CA2298015C - Production of erythropoietin by endogenous gene activation - Google Patents
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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Abstract
The invention relates to human cells which are capable, on the basis of an activation of the endogenic human EPO gene, of producing EPO in a sufficient amount and purity to make possible a cost-effective production of human EPO as a pharmaceutical preparation. The invention furthermore relates to a method for the preparation of such human EPO-producing cells, DNA constructs for the activation of the endogenic EPO in human cells, and method for the large technical production of EPO in human cells.
Description
Production of Erythropoietin by Endogenous Gene Activation The invention relates to human cells which are capable, on the basis of an activation of the endogenic human EPO gene, of producing EPO in sufficient amount and purity to permit economical preparation of human EPO as a pharmaceutical preparation. The invention furthermore relates to a method of preparing such human EPO-producing cells, DNA
constructs for activating the endogenic EPO gene in human cells, and methods for the large-scale production of EPO in human cells.
Erythropoietin (EPO) is a human glycoprotein which stimulates the production of red blood cells. EPO occurs in the blood plasma of healthy persons only in very low concentrations, so that preparation in large amounts is not possible in this manner. EP¨B-0148 605 and EP-B-0205 564 describe the preparation of recombinant human EPO in CHO cells. The EPO
described in EP-B-0148 605 has a higher molecular weight than urinary EPO and no 0-glycosylation. Meantime, the EPO from CHO cells, described in EP-B- 0 205 564 is available in large amounts and in pure form, but it originates from nonhuman cells.
Moreover, the ability of CHO cells to produce is often relatively limited.
Furthermore, the harvesting of human EPO from the urine of patients with aplastic anemia is known (Miyake et al., J. Biol. Chem. 252 (1977), 5558 - 5564). Therein a seven-step process is disclosed which includes ion exchanger chromatography, ethanol precipitation, gel filtration and adsorption chromatography. An EPO preparation with a specific activity of about 70,000 U/mg of protein is obtained in a 21% yield. Disadvantages of this process and other methods of obtaining urinary EPO consist in the procurement of starting material in sufficient amounts and in repeatable quality. Furthermore, the purification from urine is difficult and even a purified product is not free of urinary contaminants.
UK patent application No. 2,085,887 describes a method for the preparation of human lymphoblastoid cells which are capable of producing EPO in small amounts. The economical production of a pharmaceutical with these cells is not possible.
WO 91/06667 describes a method for the recombinant preparation of EPO. In a first process step in primary human embryo kidney cells the endogenic EPO gene is brought by homologous recombination into operative linkage with a viral promoter and the DNA is isolated from these cells. In a second step the DNA thus isolated is transformed to nonhuman CHO cells and the expression of EPO in these cells is analyzed. No mention is found that production of EPO in human cells is possible.
constructs for activating the endogenic EPO gene in human cells, and methods for the large-scale production of EPO in human cells.
Erythropoietin (EPO) is a human glycoprotein which stimulates the production of red blood cells. EPO occurs in the blood plasma of healthy persons only in very low concentrations, so that preparation in large amounts is not possible in this manner. EP¨B-0148 605 and EP-B-0205 564 describe the preparation of recombinant human EPO in CHO cells. The EPO
described in EP-B-0148 605 has a higher molecular weight than urinary EPO and no 0-glycosylation. Meantime, the EPO from CHO cells, described in EP-B- 0 205 564 is available in large amounts and in pure form, but it originates from nonhuman cells.
Moreover, the ability of CHO cells to produce is often relatively limited.
Furthermore, the harvesting of human EPO from the urine of patients with aplastic anemia is known (Miyake et al., J. Biol. Chem. 252 (1977), 5558 - 5564). Therein a seven-step process is disclosed which includes ion exchanger chromatography, ethanol precipitation, gel filtration and adsorption chromatography. An EPO preparation with a specific activity of about 70,000 U/mg of protein is obtained in a 21% yield. Disadvantages of this process and other methods of obtaining urinary EPO consist in the procurement of starting material in sufficient amounts and in repeatable quality. Furthermore, the purification from urine is difficult and even a purified product is not free of urinary contaminants.
UK patent application No. 2,085,887 describes a method for the preparation of human lymphoblastoid cells which are capable of producing EPO in small amounts. The economical production of a pharmaceutical with these cells is not possible.
WO 91/06667 describes a method for the recombinant preparation of EPO. In a first process step in primary human embryo kidney cells the endogenic EPO gene is brought by homologous recombination into operative linkage with a viral promoter and the DNA is isolated from these cells. In a second step the DNA thus isolated is transformed to nonhuman CHO cells and the expression of EPO in these cells is analyzed. No mention is found that production of EPO in human cells is possible.
2 describes the production of EPO in human cells, wherein a relatively high EPO production of up to 960,620 mU/106 cells/24 h is found in human fibroblasts which had been transfected with a vector containing the complete EPO gene. These cells contain an exogenic EPO gene which is not at the correct EPO gene locus, so that problems are to be expected with regard to the stability of the cell line. No information on a constitutive EPO production is found in WO 93/092222. Furthermore, neither is there any information as to whether the EPO produced can be obtained in a quality sufficient for pharmaceutical purposes.
Furthermore, in WO 93/09222 an activation of the endogenic EPO gene in human cells is described. In it an EPO production is found of only 2,500 mU/106 cells in 24 h (corresponding approximately to 16 ng/106 cells/24 h). Such low production is entirely unsuited for the economical production of a pharmaceutical preparation.
W094/12650 and WO 95/31560 describe how a human cell with an endogenic EPO
gene activated by a viral promoter is able, after amplification of the endogenic EPO gene, to produce up to approximately 100,000 mU/106 cells/24 h (corresponding to about 0.6 Ag/106 cells/24 h). This amount too is still not sufficient for the economical production of a pharmaceutical.
The problem on which the present invention is based thus consisted in eliminating at least partially the above-described disadvantage of the state of the art and to offer a technologically better method for the preparation of EPO in human cells.
Especially, this is to make it possible to obtain a product in sufficient quantity and purity to permit economical production for pharmaceutical purposes.
This problem is solved by activation of the endogenic EPO gene in human cells and if optionally by subsequent amplification of the activated human EPO gene. In this manner it has surprisingly been possible, by the selection of suitable starting cells, DNA constructs and selection strategies, to provide human cells which are capable of producing EPO in sufficient quantity, quality and purity to permit the economical production of pharmaceutical preparations. Especially after amplification of the activated endogenic EPO
gene, cells can be obtained which have a definitely higher production output than the CHO
production cells used previously for the preparation of recombinant EPO.
One subject matter of the invention is a human cell which contains a copy of an endogenic EPO gene in operative linkage with a heterologous expression control sequence active in the human cell and is capable without prior gene amplification of producing at least 200 ng of EPO/106 cells per 24 hours. Preferably the human cell according to the invention is capable of the production of 200 to 3000 ng EPO/106 cells/24h, and most preferably for the production of 1000 to 3000 ng EPO/106 cells/24h.
Another subject matter of the present invention is a human cell which is obtainable by gene amplification from the cell previously stated and contains several copies of an endogenic EPO gene, each in operative linkage with a heterologous expression control sequence active in the human cell, and is capable of the production of at least 1,000 ng EPO/196 cells/24h.
With special preference the human cell line obtainable by gene amplification is capable of producing 1,000 to 25,000 ng EPO/106 cells/24h, and most preferably for the production of 5,000 to 25,000 ng EPO/106 cells/24h.
The human cell is any cell, provided it can be cultured in vitro. Especially preferred are human cells which can be cultured in a serum-free medium, and especially in suspension.
In this manner the production of EPO can be performed in a large fermenter with a culture capacity of, for example, 1,000 liters.
Especially preferred is a human cell which is an immortalized cell, e.g., a HT1080 cell (Rasheed et al., Cancer 33 (1984), 1027-1033), a HeLaS3 cell (Puck et al., J.Exp. Med.
103(1956), 273-286), a Namalwa cell (Nadkarni et al., Cancer 23 (1969), 66-79) or a cell derived therefrom.
An example of cell according to the invention is the clone "Aladin" which was deposited on
Furthermore, in WO 93/09222 an activation of the endogenic EPO gene in human cells is described. In it an EPO production is found of only 2,500 mU/106 cells in 24 h (corresponding approximately to 16 ng/106 cells/24 h). Such low production is entirely unsuited for the economical production of a pharmaceutical preparation.
W094/12650 and WO 95/31560 describe how a human cell with an endogenic EPO
gene activated by a viral promoter is able, after amplification of the endogenic EPO gene, to produce up to approximately 100,000 mU/106 cells/24 h (corresponding to about 0.6 Ag/106 cells/24 h). This amount too is still not sufficient for the economical production of a pharmaceutical.
The problem on which the present invention is based thus consisted in eliminating at least partially the above-described disadvantage of the state of the art and to offer a technologically better method for the preparation of EPO in human cells.
Especially, this is to make it possible to obtain a product in sufficient quantity and purity to permit economical production for pharmaceutical purposes.
This problem is solved by activation of the endogenic EPO gene in human cells and if optionally by subsequent amplification of the activated human EPO gene. In this manner it has surprisingly been possible, by the selection of suitable starting cells, DNA constructs and selection strategies, to provide human cells which are capable of producing EPO in sufficient quantity, quality and purity to permit the economical production of pharmaceutical preparations. Especially after amplification of the activated endogenic EPO
gene, cells can be obtained which have a definitely higher production output than the CHO
production cells used previously for the preparation of recombinant EPO.
One subject matter of the invention is a human cell which contains a copy of an endogenic EPO gene in operative linkage with a heterologous expression control sequence active in the human cell and is capable without prior gene amplification of producing at least 200 ng of EPO/106 cells per 24 hours. Preferably the human cell according to the invention is capable of the production of 200 to 3000 ng EPO/106 cells/24h, and most preferably for the production of 1000 to 3000 ng EPO/106 cells/24h.
Another subject matter of the present invention is a human cell which is obtainable by gene amplification from the cell previously stated and contains several copies of an endogenic EPO gene, each in operative linkage with a heterologous expression control sequence active in the human cell, and is capable of the production of at least 1,000 ng EPO/196 cells/24h.
With special preference the human cell line obtainable by gene amplification is capable of producing 1,000 to 25,000 ng EPO/106 cells/24h, and most preferably for the production of 5,000 to 25,000 ng EPO/106 cells/24h.
The human cell is any cell, provided it can be cultured in vitro. Especially preferred are human cells which can be cultured in a serum-free medium, and especially in suspension.
