc6 produced by an E.coli host.
Fig. 3, Fig.4, Fig. 5 are images showing the results of 2-Dimensional electrophoresis Western blot test of EPO and FSH produced by the 30Kc6 expressed CHO cell.
Fig.6 is an image showing the results of 2-Dimensional electrophoresis Western blot test of FSH produced by the 30Kc6 expressed CHO cell.
Fig.7 is a picture showing the results of MAA lectin assay of EPO produced by the 30Kc6 expressed CHO cell.
Fig.8 is a reaction scheme of the sialylation.
Fig.9 shows the results of lectin assay of the influence of silkworm hemolymph and 3OK protein.
Fig.10 shows the mass spectrometer analysis results of the reaction product of sialylation with 30Kcl9. [Best Mode]
The object of the present invention can be achieved by providing a method for producing a recombinant protein by using a host cell, characteriszed in that the method further comprises adding a 3OK protein to a host cell culture medium to enhance the glycosylation of the recombinant proteins.
Another object of the present invention can be achieved by providing a method for producing recombinant proteins using a CHO cell as the host cell which is transformed with the 3OK gene.
Hereinafter, the present invention will be described in greater detail with reference to the following examples. The examples are given only for illustration of the present invention and not to limit the scope of the present invention. [Mode for Invention] [Example 1] Transfection with 3OK gene expressing plasmid clone selection
Of the five 3OK proteins of the aforementioned 3OK protein family group, a 30Kc6 gene was inserted between the Baml and Not I site of the pIRES-Hyg vector. Said plasmid was used to transfect a CHO host cell for producing FSH, with Lipofectamine 2000 (Invirtrogen) reagent, and the CHO host cell was cultured in 200(micro[upsilon]gram)/ml concentration of hygromicine, whereupon a large number of 30Kc6 expressing CHO cell clones for FSH production were sorted.
For the expression of the 3OK protein in a CHO cell which produces EPO, a 30Kc6 gene was inserted in a pcDNA3 vector, transfected using Lipofectamine plus (Invitrogen) reagent, whereupon the clones were sorted in the same aforementioned fashion using G418 (Invitrogen).
In order to confirm the validity of 30Kc6 gene expression of the sorted clones, RNA was extracted from the 30K6 expressed CHO cell, and cDNA synthesis was carried out using reverse transcriptase and anti-sense primer, whereupon said cDNA underwent PCR using the RT-PCR method. Fig. 1 shows the result product of RT-PCR of DNA electrophoresis.
Fig. 1 is an image showing the results after RT-PCR used to confirm the successful expression of the 30Kc6 gene in the EPO producing CHO cells. The size of the 30Kc6 gene is approximately 770bp, and as shown in the image, lane 1 is the result of an empty vector with no 30Kc6 insertion and showed no band, while with proper 30Kc6 gene insertion and 30Kc6 expression, lane 2 shows a band at an accurate size. The PCR product was cloned with pBluescript SK+ vector, and using T3 and T7 primer, the resulting read 30Kc6 gene DNA sequence of the silkworm hemolymph appeared clearly.
[Example 2] 3OK proteins produced by E.coli Of the five 3OK proteins of the aforementioned 3OK protein family group, the 30Kcl9 gene was inserted into E.coli expressed vector pET22b(+), and by using said vector, E.coli BL2KDE3) was transformed and consequently a 3OK protein producing recombinant strain was produced. His6-tag was then added to the carboxylic end of the produced 30Kcl9 so they could be easily purified. After culturing said strain in a LB culture medium, ImM of isopropyl-(beta)-D-thiogalactopranoside: IPTG was added and the prepared 30Kcl9 proteins were finally purified by HisTrap HP column (ImL, Amersham). The purified 30Kcl9 was exchanged in buffer solution with PBS and stored and used for experimentation.
