KR0139168B1 - Process for preparation of erythropoietin - Google Patents

Abstract

The present invention transforms CHO cells, a mammalian cell type, into recombinant plasmid pSVEp2neo, which is designed to express EPO at high titers. cfc33, and a method for producing active EPO in large quantities using this cell line.

Description

Method for preparing erythropoietin

1 shows the results of Western analysis of the expressed erythropoietin.

The present invention relates to novel mammalian cell lines that express human erythropoietin (hereinafter abbreviated as EPO) at high titers and to methods of producing EPO having activity using the cell lines.

More specifically, the present invention transforms CHO cells, which are a kind of mammalian cells, with recombinant plasmid pSVEp2neo designed to express EPO at high titers, and then expresses the high titers of EPO through HAT medium selection for the transformed cells. At the same time, the present invention relates to a novel cell line, CHOEp-cfc33, which stably expresses, and a method for producing active EPO in large quantities.

EPO is a circulating glycoprotein that stimulates red blood cell formation in higher organisms and is called erythropoietin.

Since the lifespan of human erythrocytes is about 120 days, about 1/120 of the total erythrocyte deicing vessels are destroyed in the semantic endothelial tissue system every day, and a certain number of erythrocytes are produced every day, so that the healthy human blood cells maintain a constant level of erythrocytes. Erythrocytes are produced by the maturation and differentiation of erythrocytes in the bone marrow, where EPO acts on less differentiated cells to induce differentiation into erythrocytes (see Guyton. Texbook of Medical Physiology, pp 42-). 50, 1986). Therefore, EPO is used as a therapeutic agent useful for the clinical treatment of anemia, especially renal anemia.

However, in the use of EPO as a clinical therapeutic agent, since antigenic problems may occur, it is preferable to manufacture it from human-derived raw materials as much as possible. Therefore, in the past, diseases such as aplastic anemia that secrete large amounts of EPO are used. EPO has been prepared through concentration and purification using blood or urine from patients suffering from it. However, since the supply of urine is extremely proposed, it is impeded to manufacture EPO products from urine of healthy people because it impedes the practical use of EPO. In addition to low levels, healthy urine still contains certain inhibitors that inhibit red blood cell formation at very high concentrations of EPO. exist. Therefore, it is not sufficient to mass produce this compound by known separation and purification techniques using natural sources.

Sugimoto et al. (See Sugimoto et al.) In U.S. Patent No. 4,377,513 as a part of solving this problem, because of the difficulty described above for the production of EPO using natural sources. 1, describes a method for mass production of EPO characterized by the in vivo proliferation of human LAMPA cells, including NALL-1, TALL-1, and JBL, and also studies for producing EPO by genetic engineering techniques. Subsequently reported in the literature, for example, published international patent WO 85/02610 describes a method for preparing EPO using human genomic DNA, and WO 85/03520 uses human genomic DNA and cDNA. Each method for preparing EPO is described.

However, the preceding patent still had room for further improvement. That is, in the case of patent WO 85/02610, a BstEII / BamHI fragment of about 4.8 kb is used as a gene for expression by forming an expression control site necessary for the production of EPO in the human cloned EPO genomic gene. Patent No. 85/03520 uses a 4.0 kb fragment of EcoRI / HindIII in a cloned gene. In fact, such expression control sites in genes maintain EPO at specific concentrations in humans, or only when needed. It plays a very important role, such as controlling the timing of expression, while in the system of artificially mass-producing EPO in cell lines other than the human body as in the patent invention or the present invention, it is unnecessary or in some cases prevents the production of EPO. It can also work.

Therefore, the present inventors have diligently studied to improve the problems of the prior art using the genetic engineering method. As a result, the cloned EPO genomic genes are smaller than the genes used in the two prior patents and only 2.4 kb of which the self-regulatory site is almost eliminated. The expression is controlled by the components of the vector itself, and the SV40 early promoter is used as a promoter and the LTR of muronimurine leukemia virus as an enhancer is introduced by about 3 kb away from the EPO gene. The use of the recombinant vector pS Ep2neo (Accession No .: KFCC-10831) has solved this problem and found that EPO can be expressed at a much higher titer and has completed a domestic application in this regard (Korean Patent Application No. 94- 20595).