In this manner the production of EPO can be performed in a large fermenter with a culture capacity of, for example, 1,000 liters.
Especially preferred is a human cell which is an immortalized cell, e.g., a HT1080 cell (Rasheed et al., Cancer 33 (1984), 1027-1033), a HeLaS3 cell (Puck et al., J.Exp. Med.
103(1956), 273-286), a Namalwa cell (Nadkarni et al., Cancer 23 (1969), 66-79) or a cell derived therefrom.
An example of cell according to the invention is the clone "Aladin" which was deposited on
- 3 -July 15, 1997, according to the prescriptions of the Budapest Agreement at the Deutsche Sammlung von Milcroorganismen und Zellkulturen (DSMZ), Mascherosder Weg lb, Braunschweig, under number DSM ACC 2320.
In the cell according to the invention, the endogenic EPO gene is linked with a heterologous expression control sequence which is active in the human cell. The expression control sequence comprises a promoter and preferably additional expression-improving sequences, e.g., an enhancer. The promoter can be a regulatable or a constitutive promoter. Preferably the promoter is a strong viral promoter, e.g., an SV40 or a CMV promoter. The CMV
promoter/enhancer is especially preferred.
Furthermore, to optimize the EPO expression it may be preferred for the endogenic EPO
gene in the human cell, which is in operative association with the heterologous promoter, to have a signal peptide-coding sequence which is different from the natural signal peptide-coding sequence and codes preferably for a signal peptide with a modified amino acid sequence. Especially preferred is a signal peptide-coding sequence which codes for a signal peptide sequence modified in the area of the first four amino acids which is selected from Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His, and especially from (a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His (c) Met-Gly-Val-Pro or (d) Met-Ser-Val-His.
With special preference the sequence of the first four amino acids of the signal peptide is Met-Ser-Ala-His.
In an additional aspect, the present invention relates to a method for preparing a human EPO-producing cell, as previously stated, comprising the steps:
In the cell according to the invention, the endogenic EPO gene is linked with a heterologous expression control sequence which is active in the human cell. The expression control sequence comprises a promoter and preferably additional expression-improving sequences, e.g., an enhancer. The promoter can be a regulatable or a constitutive promoter. Preferably the promoter is a strong viral promoter, e.g., an SV40 or a CMV promoter. The CMV
promoter/enhancer is especially preferred.
Furthermore, to optimize the EPO expression it may be preferred for the endogenic EPO
gene in the human cell, which is in operative association with the heterologous promoter, to have a signal peptide-coding sequence which is different from the natural signal peptide-coding sequence and codes preferably for a signal peptide with a modified amino acid sequence. Especially preferred is a signal peptide-coding sequence which codes for a signal peptide sequence modified in the area of the first four amino acids which is selected from Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His, and especially from (a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His (c) Met-Gly-Val-Pro or (d) Met-Ser-Val-His.
With special preference the sequence of the first four amino acids of the signal peptide is Met-Ser-Ala-His.
In an additional aspect, the present invention relates to a method for preparing a human EPO-producing cell, as previously stated, comprising the steps:
- 4 -(a) Providing human starting cells which contain at least a copy of an endogenic EPO
gene, (b) Transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous with regions of the human EPO gene locus, to permit a homologous recombination, (ii) a positive selection marker gene, and (iii) a heterologous expression control sequence which is active in the human cell, (c) Culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) Analyzing the cells selectable according to step (c), and (e) Identifying the EPO-producing cells.
The DNA construct used in preparing the human EPO-producing cell contains two flanking DNA sequences which are homologous with areas of the human EPO gene locus in order to permit a homologous recombination. The selection of suitable flanking sequences is performed, for example, by the methods described in WO 90/11 354 and WO 91/09 955.
Preferably the flanking sequences have each a length of at least 150 bp. With special preference the homologous DNA sequences are selected from the area of the 5'-untranslated sequences, exon 1 and intron 1 of the EPO gene. It is especially preferred to use a DNA
sequence modified in the area of exon 1, which codes for a signal peptide modified in the first amino acids. The modifications in the exon 1 area are preferably as stated above.
The selection marker gene can be any selection marker gene suitable for eucaryotic cells, which upon expression leads to a selectable phenotype, e.g., antibiotic resistance, auxotrophy, etc. An especially preferred positive selection marker gene is the neomycin phosphotransferase gene.
Optionally, a negative selection marker gene such as, say, the HSV-thymidine kinase gene,
gene, (b) Transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous with regions of the human EPO gene locus, to permit a homologous recombination, (ii) a positive selection marker gene, and (iii) a heterologous expression control sequence which is active in the human cell, (c) Culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) Analyzing the cells selectable according to step (c), and (e) Identifying the EPO-producing cells.
The DNA construct used in preparing the human EPO-producing cell contains two flanking DNA sequences which are homologous with areas of the human EPO gene locus in order to permit a homologous recombination. The selection of suitable flanking sequences is performed, for example, by the methods described in WO 90/11 354 and WO 91/09 955.
Preferably the flanking sequences have each a length of at least 150 bp. With special preference the homologous DNA sequences are selected from the area of the 5'-untranslated sequences, exon 1 and intron 1 of the EPO gene. It is especially preferred to use a DNA
sequence modified in the area of exon 1, which codes for a signal peptide modified in the first amino acids. The modifications in the exon 1 area are preferably as stated above.
The selection marker gene can be any selection marker gene suitable for eucaryotic cells, which upon expression leads to a selectable phenotype, e.g., antibiotic resistance, auxotrophy, etc. An especially preferred positive selection marker gene is the neomycin phosphotransferase gene.
Optionally, a negative selection marker gene such as, say, the HSV-thymidine kinase gene,
- 5 -can also be present, by whose expression cells are destroyed in the presence of a selection agent.
If an amplification of the EPO gene endogenously activated in the human cell is desired, the DNA construct contains an amplification gene. Examples of suitable amplification genes are dihydrofolate reductase, adenosine deaminase, ornithine decarboxylase, etc. An especially preferred amplification gene is the dihydrofolate reductase gene, especially a dihydrofolate reductase arginine mutant which has a lower sensitivity to the selective agent (methotrexate) than the wild type gene (Simonsen et al., Proc. Natl. Acad.
Sci. USA 80 (1983); 2495).
If an amplification gene is present in the DNA construct used for activating the EPO gene, the method of the invention can also comprise the following steps:
(f) Amplification of the DNA sequence coding for EPO, and (g) Obtaining EPO-producing cells which contain a number of copies, greater in comparison with the starting cell, of an endogenic DNA sequence coding for mature EPO in operative linkage with a heterologous expression control sequence.
Appropriate DNA constructs for the activation of the endogenic EPO gene present in the human starting cell are the plasmids listed in the examples: p187, p189, p190 and p192 or a plasmid derived therefrom. Especially preferred is the plasmid p189 which was deposited at the DSMZ in accord with the provisions of the Budapest Agreement on July 16, 1997, under the accession number DSM 11661 or a plasmid derived therefrom.
Preferably, the DNA constructs are circular plasmid molecules which are used for the transfection of the human cell in a linearized form.
An additional subject matter of the present invention is a DNA construct for the activation of an endogenic EPO gene in a human cell, comprising:
(i) two flanking DNA sequences which are chosen homologous to regions of the human EPO gene locus selected from 5'-untranslated sequences, exon 1 and intron 1, in order to permit a homologous recombination, a modified sequence
If an amplification of the EPO gene endogenously activated in the human cell is desired, the DNA construct contains an amplification gene. Examples of suitable amplification genes are dihydrofolate reductase, adenosine deaminase, ornithine decarboxylase, etc. An especially preferred amplification gene is the dihydrofolate reductase gene, especially a dihydrofolate reductase arginine mutant which has a lower sensitivity to the selective agent (methotrexate) than the wild type gene (Simonsen et al., Proc. Natl. Acad.
Sci. USA 80 (1983); 2495).
If an amplification gene is present in the DNA construct used for activating the EPO gene, the method of the invention can also comprise the following steps:
(f) Amplification of the DNA sequence coding for EPO, and (g) Obtaining EPO-producing cells which contain a number of copies, greater in comparison with the starting cell, of an endogenic DNA sequence coding for mature EPO in operative linkage with a heterologous expression control sequence.
Appropriate DNA constructs for the activation of the endogenic EPO gene present in the human starting cell are the plasmids listed in the examples: p187, p189, p190 and p192 or a plasmid derived therefrom. Especially preferred is the plasmid p189 which was deposited at the DSMZ in accord with the provisions of the Budapest Agreement on July 16, 1997, under the accession number DSM 11661 or a plasmid derived therefrom.
Preferably, the DNA constructs are circular plasmid molecules which are used for the transfection of the human cell in a linearized form.
An additional subject matter of the present invention is a DNA construct for the activation of an endogenic EPO gene in a human cell, comprising:
(i) two flanking DNA sequences which are chosen homologous to regions of the human EPO gene locus selected from 5'-untranslated sequences, exon 1 and intron 1, in order to permit a homologous recombination, a modified sequence
- 6 -being present in the exon 1 region coding for the amino acids:
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His, and especially for the amino acids:
(a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His, (c) Met-Gly-Val-Pro or (d) Met-Ser-Val-His, (ii) a positive selection marker gene, (iii) a heterologous expression control sequence which is active in a human cell, and (iv) optionally an amplification gene.
Still another subject of the present invention is a DNA construct for the activation of an endogenous EPO gene in a human cell, comprising;
(i) two flanking DNA sequences which are homologous to regions of the human EPO gene locus, selected from 5'-untranslated sequences, exon 1 and intron 1, to permit a homologous recombination, (ii) a positive selection marker gene, (iii) a heterologous expression control sequence which is active in a human cell, the distance between the heterologous expression control sequence and the translation start of the EPO gene being no greater than 1100 bp, and (iv) optionally, an amplification gene.
Surprisingly it was found that, in the case of a modification of the EPO
signal sequence and/or a shortening of the distance between the heterologous expression control sequence and the start of the EPO gene, an optimized expression is obtained. Preferably the distance between the promoter of the heterologous expression control sequence and the translation
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His, and especially for the amino acids:
(a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His, (c) Met-Gly-Val-Pro or (d) Met-Ser-Val-His, (ii) a positive selection marker gene, (iii) a heterologous expression control sequence which is active in a human cell, and (iv) optionally an amplification gene.