Fig. 2 shows the SDS-PAGE results of the 30Kcl9 produced by the gene recombination method in E.coli. As shown in the image, 28KDa of the 30Kcl9 protein was produced and a high level of purity could be determined. After freeze drying and storage of said produced recombinant 30Kcl9, it was melted and filtrated in a culture when used for experimentation.
[Example 3] CHO cell culture in a serum free medium and 2-Dementional electrophoresis Western blot analysis
For serum free medium culture in an adhesive state, cultivation first took place in DMEM/F12 or IMDM (JBI) culture supplemented with 10% fetal bovine serum (FBS, Gibco) and then after removing culture medium during exponential growth phase, it was washed with PBS and replaced with serum free culture Excel 1-301 (JRH) or SF CHO (JBI). The cell culture was basically performed using a T-25 flask under 37°C, 5% C02 conditions.
For the analysis of the EPO produced by the CHO cells, the medium was collected 3 days after replacement, and in the case of FSH, medium was collected 4 days after replacement. Furthermore, suspension culture was carried out for the CHO cell which produced FSH. After innoculating the CHO cells, which were being grown by the adhesion culture, in a spinner flask containing 5OmL Ex-cell 301 medium, said medium was collected after 4 days of culture. To observe the effectiveness of the 30Kcl9 protein production by E.coli, in the case of adhesion culture, 0.2mg/mL 30Kcl9 protein was added at an early growth stage with serum and a protein production stage with serum free culture medium and experiments were carried out. The collected culture medium was concentrated 10 fold using CentriPlus (Millipore) and used for analysis.
When analyzing the protein, the degree of glycosylation was analyzed using the basic 2D electrophoresis Western blot method. Complete glycosylation of the glycoprotein was determined by the attachement of sialic acid at the terminal end of the carbohydrate chain. Incomplete glycosylation was indicated by a comparatively decreased value of the pi (isoelectric point).
For the present experiment, IEF (isoelectro focusing) of the collected and concentrated culture was carried out following the method provided by the manufacturing company mixing with rehydration solution (8.0M urea, 2% CHAPS, 0.3% DTT, 0.5% IPG buffer, small quantity of bromophenol blue), in 7cm of pH3-10 or pH4-7 IPG strip (Amersham). After the strip had reached the equilibrium in equilibrium solution, 6.0M Urea, 30% glycerol, 2% SDS, 0.05M Tris-HCL, 0.1% DTT, electrophoresis was performed on a 12% polyacrylamide gel. The separated proteins by the electrophoresis were transferred to PVDF membrane and then underwent first round, second round antibody processing using the Western blot test.
Fig. 3 - Fig. 5 show the results of 2-Demensional electrophoresis Western blot test of EPO and FSH produced by 30Kc6 expressed Chinese Hamster Ovarian cell .
Fig. 3 is an image showing the results of 2-Dimensional electrophoresis Western blot test of the influence of glyosylation on the EPO produced by 30Kc6 expressed CHO cell. For this present experiment, the CHO cell underwent adhesion culture in a DMEM/F12 culture supplemented with 10% fetal bovine serum, and was then re-cultivated in replacement serum-free culture medium Ex-cell 301. As shown in Fig. 3, EPO which was produced in control group cell which did not express the 30Kc6 gene displayed 6 pi spots of various values (above) while EPO produced in a cell with proper expression of the 30Kc6 gene showed by a singular spot with lower pi value (below).
Fig. 4 is an image showing the results of 2-Dimensional electrophoresis Western blot test of the influence of glyosylation on the FHS produced by 30Kc6 expressed CHO cell. For this particular experiment, the CHO cell underwent adhesion culture in a IMDM culture supplemented with 10% fetal bovine serum, and was then re-cultured in replacement serum-free culture SF CHO. Although it does not appear as a completely singular spot like EPO, proper expression of the 30Kc6 gene could be determined by a much stronger and more defined spot with lower pi value.