According to the preceding invention by the present inventors, the system developed in the present invention expresses about 4,000mU / ml (when using the PSVEP2neo plasmid) for 2 days in COS-1 cells, whereas the prior patent WO In the expression system of 85/02610, when expressing COS-1 cells as host cells temporarily for 5 days, the expression rate was about 392mU / ml, and the prior patent WO 85/03520 also used COS-1 cells as a host. When expressed by expressing about 2,000mU / ml EPO it can be seen that the expression system by the present inventors is much more effective.

As such, we have developed an expression system that is much more efficient in terms of production efficiency and potency than those mentioned in the prior literature, and the present inventors have found the next industrial use method for mass production of EPO. After transforming the recombinant plasmid pSVEp2neo designed for transverse expression into CHO cells, a kind of mammalian cells, EPO was stably expressed at high titers by culturing and selecting several times in HAT medium. A new cell line CHOEp-cfc33 was prepared.

Accordingly, an object of the present invention is to provide such a novel cell line CHOEp-cfc33 for industrial use, and the formation of this cell line and a method for producing EPO in large quantities using the cell line will be described in detail below. Same as

The process of preparing the recombinant vector pSVEp2neo may be referred to because it is described in detail in Korean Patent Application No. 94-20595 and the following reference example. Therefore, hereinafter, a method for preparing a novel cell line CHOEp-cfc33, which is the final object of the present invention, from the vector pSVEp2neo will be described.

CHO cells, which are a type of mammalian cell, are intended for the production of foreign proteins by genetic engineering bar, especially when gene expression is required in higher animals due to differences in protein formation process in prokaryotes and higher animals. CHO cells were selected as cell lines for mass production of EPO in the present invention because they are commonly used cell lines.

In other words, pSVEp2neo (20 μg), an expression vector, was co-infected with plasmid pBRTK with tk-negative CHO cells by the calcium-phosphate method (Graham, 1973) to express EPO, followed by G418 (Gibco) for 2-3 weeks. Only cells transformed with at least one neo gene were selected by culturing in the medium. The plasmid pBRTK is used to introduce tk genes into tk-negative CHO cells for cell line selection. The plasmid pBRTK encodes a gene (tk gene) encoding thymidine kinase derived from Herpes simplex virus (HSV). In addition, the plasmid pTK2 (F. Colbere-Garapin, PNAS 76: 3755, 1979) was completely isolated with Bg1II / PvuII to include the tk gene. The resulting fragments were formed by gel separation and then ligation of the two separated fragments with ligase.

The colonies obtained by selecting only the transformed cells were divided into several groups to have 10-100 colonies per pool, and then cultured in HAT culture medium (hypoxanthin and thymidine at normal concentration and 100-fold dilution of aminopterin). It was. To measure the in vitro biological activity of EPO by taking only the supernatant of the medium in which the cells grow, Krystal method using H-thymidine uptake of mouse splenocytes prepared with phenylhydrazine (see Kry-stal.Exp. Hematol. 11: 649, 1983), the EPO activity showed 18.5 units / ml and 11.4 units / ml, respectively, in two randomly selected groups in 2-day culture.

The expression rate of EPO is an improvement compared to the results obtained when the EPO is manufactured by the conventional genetic engineering method, but the present inventors are still somewhat inadequate for industrial use, so that the inventors of the present invention have a certain component of the medium. In order to increase the expression rate by repeating the cell culture by continuously changing the concentration, specifically, the above-mentioned HAT medium was used, but it was repeated while increasing the concentration of aminopterin, which is one component in the medium, by 2 times. By culturing and checking the expression rate of EPO in each culture step, a method of continuously selecting cells expressing EPO at a high concentration among them was attempted.

As such, the cell lines stably expressing high concentrations of EPO were isolated even under normal culture conditions and in the absence of a selection marker during the repeated culturing and selection processes. The cell lines were named CHOEp-cfc33 and dated November 14, 1994. The KCLRF-BP-00007 was deposited with the Korea Cell Line Research Foundation. EPO expressed from this cell line not only showed activity in vitro and in vivo, but also exhibited a high expression rate of 2400 U / ml or more, thus achieving the object of the present invention to seek a method for mass production of EPO industrially. It became.

Hereinafter, the present invention will be described in more detail with reference to Examples and Examples. However, the scope of the present invention in any form is not limited thereto.