Still another subject of the present invention is a DNA construct for the activation of an endogenous EPO gene in a human cell, comprising;
(i) two flanking DNA sequences which are homologous to regions of the human EPO gene locus, selected from 5'-untranslated sequences, exon 1 and intron 1, to permit a homologous recombination, (ii) a positive selection marker gene, (iii) a heterologous expression control sequence which is active in a human cell, the distance between the heterologous expression control sequence and the translation start of the EPO gene being no greater than 1100 bp, and (iv) optionally, an amplification gene.
Surprisingly it was found that, in the case of a modification of the EPO
signal sequence and/or a shortening of the distance between the heterologous expression control sequence and the start of the EPO gene, an optimized expression is obtained. Preferably the distance between the promoter of the heterologous expression control sequence and the translation
- 7 -start of the EPO gene is not greater than 1100 bp, with special preference not more than 150 bp, and most preferentially not more than 100 bp. An especially preferred example of a DNA construct to be used according to the invention is the plasmid p189 (DSM
11661) or a plasmid derived therefrom.
Still another aspect of the present invention is a method for preparing human EPO, wherein a human cell according to the invention is cultured in a suitable medium under conditions in which a production of EPO takes place and the EPO is harvested from the culture medium.
A serum-free medium is used preferentially. The cells are preferably cultured in suspension. The culturing is performed especially preferentially in a fermenter, especially in a large fermenter with a capacity of, for example, 10 1 - 50,000 1.
The harvesting of human EPO from the culture medium of human cell lines comprises preferably the following steps:
(a) Passing the cell supernate over an affinity chromatography medium and harvesting the fractions containing EPO, (b) optionally passing the EPO-containing fractions over a hydrophobic interaction chromatography medium and harvesting the EPO-containing fractions, (c) passing the EPO-containing fractions over hydroxy apatite and harvesting the EPO-containing fractions, and (d) concentrating and/or passing over a reverse-phase (RP) HPLC medium.
Step (a) of the purification process includes passing the cell supernate, which in some cases can be pre-treated, over an affinity chromatography medium. Preferred affinity chromatography media are those to which a blue dye is coupled. An especially preferred example is blue sepharoseTM.
After elution from the affinity chromatography medium the EPO-containing eluate is optionally passed over a hydrophobic interaction chromatography medium. This step is expedient if a culture medium with a serum content _?:2% (v Iv) is used. If a culture medium with a low serum content is used, e.g., 1% (v/v), or a serum-free medium is used, this step can be omitted. A preferred hydrophobic interaction chromatography medium is butyl-
11661) or a plasmid derived therefrom.
Still another aspect of the present invention is a method for preparing human EPO, wherein a human cell according to the invention is cultured in a suitable medium under conditions in which a production of EPO takes place and the EPO is harvested from the culture medium.
A serum-free medium is used preferentially. The cells are preferably cultured in suspension. The culturing is performed especially preferentially in a fermenter, especially in a large fermenter with a capacity of, for example, 10 1 - 50,000 1.
The harvesting of human EPO from the culture medium of human cell lines comprises preferably the following steps:
(a) Passing the cell supernate over an affinity chromatography medium and harvesting the fractions containing EPO, (b) optionally passing the EPO-containing fractions over a hydrophobic interaction chromatography medium and harvesting the EPO-containing fractions, (c) passing the EPO-containing fractions over hydroxy apatite and harvesting the EPO-containing fractions, and (d) concentrating and/or passing over a reverse-phase (RP) HPLC medium.
Step (a) of the purification process includes passing the cell supernate, which in some cases can be pre-treated, over an affinity chromatography medium. Preferred affinity chromatography media are those to which a blue dye is coupled. An especially preferred example is blue sepharoseTM.
After elution from the affinity chromatography medium the EPO-containing eluate is optionally passed over a hydrophobic interaction chromatography medium. This step is expedient if a culture medium with a serum content _?:2% (v Iv) is used. If a culture medium with a low serum content is used, e.g., 1% (v/v), or a serum-free medium is used, this step can be omitted. A preferred hydrophobic interaction chromatography medium is butyl-
- 8 -sepharoseTM.
The eluate from step (a) or, if used, step (b), is passed in step (c) of the method of the invention over hydroxyapatite and the EPO-containing eluate is subjected to a concentrating step and/or a reverse-phase HPLC purification step. The concentration is performed preferably by exclusion chromatography, e.g., membrane filtration, the use of a medium, such as a membrane with an exclusion size of 10 kD having proven desirable.
By the method according to the invention an isolated human EPO with a specific activity of at least 100,000 U/mg protein in vivo (normocythemic mouse) is obtainable, which is free from urinary contaminants and can differ in its glycosilation from recombinant EPO from CHO cells. Preferably, the EPO of the invention has a specific activity of at least 175,000, and with special preference at least 200,000 to 400,000 or 450,000 IU/mg protein. The human EPO obtainable by the method of the invention can contain a-2, 3- and/or a-2, 6-linked sialic acid residues. In studies of EPO from cells which contain an endogenically activated EPO gene, on the basis of the present preliminary results, the presence of a-2, 3-and a-2, 6 linked sialic acid residues were found. Furthermore, it was found on the basis of the present preliminary results that the human EPO of the invention has a content of less than 0.2% N-glycol neuraminic acid based on the content of N-acetyl neuraminic acid The purity of the human EPO of the invention amounts preferably to at least 90%, with special preference at least 95%, and most preferably at least 98% of the total protein content. The determination of the total protein content can be performed by reverse phase HPLC, e.g., with a Poros R/2HTM column.
Furthermore, by the method of the invention, human EPO species are obtainable which differ in their amino acid sequence. Thus it was found by mass spectrometric analysis (MALDI-MS) that a human EPO can be isolated from HeLa S3 cells, which is mainly a polypeptide with a length of 165 amino acids, which is formed by C-terminal processing of an arginine residue, and in some cases includes up to 15% of an EPO with 166 amino acids.
Also, a human EPO is obtainable which includes a polypeptide with a length of 166 amino acids, i.e., a non-processed EPO. From Namalwa cells, for example, a human EPO
has been isolated which comprises a mixture of polypeptides with a length of 165 and 166
The eluate from step (a) or, if used, step (b), is passed in step (c) of the method of the invention over hydroxyapatite and the EPO-containing eluate is subjected to a concentrating step and/or a reverse-phase HPLC purification step. The concentration is performed preferably by exclusion chromatography, e.g., membrane filtration, the use of a medium, such as a membrane with an exclusion size of 10 kD having proven desirable.
By the method according to the invention an isolated human EPO with a specific activity of at least 100,000 U/mg protein in vivo (normocythemic mouse) is obtainable, which is free from urinary contaminants and can differ in its glycosilation from recombinant EPO from CHO cells. Preferably, the EPO of the invention has a specific activity of at least 175,000, and with special preference at least 200,000 to 400,000 or 450,000 IU/mg protein. The human EPO obtainable by the method of the invention can contain a-2, 3- and/or a-2, 6-linked sialic acid residues. In studies of EPO from cells which contain an endogenically activated EPO gene, on the basis of the present preliminary results, the presence of a-2, 3-and a-2, 6 linked sialic acid residues were found. Furthermore, it was found on the basis of the present preliminary results that the human EPO of the invention has a content of less than 0.2% N-glycol neuraminic acid based on the content of N-acetyl neuraminic acid The purity of the human EPO of the invention amounts preferably to at least 90%, with special preference at least 95%, and most preferably at least 98% of the total protein content. The determination of the total protein content can be performed by reverse phase HPLC, e.g., with a Poros R/2HTM column.
Furthermore, by the method of the invention, human EPO species are obtainable which differ in their amino acid sequence. Thus it was found by mass spectrometric analysis (MALDI-MS) that a human EPO can be isolated from HeLa S3 cells, which is mainly a polypeptide with a length of 165 amino acids, which is formed by C-terminal processing of an arginine residue, and in some cases includes up to 15% of an EPO with 166 amino acids.
Also, a human EPO is obtainable which includes a polypeptide with a length of 166 amino acids, i.e., a non-processed EPO. From Namalwa cells, for example, a human EPO
has been isolated which comprises a mixture of polypeptides with a length of 165 and 166
- 9 -amino acids.
It is provided a human cell, characterized in that it contains a copy of an endogenous EPO gene in operative linkage with an heterologous promoter that is active in the human cell and is able to produce at least 200 ng EPO/106 cells/24h, the distance between the heterologous promoter and the translation start of the EPO gene being less than 1100 bp, and said human cell is obtained by a process comprising the steps:
(a) providing human starting cells which contain at least one copy of an endogenous EPO
gene, provided that the human starting cells are not germ line cells, (b) transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous to regions of the human EPO
gene locus in order to permit a homologous recombination, (ii) a positive selection marker gene and (iii) an heterologous expression control sequence which is active in the human cell, wherein the heterologous expression control sequence comprises a viral promoter and the distance between the heterologous promoter and the translation start of the EPO gene is less than 1100 bp, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selected according to step (c), and (e) identifying the EPO-producing cells.
It is also provided a human cell, characterized in that it produces 200 - 3000 ng EPO/106 cells/24 h.
It is further provided a method for the preparation of a human EPO-producing cell as described herein, comprising the steps of:
(a) providing human starting cells which contain one copy of an endogenic EPO gene, (b) transfecting the cells with a DNA construct comprising:
(I) two flanking DNA sequences which are homologous to regions of the human EPO
gene locus, to permit a homologous recombination, (ii) a positive selection marker gene, and (iii) an heterologous expression control sequence which is active in the human cell, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selectable according to step (c), and (e) identifying of the EPO-producing cells.
9a This human EPO can be used as an active substance for a pharmaceutic preparation which can contain additional active substances, as well as pharmaceutically common adjuvants, vehicles and additives.
In still another aspect the present invention relates to an isolated DNA which codes for a human EPO with a sequence modified in the region of the first four amino acids of the signal peptide, which is selected from:
Met-X1-X2-X3 wherein XI is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X7-X3 is not the sequence Gly-Val-His, and especially for the amino acids:
(a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His (c) Met-Gly-Val-Pro or (d) Met-Ser-Val-His.
The DNA can be for example a genomic DNA or a cDNA.
The invention will continue to be explained by the following examples and figures and sequence listings.
Figure 1 is a schematic representation of the amplification of homology regions of the EPO gene from the area of the 5'-untranslated sequences, exon 1 and intron 1 and of primer products 1 and 2.
Figure 2 is a schematic representation of a plasmid which contains EPO
homology regions from the area of the 5'-untranslated sequences, exon 1 and intron 1.