Fig. 5 shows the results of FSH producing CHO cells after suspension culture. In this case, an equal number of FSHs produced by CHO cells which expressed the 30Kc6 gene and by CHO cells which did not express the 30Kc6 gene were analyzed by 2-D electrophoresis Western blot test. The resulting spot indicated the increased uniformity of the FSH, lowered pi, and namely the high degree of sailic acid bonded. These results show is that there is enhancement of glycosylation regardless of the type of protein or culturing method used, and possibly the most meaningful implication of the results is the enhanced binding of sialic acid.
Namely, as shown in Fig. 3, Fig. 4, and Fig. 5 the definitive spot with lower pi indicates that regardless of the type of protein and method of cell culture, glycosylation is enhanced when expressing 30Kc6 genes which is depicted by a stronger and more definitive spot with lower pi.
Fig. 6 is an image showing the 2-D electrophoresis Western blot test results which indicate the influence of the addition of recombinant 30Kcl9 produced by E.coli on the glycosylation of FSH produced in CHO cells. For the present experiment the CHO cell underwent adhesion culture in a IMDM culture medium supplemented with 10% FBS, re-cultured in replacement serum- free culture SF CHO, and then cultured again four days later. 30Kcl9 was added both in serum-included and serum-free cultures for the experiment. As shown in Fig. 6, it can be appreciated that when 30Kcl9 is added, the pi spot is relatively lower and much more distinct, and it can be specifically appreciated with the addition of 30Kcl9 that the 2 spots with the lower pi value have lower pi value compared to that of E.coli. The increase in spots with high pi when handling with 0.3mg/ml density compared to that when handling 0.15mg/ml indicates the appropriate concentration of 30Kcl9 for glycosylation.
[Example 4] Analysis of Degree of sialic acid bond
The most important factor of glycosylation is the degree of bonding of the sialic acid. In order to analyze the degree of bonding, lectin assay was carried out. The culture solution underwent SDS-PAGE electrophoresis and was then transferred onto PVDF membrane, whereupon MAA (Maackia amurensis agglutinin),which among sugars specifically binds to sialic acid, was used for assay.
Fig. 7 is an image showing the MAA lectin assay of EPO produced by 30Kc6 expressed CHO cell. As shown in Fig. 7, in the case that the 30Kc6 gene is expressed, the lectin band appears larger, which indicates that the expression of 30Kc6 has increased the sialylation of the recombinant protein.
[Example 5] Analysis of influence on sialyltransferase activity
In order to analyze the influence on sialyltransferase which induces sialylation in the 3OK protein, in vitro experiments were carried out using recombinant sialyltransferase.
3OK proteins were added during the reaction of producing fetuin. The fetuin can be produced by adding CMP-sialic acid, a sialylation precursor, and recombinant sialyltransferase to asialofetuin produced by the asialylation of fetuin which is a typical glycoprotein. The degree of influence of the addition of the 3OK proteins to the reaction was analyzed. For this analysis, lectin assay and mass spectrometry were carried out. Sialylation of asialofetuin used by sialyltransferase appears in Fig. 8. If sialyltransferase is added to the sialylation pre-cursor, CMP-sialic acid, and asialofetuin during sialylation then sialylation advances and the complete fetuin glycoprotein is produced.
Fig. 9 is an image showing MAA lectin assay of the influence that silk worm hemolymph and recombinant 30Kcl9 has on the aforementioned reaction. If sialic acid is binded with asialofetuin and fetuin is made, the MAA lectin indicates the binding and a band appears (Fig. 9).
As shown in Fig. 9, the basic sialic acid reaction without any additives shows almost no band (lane 1), however with the addition of silkworm hemolymph, sialic acid binds with fetuin, and appeares as a band (lane 3). After boiling silkworm hemolymph and removing proteins, almost no band was visible after adding the boiled silkworm hemolymph(lane 2), which indicates that silk worm hemolymph proteins promote sialylation. In order to test the influence of silkworm hemolymph on the 3OK protein, 0.2 mg/ml of 30Kcl9 recombinant protein produced by E.coli was added to the reaction. Fig. 9 shows that a band appeared (lane 4). As shown in Fig. 9, sialylation is increased by the addition of silkworm hemolymph and recombinant 30Kcl9 protein as indicated by the distinctive band.