Reference Example: Preparation of Vector pSVEp2neo

A. Isolation of Human EPO Genomic Clones:

Oligonucleotides labeled with the 5'-terminus of 32-P were prepared using polynucleotide kinase (BRL) and gamma 32-P-ATP (GCAGGCCGTAGAAGTCT). The specific activity of the oligonucleotides was 1000-2000 Ci / mmol.

A modification of the in-situ proliferation method described by Woo et al. (Woo et al., PNAS 75; 3688, 1988) was used to modify the library of human genomes of bacteriophage lambda (Lawn et al., Cell 15; 1158, 1978). ) Was screened. About 800,000 phages were plated at a density of 150,000 phages per 20 cm Petri dishes (NZCYM medium) and then incubated at 37 ° C. until small plaques (about 0.5 mm) were visible to the naked eye. After cooling for one hour at 4 ° C., duplicate replicas of plaque patterns were transferred to nylon membranes (Amersham) and incubated overnight in fresh NZCYM medium plates (37 ° C.). The filter was denatured in 0.5M NaOH / 1M NaCl solution for 10 minutes and then neutralized for 10 minutes in 0.5M Tris (pH 8.0) / 1M NaCl solution. Vacuum baked at 80 ° C. for 2 hours and then washed for 1 hour in 5 × SSC / 0.5% SDS. Cell fragments adhered to the filter surface reduced nonspecific binding to the filter by gently rubbing away with wet tissue. The filter was washed with distilled water and then hybridized to 49 ° C. for 4 hours in a 5 × SSC / 5 × tenhard / 0.5% SDX / 10mM EDTA solution. 17mer labeled 32-P was added at 0.1 pmol / ml and hybridized at 49 ° C. for 22 hours. After hybridization, the filter was strongly washed with 5 × SSC / 0.1% SDS solution three times at room temperature for 10 minutes and twice at 49 ° C. for 10 minutes.

Since approximately 40 strong duplicate signals were obtained by autoradiography for 2 days with a strengthening screen, the double positive phages were plated at a density of 200 phages per 9 cm Petri dish and then rescreened in the manner described above.

Restriction enzyme mapping and Southern analysis revealed gene fragments from two of the seven positive phage clones. EPO DNA fragments were obtained from one of them (lambda Ep10) and subcloned in M13 for DNA sequencing by the dideoxy chain termination method (Sanger and Coulson, PNAS 74; 5463, 1977). The restriction enzyme, map and DNA sequence of the isolated EPO gene were confirmed to be identical to those already published and known (Lin et al., PNAS 82; 5463, 1985), followed by lambda-Ep10 carrying the EPO gene. The EcoR1 fragment was cloned into pB322 and the resulting vector was designated pBREpo.

B. Preparation of the vector pSEVp1:

One of the transformation vectors, pSVgpt (Mulligan and Berg, PNAS 78; 2072, 1981), has a gpt gene under transcriptional control of the SV40 early promoter, and downstream of the gpt coding sequence is derived from the SV40 T-antigen gene. There is a termination signal and a polyadenylation signal, and the prokaryotic site of pSVgpt does not have a pBR322 sequence that interferes with replication in mammalian cells (Lusky et al., Nature 293: 79, 1981). This is known.

Therefore, the pBREpo plasmid was completely digested with restriction enzymes HindII and BalII, followed by gel separation of a 3 kb length HindIII / BglII fragment containing the entire EPO gene, complete cleavage of the pSVgpt plasmid with restriction enzymes HindIII and BamHl, followed by SV40 early The fragments containing the promoter were gel separated and the genes of the two isolated fragments ligated with each other with ligase to obtain a pSVEpl plasmid with the EPO gene under the control of the SV40 early promoter.

C. Preparation of the vector pSVEp2:

The HindIII / BglII fragment of the EPO genomic gene contains the 5'-adjacent sequence (about 0.57 kb) as well as the coating sequence of the EPO gene. In general, 5'-contiguous sequences are located in regulators of gene expression, such as enhancers and silencers, and these factors regulate tissue-specific transcription. Therefore, when expressing the EPO gene in other cellular systems, it is desirable to remove its own regulator, so for this purpose, the vector pSVEp1 was completely cleaved with HindIII / BstEII to remove the large 5'-adjacent sequence 570 bp fragment of the EPO gene. The sections were gel isolated, treated with Klenow of DNA polymerase, and then re-ligated by ligase treatment to prepare a new vector pSVEp2 with a 2.4 kb EPO gene.