Figure 3 is a schematic representation of a gene-activation sequence which contains
It is provided a human cell, characterized in that it contains a copy of an endogenous EPO gene in operative linkage with an heterologous promoter that is active in the human cell and is able to produce at least 200 ng EPO/106 cells/24h, the distance between the heterologous promoter and the translation start of the EPO gene being less than 1100 bp, and said human cell is obtained by a process comprising the steps:
(a) providing human starting cells which contain at least one copy of an endogenous EPO
gene, provided that the human starting cells are not germ line cells, (b) transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous to regions of the human EPO
gene locus in order to permit a homologous recombination, (ii) a positive selection marker gene and (iii) an heterologous expression control sequence which is active in the human cell, wherein the heterologous expression control sequence comprises a viral promoter and the distance between the heterologous promoter and the translation start of the EPO gene is less than 1100 bp, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selected according to step (c), and (e) identifying the EPO-producing cells.
It is also provided a human cell, characterized in that it produces 200 - 3000 ng EPO/106 cells/24 h.
It is further provided a method for the preparation of a human EPO-producing cell as described herein, comprising the steps of:
(a) providing human starting cells which contain one copy of an endogenic EPO gene, (b) transfecting the cells with a DNA construct comprising:
(I) two flanking DNA sequences which are homologous to regions of the human EPO
gene locus, to permit a homologous recombination, (ii) a positive selection marker gene, and (iii) an heterologous expression control sequence which is active in the human cell, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selectable according to step (c), and (e) identifying of the EPO-producing cells.
9a This human EPO can be used as an active substance for a pharmaceutic preparation which can contain additional active substances, as well as pharmaceutically common adjuvants, vehicles and additives.
In still another aspect the present invention relates to an isolated DNA which codes for a human EPO with a sequence modified in the region of the first four amino acids of the signal peptide, which is selected from:
Met-X1-X2-X3 wherein XI is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X7-X3 is not the sequence Gly-Val-His, and especially for the amino acids:
(a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His (c) Met-Gly-Val-Pro or (d) Met-Ser-Val-His.
The DNA can be for example a genomic DNA or a cDNA.
The invention will continue to be explained by the following examples and figures and sequence listings.
Figure 1 is a schematic representation of the amplification of homology regions of the EPO gene from the area of the 5'-untranslated sequences, exon 1 and intron 1 and of primer products 1 and 2.
Figure 2 is a schematic representation of a plasmid which contains EPO
homology regions from the area of the 5'-untranslated sequences, exon 1 and intron 1.
Figure 3 is a schematic representation of a gene-activation sequence which contains
- 10 -=
the Rous sarcoma virus promoter (RSV), the neomycin phosphotransferase gene (NEO), the early polyadenylation region of SV40 (SVI pA), the early SV40 promoter (SVI), the dihydrofolate reductase gene (DHFR), an additional early SV40 polyadenylation region, and the cytomegalovirus immediate early promoter and enhancer (MCMV).
Figure 4a The preparation of the EPO gene targeting vector p176.
Figure 4b The preparation of the EPO gene targeting vectors p179 and p187.
Figure 4c The preparation of the EPO gene targeting vector p189.
Figure 4d The preparation of the EPO gene targeting vector p190.
Figure 4e The preparation of the EPO gene targeting vector p192.
Figure 5 A schematic representation of the preparation of EPO cDNA with signal sequence mutations.
Figure 6a The hybridization of cellular DNA with a probe from the CMV area of the gene cassette represented in Fig. 3; the lanes 1 to 4 are each of DNA from human cells cleaved with the restriction enzymes AgeI and AscI; lane 1:
EPO-producing HeLa S3 cell amplified with 1,000 nM MTX; lane 2: EPO-producing HeLa S3 cell amplified with 500 nM MTX; lane 3; EPO-producing HeLa S3 Cell without amplification; lane 4: HeLa S3 cell without activated EPO gene; lane 5: digoxigenin-marked length marker; the size of the hybridizing fragment in lanes 1 to 3 is approximately 5,200 bp, and Figure6b The hybridization of a probe from the coding area of EPO with DNA
from human cells; lane 1: digoxigenin-marked length marker; lanes 2 to 4: DNA
from human cells cleaved with the restriction enzymes BamHI, Hind!!! and SalI; lane 2: EPO producing HeLa S3 cell amplified with 500 nM MTX
(length of the band produced by the non-activated endogenic gene: 3,200 bp;
the Rous sarcoma virus promoter (RSV), the neomycin phosphotransferase gene (NEO), the early polyadenylation region of SV40 (SVI pA), the early SV40 promoter (SVI), the dihydrofolate reductase gene (DHFR), an additional early SV40 polyadenylation region, and the cytomegalovirus immediate early promoter and enhancer (MCMV).
Figure 4a The preparation of the EPO gene targeting vector p176.
Figure 4b The preparation of the EPO gene targeting vectors p179 and p187.
Figure 4c The preparation of the EPO gene targeting vector p189.
Figure 4d The preparation of the EPO gene targeting vector p190.
Figure 4e The preparation of the EPO gene targeting vector p192.
Figure 5 A schematic representation of the preparation of EPO cDNA with signal sequence mutations.
Figure 6a The hybridization of cellular DNA with a probe from the CMV area of the gene cassette represented in Fig. 3; the lanes 1 to 4 are each of DNA from human cells cleaved with the restriction enzymes AgeI and AscI; lane 1:
EPO-producing HeLa S3 cell amplified with 1,000 nM MTX; lane 2: EPO-producing HeLa S3 cell amplified with 500 nM MTX; lane 3; EPO-producing HeLa S3 Cell without amplification; lane 4: HeLa S3 cell without activated EPO gene; lane 5: digoxigenin-marked length marker; the size of the hybridizing fragment in lanes 1 to 3 is approximately 5,200 bp, and Figure6b The hybridization of a probe from the coding area of EPO with DNA
from human cells; lane 1: digoxigenin-marked length marker; lanes 2 to 4: DNA
from human cells cleaved with the restriction enzymes BamHI, Hind!!! and SalI; lane 2: EPO producing HeLa S3 cell amplified with 500 nM MTX
(length of the band produced by the non-activated endogenic gene: 3,200 bp;
-11 -length of the copy of the EPO gene activated by gene targeting: 2,600 bp);
lane 3: DNA from an EPO producing HeLa S3 cell not amplified; lane 4:
DNA from an HeLa S3 control cell.
SEQ ID No. 1 and No. 2 Nucleotide sequences of the primers used for preparing PCR
Product 1 (Fig. 1).
SEQ ID No. 3 and No. 4 Sequences of the primers used for preparing PCR Product 2 (Fig.
1).
SEQ ID No. 5 Sequence of the primer EPO EX1.
SEQ ID No. 6 Sequence of the primer EX2.
SEQ ID No. 7 Sequence of the primer EX3 (Met-Gly-Ala-His).
SEQ ID No. 8 Sequence of the modified signal peptide start encoded by primer EX3.
SEQ ID No. 9 Sequence of primer EX4 (Met-Ser-Ala-His).
SEQ ID No. 10 Sequence of the modified signal peptide start encoded by primer EX4.
SEQ ID No. 11 Sequence of primer EX5 (Met-Gly-Val-Pro).
SEQ ID No. 12 Sequence of the modified signal peptide start encoded by primer EX5.
SEQ ID No. 13 Sequence of primer EX6 (Met-Ser-Val-His).
SEQ ID No. 14 Sequence of the modified signal peptide start encoded by primer EX6.
SEQ ID No. 15 Sequence of primer EX genome 1.
lane 3: DNA from an EPO producing HeLa S3 cell not amplified; lane 4:
DNA from an HeLa S3 control cell.
SEQ ID No. 1 and No. 2 Nucleotide sequences of the primers used for preparing PCR
Product 1 (Fig. 1).
SEQ ID No. 3 and No. 4 Sequences of the primers used for preparing PCR Product 2 (Fig.
1).
SEQ ID No. 5 Sequence of the primer EPO EX1.
SEQ ID No. 6 Sequence of the primer EX2.
SEQ ID No. 7 Sequence of the primer EX3 (Met-Gly-Ala-His).
SEQ ID No. 8 Sequence of the modified signal peptide start encoded by primer EX3.
SEQ ID No. 9 Sequence of primer EX4 (Met-Ser-Ala-His).
SEQ ID No. 10 Sequence of the modified signal peptide start encoded by primer EX4.
SEQ ID No. 11 Sequence of primer EX5 (Met-Gly-Val-Pro).
SEQ ID No. 12 Sequence of the modified signal peptide start encoded by primer EX5.
SEQ ID No. 13 Sequence of primer EX6 (Met-Ser-Val-His).
SEQ ID No. 14 Sequence of the modified signal peptide start encoded by primer EX6.
SEQ ID No. 15 Sequence of primer EX genome 1.
- 12 -SEQ ID No. 16 Sequence of primer EX13.
SEQ ID No. 17 Sequence of primer EPO EX 17.
Examples The activation of the EPO gene locus for protein production in an industrial scale was achieved by homologous integration of a gene activation sequence which contains the neomycin phosphotransferase (NEO) gene for the selection (G-418 resistance), the murine dihydrofolate reductase (DHFR) gene (for gene amplification by MTX) and the cytomegalovirus (CMV) immediate early promoter and enhancer for gene activation.
Example 1 Cloning of EPO Homology Regions Homology regions of the EPO gene were amplified by using a genomic placenta DNA
(Boehringer Mannheim). Two PCR products were prepared from a homology region 6.3 1c13 long from the area of the 5'-untranslated sequences of the EPO gene, exon 1 and intron 1 (cf. Figure 1). The primers used for the preparation of PCR Product 1 had the following sequences: 5'-CGC GGC GGA TCC CAG GGA GCT GGG TTG ACC GG-3' (SEQ ID No.
1) and 5'-GGC CGC GAA TTC TCC GCG CCT GGC CGG GGT CCC TCA GC-3' (SEQ
ID No. 2). The primers used for the preparation of PCR Product 2 had the following sequences: 5'-CGC GGC GGA TCC TCT CCT CCC TCC CAA GCT GCA ATC-3' (SEQ
ID No. 3) and 5'GGC CGC GAA TTC TAG AAC AGA TAG CCA GGC TGA GAG-3' (SEQ ID No. 4).
The desired segments were cut out of PCR Products 1 and 2 by restriction cleavage (PCR
Product 1: HindIII, PCR Product 2: HindIII and Eco RV) and cloned into the vector pCRII
(Invitrogen) which had been cleaved with Hind III and Eco RV. The recombinant vector obtained in this manner was named 5epoper1000 (cf. Figure 2).