Fig. 10 shows the results of mass spectrometry analysis after adding 30Kcl9 to the reactions shown in Fig. 8. As shown in Fig. 10, with the addition of a 3OK protein a large new peak in molecular weight was observed (red circle), which was due to the fetuin bound with sialic acid. Namely, it was observed that sialylation was promoted by the 3OK protein.
These results indicate that 3OK proteins enhance the glycosylation of glycoproteins in CHO cells, and more specifically promote the sialylation, by increasing the activity of sialyltransferase. [Industrial Applicability]
The method of the present invention for producing recombinant proteins will allow the advancement and increased value of the qualitative activity of biomedical drugs. Therefore, the present invention is widely applicable for the production of bio-medical drugs which market share is presently 10% of the pharmaceutical drug market with 20% yearly growth. [Sequence List Text]
Attached hereto is the Sequence Lists 1 to 5 of amino acid sequence of the 3OK proteins, specifically 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23, respectively.
Also attached hereto is the Sequence List 6 to 10 of base sequence of the 3OK protein gene, specifically 30Kc6, 30Kcl2, 30Kcl9, 30Kc21 and 30Kc23, respectively.

Claims

[CLAIMS] [Claim 1]
A method for producing a recombinant protein by using a Chinse Hamster Ovarian (CHO) cell, characteriszed in that the method further comprises adding a 3OK protein to a CHO cell culture medium to enhance the glycosylation of the recombinant proteins. [Claim 2]
The method according to claim 1, wherein said recombinant protein is follicle stimulating hormone (FSH). [Claim 3]
The method according to claim 1, wherein said recombinant protein is erythropoietin (EPO). [Claim 4]
A glycosylation enhancer comprising a 3OK protein to enhance the glycosylation of the recombinant protein produced by a CHO cell. [Claim 5]
A glycosylation enhancer comprising a 3OK protein to enhance the glycosylation of the recombinant protein by using a CHO host cell. [Claim 6]
The glycosylation enhancer according to claim 5, wherein said recombinant protein is FSH. [Claim 7]
The glycosylation enhancer according to claim 5, wherein said recombinant protein is EPO. [Claim 8]
The glycosylation enhancer according to claim 5, wherein said 3OK protein is the recombinant protein produced by an E.coli host cell. [Claim 9]
A method for producing a recombinant protein using a CHO host cell, characterized in that the CHO cell is transformed with a 3OK gene. [Claim 10] The method according to claim 9, wherein said recombinant protein is follicle stimulating hormone (FSH). [Claim 11]
The method according to claim 9, wherein said recombinant protein is erythropoietin (EPO). [Claim 12]
A CHO host cell line for producing a recombinant protein, characterized in that said CHO host cell line is a cell line transformed with a 3OK gene to produce a recombinant protein with enhanced glycosylation. [Claim 13]
The CHO host cell line, according to claim 12, wherein said recombinant protein is FSH. [Claim 14]
The CHO host cell line according to Claim 12, wherein said recombinant protein is EPO. [Claim 15]
A culture medium for enhancing the glycosylation of recombinant protein produced by using a CHO host cell, characterized in that said culture medium comprises 3OK protein. [Claim 16]
The culture medium according to Claim 15, wherein said recombinant protein is FSH. [Claim 17]
The culture medium according to Claim 15, wherein said recombinant protein is erythropoietin. [Claim 18]
The culture medium according to Claim 16, wherein said recombinant protein is the recombinant protein produced by an E.coli host cell.
PCT/KR2006/004523 2006-10-20 2006-11-01 Method for enhancing glycosylation of recombinant protein WO2008047966A2 (en)

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