D. Preparation of the vector pSVEp2neo:

In view of the fact that long terminal repeats (LTRs) of retroviruses containing powerful enhancers can increase transcription in other cells, the introduction of the moleonimurin as a supply gene for LTR is intended to be incorporated into the present invention. Leucemia virus (MoMuLV) was selected.

Thus, to obtain LTR sequences and antineomycin genes derived from the pZIP-neoSV (X) 1 plasmid (Cepko et al., Cell 37: 1053, 1984), and to insert into the pSVEp2 vector, the recombinant pSVEp2 Was completely digested with EcoR I and linearized, while pNeoLTR (Accession No. KFCC-10830), an expression vector derived from the pZIP-neoSV (X) 1 plasmid, was completely digested with EcoR I to fragment 6.3kb containing the neogene and LTR sequence. The new vector pSVEp2neo was formed by gel separation and ligase treatment of both gene sections (Accession No. KFCC-10831). At this time, the LTR and EPO genes are about 3 kb apart, and the formed vector is regulated by the SV40 early promoter and the LTR MoMuLV enhancer to regulate the expression of the 2.4 kb EPO gene.

Example 1

A. Expression of EPO in CHO Cells:

PSVEp2neo (20 μg) of the expression vector of EPO and plasmid pBRTK (10 μg) containing the tk gene were simultaneously infected with tk negative CHO cells by the calcium-phosphate method (Graham 1973). Transfected CHO cells were cultured in G418 (Gibco) medium for 2-3 weeks and only transformed cells containing at least one neo gene were selected. The colonies thus obtained were divided into two groups with 10-100 colonies per pool, followed by HAT culture medium (hypoxanthin and thymidine at normal concentrations, i.e. 5 mM and 0.8 mM, respectively, and 100-fold dilution of aminopterin, And cultured at 0.2 μM). EPO activity was measured by taking only the supernatant of the medium in which the cells grew, and the EPO activity was 18.5unit / ml and 11.4unit / ml in two randomly selected groups at 2 days of culture.

B. Preparation of CHOEp-cfc33 Cell Line:

Only the pools showing the EPO activity in each pool of cells were picked and transferred to the second stage of HAT medium (only 2 times the aminopterin concentration, ie 0.4 μM), and then cultured. Among them, the pools showing the high concentration of EPO activity were selected, and the cells expressing the high concentration of EPO were again selected by culturing in medium that raised the aminopterin concentration twice again.

By the above method, the cell lines stably expressing EPO at high concentrations even under normal culture conditions and no selection markers were isolated during 12 consecutive culturing and selection processes, and this cell line was called CHOEp-cfc33. Name and complete the deposit (Accession Number: KCLRF-BP-00007). This cell line showed an expression titer of stable expression of more than 2,400 units of EPO activity per ml of medium when cultured for 2 days for continuous titer measurement.

Example 2

Westin analysis of expressed EPO

Western assay was performed on the production medium of cell line CHOEp-cfc33 which was transformed with the vector pSVEp2neo and stably produced EPO according to the method described in Example 1.

200ng of EPO obtained by partial purification from the resulting medium using gel filtration and ion exchange resin and the like was electrophoresed on 8-16% gradient SDS polyacrylamide gel (Novex), and then electrophoresed to a nitro cellulose filter. The filter was probed with an anti-EPO monoclonal antibody (anti-EPO mAb-peroxidase conjugate, Boehringer Mannheim) marked with peroxidase, washed, and then added with a coloring reagent (40chloro-1-naphthol). Indicated. Lane 1 represents the standard marker (Novex, prestained marker), lane 2 represents the EPO in the expressed medium, lane 4 represents the EPO standard from Searak, lanes 3 and 5 represent partially purified EPO produced by the CHOEp-cfc33 cell line.

Claims (4)

Cell line prepared by HAT medium selection process from a tk negative CHO cell line transformed with recombinant vector pSVEp2neo (Accession No .: KFCC-10831). The cell line of claim 1, which is CHOEP-cfc33 (Accession Number: KCLRF-BP-00007). A method for producing human erythropoietin, comprising culturing the cell line of claim 1 and separating and purifying human erythropoietin from the cell line. The method for producing erythropoietin according to claim 3, wherein the cell line is CHOEp-cfc33 (Accession No. KCLRF-BP-00007).