Example 2 Construction of EPO Gene Targeting Vectors 2.1 A gene activation sequence which contains the NEO gene, the DHFR gene and a
SEQ ID No. 17 Sequence of primer EPO EX 17.
Examples The activation of the EPO gene locus for protein production in an industrial scale was achieved by homologous integration of a gene activation sequence which contains the neomycin phosphotransferase (NEO) gene for the selection (G-418 resistance), the murine dihydrofolate reductase (DHFR) gene (for gene amplification by MTX) and the cytomegalovirus (CMV) immediate early promoter and enhancer for gene activation.
Example 1 Cloning of EPO Homology Regions Homology regions of the EPO gene were amplified by using a genomic placenta DNA
(Boehringer Mannheim). Two PCR products were prepared from a homology region 6.3 1c13 long from the area of the 5'-untranslated sequences of the EPO gene, exon 1 and intron 1 (cf. Figure 1). The primers used for the preparation of PCR Product 1 had the following sequences: 5'-CGC GGC GGA TCC CAG GGA GCT GGG TTG ACC GG-3' (SEQ ID No.
1) and 5'-GGC CGC GAA TTC TCC GCG CCT GGC CGG GGT CCC TCA GC-3' (SEQ
ID No. 2). The primers used for the preparation of PCR Product 2 had the following sequences: 5'-CGC GGC GGA TCC TCT CCT CCC TCC CAA GCT GCA ATC-3' (SEQ
ID No. 3) and 5'GGC CGC GAA TTC TAG AAC AGA TAG CCA GGC TGA GAG-3' (SEQ ID No. 4).
The desired segments were cut out of PCR Products 1 and 2 by restriction cleavage (PCR
Product 1: HindIII, PCR Product 2: HindIII and Eco RV) and cloned into the vector pCRII
(Invitrogen) which had been cleaved with Hind III and Eco RV. The recombinant vector obtained in this manner was named 5epoper1000 (cf. Figure 2).
Example 2 Construction of EPO Gene Targeting Vectors 2.1 A gene activation sequence which contains the NEO gene, the DHFR gene and a
- 13 -CMV promoter/enhancer (cf. Fig. 3) was inserted into the AgeI site of the plasmid 5cpocr1000 containing the EPO homology region, and the plasmid p176 was obtained (cf. Fig. 4a). To bring the CMV promoter as close as possible to the translation start locus of the EPO gene, a segment 963 bp long was deleted between the restriction sites AscI and AgeI (partial cleavage), whereupon the plasmid p179 was obtained (Fig. 4b).
2.2 To achieve an optimization of the expression, nucleotides in exon 1, which code for the beginning of the EPO leader sequence Met-Gly-Val-His, were replaced by the synthetic sequence Met-Ser-Ala-His (cf. also Example 6). This sequence was obtained by amplification of a genomic EPO-DNA sequence, e.g., of the plasmid pEP0148, which contains a 3,5 kB BstEII/EcoRI fragment (including the exons 1-5) of the human EPO gene sequence under the control of the SV40 promoter (Jacobs et al., Nature 313 (1985), 806 and Lee-Huang et al., Gene 128 (1993), 227) as template with the primers Ex4 (SEQ ID No. 9) and Ex17 (SEQ ID No. 17) (Table 1). The plasmid p187 was thus obtained (Fig. 4b).
2.3 The plasmid p189 was prepared from the plasmid p187 by insertion of the herpes simplex virus thymidine kinase gene (HSV-TK) which originated from Psvtk-1 (Pvull/NarI fragment) (Fig. 4c). The HSV-TK gene is under control of the SV40 promoter at the 3' end of intron 1 (Eco RV/ClaI) in an opposite orientation relative to the CMV promoter and should serve for negative selection for a homologous recombination.
2.4 For the construction of plasmid p190, an SfiI/BglII fragment of pHEAVY, a plasmid which contains the cDNA of an arginine mutant of DHFR described in Simonsen et al.
(Proc. Natl. Acad. Sci, USA 80 (1983), 2495) was subcloned into the plasmid pGenak-1 cut with SfiI and BglII, which contains the NEO gene under control of the RSV promoter and the late SV40 polyadenylation site as terminator, the murine DHFR gene under control of the early SV40 promoter and of the early SV40 polyadenylation site as terminator (Kaufmann et al., Mol. Cell. Biol. 2 (1982), 1304;
Okayama et al., Mol. Cell. Biol. 3 (1983), 280, and Schimke, J. Biol. Chem.
(1988), 5989) and the CMV promoter (Boshart et al., Cell 41(1995) 521). Then an
2.2 To achieve an optimization of the expression, nucleotides in exon 1, which code for the beginning of the EPO leader sequence Met-Gly-Val-His, were replaced by the synthetic sequence Met-Ser-Ala-His (cf. also Example 6). This sequence was obtained by amplification of a genomic EPO-DNA sequence, e.g., of the plasmid pEP0148, which contains a 3,5 kB BstEII/EcoRI fragment (including the exons 1-5) of the human EPO gene sequence under the control of the SV40 promoter (Jacobs et al., Nature 313 (1985), 806 and Lee-Huang et al., Gene 128 (1993), 227) as template with the primers Ex4 (SEQ ID No. 9) and Ex17 (SEQ ID No. 17) (Table 1). The plasmid p187 was thus obtained (Fig. 4b).
2.3 The plasmid p189 was prepared from the plasmid p187 by insertion of the herpes simplex virus thymidine kinase gene (HSV-TK) which originated from Psvtk-1 (Pvull/NarI fragment) (Fig. 4c). The HSV-TK gene is under control of the SV40 promoter at the 3' end of intron 1 (Eco RV/ClaI) in an opposite orientation relative to the CMV promoter and should serve for negative selection for a homologous recombination.
2.4 For the construction of plasmid p190, an SfiI/BglII fragment of pHEAVY, a plasmid which contains the cDNA of an arginine mutant of DHFR described in Simonsen et al.
(Proc. Natl. Acad. Sci, USA 80 (1983), 2495) was subcloned into the plasmid pGenak-1 cut with SfiI and BglII, which contains the NEO gene under control of the RSV promoter and the late SV40 polyadenylation site as terminator, the murine DHFR gene under control of the early SV40 promoter and of the early SV40 polyadenylation site as terminator (Kaufmann et al., Mol. Cell. Biol. 2 (1982), 1304;
Okayama et al., Mol. Cell. Biol. 3 (1983), 280, and Schimke, J. Biol. Chem.
(1988), 5989) and the CMV promoter (Boshart et al., Cell 41(1995) 521). Then an
- 14 -HpaI fragment which contained the cDNA coding for the DHFR arginine mutant was ligated into the plasmid p189 cut with HpaI, whereupon the plasmid p190 was obtained (Fig. 4d).
2.5 To obtain a transfection vector without the HSV-TK gene, an AscI/NheI
fragment of the plasmid p190, which contained the gene activation sequence, was ligated into an AscI/NheI fragment, containing the exon 1, of the plasmid p187. The resulting plasmid was named p192 (Fig. 4e).
Example 3 Transfection of Cells Various cell lines were selected for the production of EPO and transfected with targeting vectors.
3.1 Namalwa Cells The cells were cultured in T150 tissue culture bottles and transfected by electroporation (1 x 107 cells/800 ttl of electroporation buffer 20 mM Hepes, 138 mM NaC1, 5 mM
KC1, 0.7 mM Na2HPO4, 6 mM D-glucose monohydrate pH 7.0, 10 pig linearized DNA, 960 F, V BioRad Gene Pulser). After the electroporation the cells were cultured in RPMI 1640, 10% (v/v) of fetal calf serum (FCS), 2 mM of L-glutamine, 1 mM of sodium pyruvate in forty-six 96-well plates. After two days the cells were cultured for 10 to 20 days in 1 mg/ml of medium containing G-418. The supernate was tested in a solid-phase ELISA for the production of EPO (see Example 4). The EPO producing clones were expanded in 24-well plates and T-25 tissue culture bottles. Aliquots were frozen and the cells were subcloned by FACS (Ventage, Becton Dickinson). The subclones were repeatedly tested for EPO production.
3.2 HT 1080 Cells The conditions were as described for Namalwa cells, except that the HT1080 cells were cultivated in DMEM, 10% (v/v) FCS, 2 mM of L-glutamine, 1 mM of sodium pyruvate.
For transfection by electroporation the cells were released from the walls of the culture
2.5 To obtain a transfection vector without the HSV-TK gene, an AscI/NheI
fragment of the plasmid p190, which contained the gene activation sequence, was ligated into an AscI/NheI fragment, containing the exon 1, of the plasmid p187. The resulting plasmid was named p192 (Fig. 4e).
Example 3 Transfection of Cells Various cell lines were selected for the production of EPO and transfected with targeting vectors.
3.1 Namalwa Cells The cells were cultured in T150 tissue culture bottles and transfected by electroporation (1 x 107 cells/800 ttl of electroporation buffer 20 mM Hepes, 138 mM NaC1, 5 mM
KC1, 0.7 mM Na2HPO4, 6 mM D-glucose monohydrate pH 7.0, 10 pig linearized DNA, 960 F, V BioRad Gene Pulser). After the electroporation the cells were cultured in RPMI 1640, 10% (v/v) of fetal calf serum (FCS), 2 mM of L-glutamine, 1 mM of sodium pyruvate in forty-six 96-well plates. After two days the cells were cultured for 10 to 20 days in 1 mg/ml of medium containing G-418. The supernate was tested in a solid-phase ELISA for the production of EPO (see Example 4). The EPO producing clones were expanded in 24-well plates and T-25 tissue culture bottles. Aliquots were frozen and the cells were subcloned by FACS (Ventage, Becton Dickinson). The subclones were repeatedly tested for EPO production.
3.2 HT 1080 Cells The conditions were as described for Namalwa cells, except that the HT1080 cells were cultivated in DMEM, 10% (v/v) FCS, 2 mM of L-glutamine, 1 mM of sodium pyruvate.
For transfection by electroporation the cells were released from the walls of the culture
- 15 -vessels by trypsinization. After electroporation 1 x 107 cells were cultured in DMEM, 10%
(v/v) FCS, 2 mM L-glutamine, 1 mM sodium pyruvate in five 96-well plates.
3.3 HeLa S3 Cells The conditions were as described for Namalwa cells, except that the HeLa cells were cultured in RPM 1640, 10% (V/V) FCS, 2 mM L-glutamine, 1% (v/v) MEM
nonessential amino acids (Sigma), and 1 mM sodium pyruvate. For transfection by electroporation the cells were released from the walls of the culture vessels by trypsinization.
The conditions for the electroporation were 960 g/250 V. After the electroporation the cells were cultured in RPMI 1640, 10% (v/v) FCS, 2 mM L-glutamine, 1% (v/v) MEM, 1 mM sodium pyruvate in T75 tissue culture bottles. 24 hours after electroporation the cells were trypsinized and cultured for 10 to 15 days in a medium containing 600 ti,g/inl of G-418 in ten 96-well plates.
Example 4 Selection for EPO-Producing Clones The culture supemate of transfected cells was tested in an EPO ELISA. All steps were performed at room temperature. 96-well plates previously coated with Streptavidin were coated with biotinylated anti-EPO antibodies (Boehringer Mannheim). For coating, the plates were first washed with 50 mM of sodium phosphate pH 7.2, and 0.05%
(v/v) of TweenTm 20. Then 0.01 ml of coating buffer (4 i../g/m1 of biotinylated antibody, 10 mM
sodium phosphate pH 7.2, 3 g/1 bovine serum albumin, 20 g/I saccharose, and 9 g/INaC1 were added per well, and incubated at room temperature for 3 h. Then the plates were washed with 50 mM of sodium phosphate pH 7.2, dried and sealed.
Before the test, after washing the plates three times with 0.3 ml of phosphate-buffered salt solution (PBS) and 0.05% TweenTm 20 (Sigma), the plates were incubated overnight with 0.2 ml PBS and 1% (w/v) CrotemTM (Boehringer Mannheim) per well in order to block unspecific bonds.
After removal of the blocking solution, 0.1 ml of culture supemate was added and the plates were incubated overnight. The individual wells were washed three times with 0.3 ml of
(v/v) FCS, 2 mM L-glutamine, 1 mM sodium pyruvate in five 96-well plates.
3.3 HeLa S3 Cells The conditions were as described for Namalwa cells, except that the HeLa cells were cultured in RPM 1640, 10% (V/V) FCS, 2 mM L-glutamine, 1% (v/v) MEM
nonessential amino acids (Sigma), and 1 mM sodium pyruvate. For transfection by electroporation the cells were released from the walls of the culture vessels by trypsinization.
The conditions for the electroporation were 960 g/250 V. After the electroporation the cells were cultured in RPMI 1640, 10% (v/v) FCS, 2 mM L-glutamine, 1% (v/v) MEM, 1 mM sodium pyruvate in T75 tissue culture bottles. 24 hours after electroporation the cells were trypsinized and cultured for 10 to 15 days in a medium containing 600 ti,g/inl of G-418 in ten 96-well plates.
Example 4 Selection for EPO-Producing Clones The culture supemate of transfected cells was tested in an EPO ELISA. All steps were performed at room temperature. 96-well plates previously coated with Streptavidin were coated with biotinylated anti-EPO antibodies (Boehringer Mannheim). For coating, the plates were first washed with 50 mM of sodium phosphate pH 7.2, and 0.05%
(v/v) of TweenTm 20. Then 0.01 ml of coating buffer (4 i../g/m1 of biotinylated antibody, 10 mM
sodium phosphate pH 7.2, 3 g/1 bovine serum albumin, 20 g/I saccharose, and 9 g/INaC1 were added per well, and incubated at room temperature for 3 h. Then the plates were washed with 50 mM of sodium phosphate pH 7.2, dried and sealed.
Before the test, after washing the plates three times with 0.3 ml of phosphate-buffered salt solution (PBS) and 0.05% TweenTm 20 (Sigma), the plates were incubated overnight with 0.2 ml PBS and 1% (w/v) CrotemTM (Boehringer Mannheim) per well in order to block unspecific bonds.
After removal of the blocking solution, 0.1 ml of culture supemate was added and the plates were incubated overnight. The individual wells were washed three times with 0.3 ml of
- 16 -PBS and 0.05% TweenTivi 20 each time. Then 100 Al of peroxidase (POD) conjugated monoclonal antibody (Boehringer Mannheim, 150 mU/m1) was added for two hours.
The wells were then again washed three times with 0.3 ml of PBS and 0.05% TweenTm 20 each time. Then the peroxidase reaction was performed using ABTS as substrate in a Perkin Elmer Photometer at 405 nm. A standard calibration curve using recombinant EPO
from CHO cells (Boehringer Mannheim, 100-1000 pg/well) was used to calculate the EPO
concentrations.
Example 5 EPO Gene Amplification To increase the EPO expression the EPO-producing clones were cultured in the presence of increasing concentrations (100 pM - 1000 nM) of methotrexate (MTX). The clones were tested at each MTX concentration by an ELISA (see Example 4) for the production of EPO.
Strong producers were subcloned by limited dilution.
Example 6 Signal Sequence Mutations To optimize the leader sequence of the EPO molecule, the first amino acids coded by exon 1 were replaced. Primers with different sequences (SEQ ID No. 4-17; the 3' primer contained a Celli site for the selection of modified sequences) were used in order to obtain as template an AscI/XbaI fragment by PCR using plasmid pEP0227 which contains a 4 kb HindIII/EcoRI fragment (including exons 1-5) of the human EPO gene sequence under control of the SV40 promoter (Jacobs et al., Nature 313 (1985), 806; Lee-Huang et al., Gene 128 (1993), 227). The resulting fragments were then cloned into the plasmid pEP0148 (Example 2.2), and the plasmids pEPO 182, 183, 184 and 185 were obtained (Figure 5). The EPO gene expression was driven by a SV40 promoter. COS-7 cells were transfected transiently with the constructs (DEAE dextran method) and the cells were tested 48 h after transfection for EPO production.
The mutated leader sequence Met-Ser-Ala-His obtained in this manner with the best EPO
expression was used for constructing the gene targeting vectors (cf. Example 2.2).
The wells were then again washed three times with 0.3 ml of PBS and 0.05% TweenTm 20 each time. Then the peroxidase reaction was performed using ABTS as substrate in a Perkin Elmer Photometer at 405 nm. A standard calibration curve using recombinant EPO
from CHO cells (Boehringer Mannheim, 100-1000 pg/well) was used to calculate the EPO
concentrations.
Example 5 EPO Gene Amplification To increase the EPO expression the EPO-producing clones were cultured in the presence of increasing concentrations (100 pM - 1000 nM) of methotrexate (MTX). The clones were tested at each MTX concentration by an ELISA (see Example 4) for the production of EPO.
Strong producers were subcloned by limited dilution.
Example 6 Signal Sequence Mutations To optimize the leader sequence of the EPO molecule, the first amino acids coded by exon 1 were replaced. Primers with different sequences (SEQ ID No. 4-17; the 3' primer contained a Celli site for the selection of modified sequences) were used in order to obtain as template an AscI/XbaI fragment by PCR using plasmid pEP0227 which contains a 4 kb HindIII/EcoRI fragment (including exons 1-5) of the human EPO gene sequence under control of the SV40 promoter (Jacobs et al., Nature 313 (1985), 806; Lee-Huang et al., Gene 128 (1993), 227). The resulting fragments were then cloned into the plasmid pEP0148 (Example 2.2), and the plasmids pEPO 182, 183, 184 and 185 were obtained (Figure 5). The EPO gene expression was driven by a SV40 promoter. COS-7 cells were transfected transiently with the constructs (DEAE dextran method) and the cells were tested 48 h after transfection for EPO production.
The mutated leader sequence Met-Ser-Ala-His obtained in this manner with the best EPO
expression was used for constructing the gene targeting vectors (cf. Example 2.2).
- 17 -Example 7 Characterization of EPO-Producing Cell Lines Three different cell lines (Namalwa, HeLa S3 and HT 1080) were selected for the EPO
gene activation. EPO-producing clones were obtained by transfection with the plasmids p179, p187, p189, p-190 or p192 (cf. Examples 2 and 3).
Approximately 160,000 NEO-resistant clones were tested for EPO production, of which 12 to 15 secreted EPO reproducibly in significant yield into the cell supemate.
Of these a total of 7 EPO clones were surprisingly identified which produced without gene amplification, by MTX, EPO in sufficient amounts for a large industrial production. The EPO production of these clones ranged from 200 ng/ml to more than 1000 ng/mU106 cells/24h. An example of one such cell is the clone "Aladin" deposited at the DSMZ (DSM
ACC 2320), which was obtained from a Namalwa cell.
After gene amplification with 500 nM of MTX the EPO production of the identified EPO
clones was increased to more than 3000 ng/m1/106 cells/24h. An additional increase of the MTX concentration to 1000 nM led to a production of up to more than 7000 ng/m1/106 cells/24h.
The clones obtained also showed EPO production under serum-free culture conditions.
Example 8 Characterization of the Genome of EPO-Producing Clones 8.1 Methodology Human genomic DNA was isolated from about 108 cells and quantified (Sambrook et al., 1989). After cleaving the genomic DNA with restriction enzymes, e.g., AgeI and AscI, or BamHI, Hind III and Sall, respectively, the DNA fragments were separated according to their size by agarose gel electrophoresis and finally transferred to a nylon membrane and immobilized.
The immobilized DNA was hybridized with digoxigenin-marked EPO probes or gene
gene activation. EPO-producing clones were obtained by transfection with the plasmids p179, p187, p189, p-190 or p192 (cf. Examples 2 and 3).
Approximately 160,000 NEO-resistant clones were tested for EPO production, of which 12 to 15 secreted EPO reproducibly in significant yield into the cell supemate.
Of these a total of 7 EPO clones were surprisingly identified which produced without gene amplification, by MTX, EPO in sufficient amounts for a large industrial production. The EPO production of these clones ranged from 200 ng/ml to more than 1000 ng/mU106 cells/24h. An example of one such cell is the clone "Aladin" deposited at the DSMZ (DSM
ACC 2320), which was obtained from a Namalwa cell.
After gene amplification with 500 nM of MTX the EPO production of the identified EPO
clones was increased to more than 3000 ng/m1/106 cells/24h. An additional increase of the MTX concentration to 1000 nM led to a production of up to more than 7000 ng/m1/106 cells/24h.
The clones obtained also showed EPO production under serum-free culture conditions.
Example 8 Characterization of the Genome of EPO-Producing Clones 8.1 Methodology Human genomic DNA was isolated from about 108 cells and quantified (Sambrook et al., 1989). After cleaving the genomic DNA with restriction enzymes, e.g., AgeI and AscI, or BamHI, Hind III and Sall, respectively, the DNA fragments were separated according to their size by agarose gel electrophoresis and finally transferred to a nylon membrane and immobilized.
The immobilized DNA was hybridized with digoxigenin-marked EPO probes or gene
- 18-activation sequence-specific DNA probes (DIG DNA Labeling Kit, Boehringer Mannheim) and washed under stringent conditions. The specific hybridization signals were detected by means of a chemiluminescence process using radiation-sensitive films.
8.2 Results The treatment of cells with 500 nM of MTX led to an increase of the hybridization signal at the EPO locus by a factor of 5 to 10. Upon an additional increase to 1000 nMMTX an increase by a factor >10 was obtained (Figure 6a).
In the case of hybridization with the EPO specific probe, the copies of chromosome 7 that were unaffected by homologous recombination were also detected. As it can be seen in Figure 6b, these likewise hybridizing DNA fragments have a different, clearly distinguishable size and are not changed in their signal strength by the use of MTX.
Example 9 Purification of EPO from Culture Supernates of Human Cell Lines (HeLa S3; Namalwa and HT1080) For the purification of EPO from cell culture supernates of human cell lines, basically two methods were used, which differ in number and principle of the chromatography steps and were used depending on the composition of the medium and the EPO
concentration:
Method 1: 1st step: blue sepharoseTM column 2nd step: butyl-sepharoseTM column 3rd step: hydroxyapatite column 4th step: reconcentration Method 2: 1st step: blue sepharoseTM column 2nd step: hydroxyapatite column 3rd step: reconcentration (alternative 3rd step: RP-HPLC) Example of purification of an HeLaS3 cell culture supernate with 2% (v/v) fetal calf serum
8.2 Results The treatment of cells with 500 nM of MTX led to an increase of the hybridization signal at the EPO locus by a factor of 5 to 10. Upon an additional increase to 1000 nMMTX an increase by a factor >10 was obtained (Figure 6a).
In the case of hybridization with the EPO specific probe, the copies of chromosome 7 that were unaffected by homologous recombination were also detected. As it can be seen in Figure 6b, these likewise hybridizing DNA fragments have a different, clearly distinguishable size and are not changed in their signal strength by the use of MTX.
Example 9 Purification of EPO from Culture Supernates of Human Cell Lines (HeLa S3; Namalwa and HT1080) For the purification of EPO from cell culture supernates of human cell lines, basically two methods were used, which differ in number and principle of the chromatography steps and were used depending on the composition of the medium and the EPO
concentration:
Method 1: 1st step: blue sepharoseTM column 2nd step: butyl-sepharoseTM column 3rd step: hydroxyapatite column 4th step: reconcentration Method 2: 1st step: blue sepharoseTM column 2nd step: hydroxyapatite column 3rd step: reconcentration (alternative 3rd step: RP-HPLC) Example of purification of an HeLaS3 cell culture supernate with 2% (v/v) fetal calf serum
- 19 -(FCS) by method 1:
1. Blue SepharoseTM Column:
A 5 ml Hi-Trap-Blue column (Pharmacia's blue sepharoseTM ready-to-use column) was equilibrated with at least 5 column-volumes (SV's) of buffer A (20 mM Tris-HCL, pH 7.0;
mM CaCl2; 100 mM NaC1). Then 70 ml of HeLa cell supemate (containing approx.
jg EPO and 70-100 mg total protein) was drawn up overnight at a flow of 0.5 ml/min by the circulatory method.
The column was washed with at least 5 SV's of buffer B (20 mM of Tris-HC1, pH
7.0; 5 mM CaCl2, 250 mM NaC1) and at least 5 SV's of buffer C (20 mM Tris-HC1, pH
7.0; 0.2 mM CaC12; 250 mM NaC1) at 0.5 ml/min. The success of the washing was followed by measuring the protein content at 0D280.
The elution of EPO was performed with buffer D (100 mM Tris-HC1, pH 7.0; 0.2 mM
CaCl2; 2 M NaC1) at a flow of 0.5 ml/min. The elution solution was collected in 1 - 2 ml fractions.
The EPO content of the fractions, wash solutions and the flow through was determined by reverse phase (RP)-HPLC by applying an aliquot to a POROS R2/HTM column (Boehringer Mannheim). Alternatively, an immunological dot blot was performed for the qualitative identification of fractions containing EPO.
Fractions containing EPO (8-12 ml) were pooled and applied to a butyl-sepharoseTM
column.
The yield after the blue sepharoseTM column was about 175 fig EPO (corresponds to about 70%). In general the yield after blue sepharoseTM was between 50 and 75%.
2. Butyl SepharoseTM Column (Hydrophobic Interaction Chromatography) A self-made 2-3 ml butyl sepharoseTM column (material: Toyopearl Butyl S650) was - 19a -equilibrated with at least 5 SV's of buffer D (100 mM Tris HC1, pH 7.0; 0.2 mM
CaC12 and 2 M NaCl) and then the blue sepharoseTM pool containing EPO from I. (approx.
150 Ag EPO) was drawn up at a flow of 0.5 ml/min.
The column was washed at 0.5 mUmin with at least 5 SV's of buffer E (20 mM
Tris-HC1, pH 7.0; 2 M NaC1 and 10% isopropanol). The washing success was followed by measuring the protein content at 0D280.
The elution of EPO was performed with buffer F (20 mM tris-HC1, pH 7.0; 2 M
NaC1 and
1. Blue SepharoseTM Column:
A 5 ml Hi-Trap-Blue column (Pharmacia's blue sepharoseTM ready-to-use column) was equilibrated with at least 5 column-volumes (SV's) of buffer A (20 mM Tris-HCL, pH 7.0;
mM CaCl2; 100 mM NaC1). Then 70 ml of HeLa cell supemate (containing approx.
jg EPO and 70-100 mg total protein) was drawn up overnight at a flow of 0.5 ml/min by the circulatory method.
The column was washed with at least 5 SV's of buffer B (20 mM of Tris-HC1, pH
7.0; 5 mM CaCl2, 250 mM NaC1) and at least 5 SV's of buffer C (20 mM Tris-HC1, pH
7.0; 0.2 mM CaC12; 250 mM NaC1) at 0.5 ml/min. The success of the washing was followed by measuring the protein content at 0D280.
The elution of EPO was performed with buffer D (100 mM Tris-HC1, pH 7.0; 0.2 mM
CaCl2; 2 M NaC1) at a flow of 0.5 ml/min. The elution solution was collected in 1 - 2 ml fractions.
The EPO content of the fractions, wash solutions and the flow through was determined by reverse phase (RP)-HPLC by applying an aliquot to a POROS R2/HTM column (Boehringer Mannheim). Alternatively, an immunological dot blot was performed for the qualitative identification of fractions containing EPO.
Fractions containing EPO (8-12 ml) were pooled and applied to a butyl-sepharoseTM
column.
The yield after the blue sepharoseTM column was about 175 fig EPO (corresponds to about 70%). In general the yield after blue sepharoseTM was between 50 and 75%.
2. Butyl SepharoseTM Column (Hydrophobic Interaction Chromatography) A self-made 2-3 ml butyl sepharoseTM column (material: Toyopearl Butyl S650) was - 19a -equilibrated with at least 5 SV's of buffer D (100 mM Tris HC1, pH 7.0; 0.2 mM
CaC12 and 2 M NaCl) and then the blue sepharoseTM pool containing EPO from I. (approx.
150 Ag EPO) was drawn up at a flow of 0.5 ml/min.
The column was washed at 0.5 mUmin with at least 5 SV's of buffer E (20 mM
Tris-HC1, pH 7.0; 2 M NaC1 and 10% isopropanol). The washing success was followed by measuring the protein content at 0D280.
The elution of EPO was performed with buffer F (20 mM tris-HC1, pH 7.0; 2 M
NaC1 and
20% isopropanol) at a flow of 0.5 mUmin. The elution solution was collected in 1 - 2 ml fractions.
The EPO content of the fractions, the wash solutions and the flow-through was determined by RP-HPLC by applying an aliquot to a POROS R2/HTM column. Alternatively, an immunological dot blot was performed for the qualitative identification of EPO-containing fractions.
Fractions containing EPO (10 - 15 ml) were pooled and applied to a hydroxyapatite column.
The yield of the butyl sepharoseTm column was about 130 pg EPO (corresponds to about 85%). In general, the yield of the butyl sepharoseTM was between 60 and 85% of the amount applied from the blue sepharoseTM pool.
3. Hydroxyapatite column A 5 ml hydroxyapatite column (Econo-Pac CHT II ready-to-use column from BioRAD) was equilibrated with at least 5 SV's of buffer F (20 mM Tris-HC1, pH 7.0; 2 M
NaCI; 20%
isopropanol) and then the butyl sepharoseTM pool containing EPO from 2.
(approx. 125 n EPO) was drawn up at a flow of 0.5 mUmin.
The column was washed with at least 5 SV's of buffer G (20 mM Tris-HC1, pH
7.0; 2 M
NaCl) at 0.5 ml/min. The washing success was followed by measuring the protein content - 19b at OD280.
The elution of EPO was performed with buffer H (10 mM sodium phosphate, pH
7.0; 80 mM NaCI) at a flow of 0.5 ml/min. The elution solution was collected in 1-2 ml fractions.
The EPO content of the fractions, wash solutions and flow-through was determined through RP-HPLC by applying an aliquot to a POROS R2/HTM column.
Fractions containing EPO (3-6 ml) were pooled. The yield of the hydroxyapatite column was about 80 itg of EPO (corresponds to about 60%). In general, the yield of the hydroxyapatite column amounted to between 50 and 65% of the butyl sepharoseTm pool applied.
4. Reconcentration The pooled EPO fractions from the hydroxyapatite step were raised to a concentration of 0.1 - 0.5 mg/ml in centrifuging units with a exclusion size of 10 kD (e.g., MicrosepTM by Filtron), 0.01% of TweenTm 20 was added and stocked in aliquots at -20 C.
Table of Yields EPO (pg) Yield (%) Start 245 100 Blue sepharosen4 175 70 Butyl sepharoseTM column 130 53 Hydroxyapatite column 80 33 Reconcentration 60 25 The purity of the isolated EPO was approximately >90%, and generally even >95%.
To increase the EPO yield, Method 2 was also used, in which the butyl sepharoseTM step was missing. This method is applicable especially in the case of cell culture supemates - 19c -without or with 1% (v/v) FCS addition, and yields isolated EPO of approximately equal purity (90-95%). The presence of 5 mM CaCl2 in the equilibration buffer (buffer F) for the hydroxyapatite column led in this method to improved binding and thus also to a reproducible elution behaviour of EPO in the hydroxyapatite step. Therefore Method 2 was practiced with basically the same procedure as in Method 1, with the following buffers:
1. Blue SepharoseTM Column:
Equilibration buffer (buffer A) 20 mM Tris-HC1, pH 7.0 mM CaC12; 100 mM NaC1 Washing buffer 1 (buffer B) 20 mM Tris-HC1, pH 7.0 5 mM CaC12; 250 mM NaC1 Washing buffer 2 (buffer C) 20 mM Tris-HC1, pH 7.0 5 mM CaC12, 250 mM NaC1 Elution buffer (buffer D) 100 mM Tris-HC1, pH 7.0 5 mM CaC12; 2 M NaC1 2. Hydroxyapatite Column:
Equilibration buffer (buffer F): 50 mM Tris-HC1, pH 7.0 5 mM CaC12, 1 M NaC1 Washing buffer (buffer G): 10 mM Tris-HC1, pH 7.0 5 mM CaC12; 80 mM NaC1 Elution buffer (buffer H): 10 mM sodium phosphate, pH 7.0 0.5 mM CaC12; 80 mM NaC1 Table of Yields:
EPO ( g) Yield (%) Start 600 100 Blue SepharoseTM 450 75 Hydroxyapatite column 335 55 - 19d-Reconcentration 310 52 The addition of 5 mM CaC12 to the buffers B to G in Method 1 also resulted in better binding and more defined elution of the hydroxyapatite column.
Example 10 Determination of the Specific Activity in vivo of EPO from Human Cell Lines (Bioassay on the Normocythemic Mouse) The dose-related activity of EPO on the proliferation and differentiation of erythrocyte precursor cells was determined in vivo in mice on the basis of the increase of reticulocytes in the blood after EPO administration.
For this purpose eight mice were treated parenterally with different doses of the EPO
sample and to be analyzed and of an EPO standard (balanced against the WHO's standard EPO). The mice were then kept under constant, defined conditions. 4 days after the EPO
treatment blood was taken from the mice and the reticulocytes stained with acridine orange.
Determination of the number of reticulocytes per 30,000 erythrocytes was performed by microfluorimetry in the flow cytometer by analyzing the red fluorescence histogram.
The computation of the biological activity was made from the values of the reticulocyte counts of the specimen and those of the standard at the various doses by the method described by Linder of content determination in pairs with parallel straight lines (A. Linder, Planen und Auswerten von Versuchen, 3rd ed., 1969, Birkenhauser Verlag Basel).
Result:
EPO from cell line Specific Activity U/mg HeLa S3 (Sample 1) 100,000 HeLa S3 (Sample 2) 110,000 - 19e -SEQUENCE LISTING
<110> Roche Diagnostics GmbH
<120> Preparation of Erythropoietin by Endogenic Gene Activation <130> 17235PKR
<140> PCT/EP98/04590 <141> 1998-07-22 <150> EP97112640.4 <151> 1997-07-23 <150> DE19753681.6 <151> 1997-12-03 <150> US09/113692 <151> 1998-07-10 <160> 17 <170> PatentIn Ver. 2.1 <210> 1 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 1 cgcggcggat cccagggagc tgggttgacc gg 32 <210> 2 <211> 38 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 2 ggccgcgaat tctccgcgcc tggccggggt ccctcagc 38 <210> 3 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 3 cgcggcggat cctctcctcc ctcccaagct gcaatc 36 <210> 4 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 4 ggccgcgaat tctagaacag atagccaggc tgagag 36 <210> 5 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EPO EX1 <400> 5 tcacccggcg cgccccaggt cgct 24 <210> 6 <211> 61 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX2 <400> 6 atgctcgagc ggccgccagt gtgatggata tctgcagagc tcagcttggc cgcgaattct 60 a 61 <210> 7 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Primer EX3 <220>
<221> CDS
<222> (49)..(60) <400> 7 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg ggg gcc 57 Met Gly Ala
The EPO content of the fractions, the wash solutions and the flow-through was determined by RP-HPLC by applying an aliquot to a POROS R2/HTM column. Alternatively, an immunological dot blot was performed for the qualitative identification of EPO-containing fractions.
Fractions containing EPO (10 - 15 ml) were pooled and applied to a hydroxyapatite column.
The yield of the butyl sepharoseTm column was about 130 pg EPO (corresponds to about 85%). In general, the yield of the butyl sepharoseTM was between 60 and 85% of the amount applied from the blue sepharoseTM pool.
3. Hydroxyapatite column A 5 ml hydroxyapatite column (Econo-Pac CHT II ready-to-use column from BioRAD) was equilibrated with at least 5 SV's of buffer F (20 mM Tris-HC1, pH 7.0; 2 M
NaCI; 20%
isopropanol) and then the butyl sepharoseTM pool containing EPO from 2.
(approx. 125 n EPO) was drawn up at a flow of 0.5 mUmin.
The column was washed with at least 5 SV's of buffer G (20 mM Tris-HC1, pH
7.0; 2 M
NaCl) at 0.5 ml/min. The washing success was followed by measuring the protein content - 19b at OD280.
The elution of EPO was performed with buffer H (10 mM sodium phosphate, pH
7.0; 80 mM NaCI) at a flow of 0.5 ml/min. The elution solution was collected in 1-2 ml fractions.
The EPO content of the fractions, wash solutions and flow-through was determined through RP-HPLC by applying an aliquot to a POROS R2/HTM column.
Fractions containing EPO (3-6 ml) were pooled. The yield of the hydroxyapatite column was about 80 itg of EPO (corresponds to about 60%). In general, the yield of the hydroxyapatite column amounted to between 50 and 65% of the butyl sepharoseTm pool applied.
4. Reconcentration The pooled EPO fractions from the hydroxyapatite step were raised to a concentration of 0.1 - 0.5 mg/ml in centrifuging units with a exclusion size of 10 kD (e.g., MicrosepTM by Filtron), 0.01% of TweenTm 20 was added and stocked in aliquots at -20 C.
Table of Yields EPO (pg) Yield (%) Start 245 100 Blue sepharosen4 175 70 Butyl sepharoseTM column 130 53 Hydroxyapatite column 80 33 Reconcentration 60 25 The purity of the isolated EPO was approximately >90%, and generally even >95%.
To increase the EPO yield, Method 2 was also used, in which the butyl sepharoseTM step was missing. This method is applicable especially in the case of cell culture supemates - 19c -without or with 1% (v/v) FCS addition, and yields isolated EPO of approximately equal purity (90-95%). The presence of 5 mM CaCl2 in the equilibration buffer (buffer F) for the hydroxyapatite column led in this method to improved binding and thus also to a reproducible elution behaviour of EPO in the hydroxyapatite step. Therefore Method 2 was practiced with basically the same procedure as in Method 1, with the following buffers:
1. Blue SepharoseTM Column:
Equilibration buffer (buffer A) 20 mM Tris-HC1, pH 7.0 mM CaC12; 100 mM NaC1 Washing buffer 1 (buffer B) 20 mM Tris-HC1, pH 7.0 5 mM CaC12; 250 mM NaC1 Washing buffer 2 (buffer C) 20 mM Tris-HC1, pH 7.0 5 mM CaC12, 250 mM NaC1 Elution buffer (buffer D) 100 mM Tris-HC1, pH 7.0 5 mM CaC12; 2 M NaC1 2. Hydroxyapatite Column:
Equilibration buffer (buffer F): 50 mM Tris-HC1, pH 7.0 5 mM CaC12, 1 M NaC1 Washing buffer (buffer G): 10 mM Tris-HC1, pH 7.0 5 mM CaC12; 80 mM NaC1 Elution buffer (buffer H): 10 mM sodium phosphate, pH 7.0 0.5 mM CaC12; 80 mM NaC1 Table of Yields:
EPO ( g) Yield (%) Start 600 100 Blue SepharoseTM 450 75 Hydroxyapatite column 335 55 - 19d-Reconcentration 310 52 The addition of 5 mM CaC12 to the buffers B to G in Method 1 also resulted in better binding and more defined elution of the hydroxyapatite column.
Example 10 Determination of the Specific Activity in vivo of EPO from Human Cell Lines (Bioassay on the Normocythemic Mouse) The dose-related activity of EPO on the proliferation and differentiation of erythrocyte precursor cells was determined in vivo in mice on the basis of the increase of reticulocytes in the blood after EPO administration.
For this purpose eight mice were treated parenterally with different doses of the EPO
sample and to be analyzed and of an EPO standard (balanced against the WHO's standard EPO). The mice were then kept under constant, defined conditions. 4 days after the EPO
treatment blood was taken from the mice and the reticulocytes stained with acridine orange.
Determination of the number of reticulocytes per 30,000 erythrocytes was performed by microfluorimetry in the flow cytometer by analyzing the red fluorescence histogram.
The computation of the biological activity was made from the values of the reticulocyte counts of the specimen and those of the standard at the various doses by the method described by Linder of content determination in pairs with parallel straight lines (A. Linder, Planen und Auswerten von Versuchen, 3rd ed., 1969, Birkenhauser Verlag Basel).
Result:
EPO from cell line Specific Activity U/mg HeLa S3 (Sample 1) 100,000 HeLa S3 (Sample 2) 110,000 - 19e -SEQUENCE LISTING
<110> Roche Diagnostics GmbH
<120> Preparation of Erythropoietin by Endogenic Gene Activation <130> 17235PKR
<140> PCT/EP98/04590 <141> 1998-07-22 <150> EP97112640.4 <151> 1997-07-23 <150> DE19753681.6 <151> 1997-12-03 <150> US09/113692 <151> 1998-07-10 <160> 17 <170> PatentIn Ver. 2.1 <210> 1 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 1 cgcggcggat cccagggagc tgggttgacc gg 32 <210> 2 <211> 38 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 2 ggccgcgaat tctccgcgcc tggccggggt ccctcagc 38 <210> 3 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 3 cgcggcggat cctctcctcc ctcccaagct gcaatc 36 <210> 4 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 4 ggccgcgaat tctagaacag atagccaggc tgagag 36 <210> 5 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EPO EX1 <400> 5 tcacccggcg cgccccaggt cgct 24 <210> 6 <211> 61 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX2 <400> 6 atgctcgagc ggccgccagt gtgatggata tctgcagagc tcagcttggc cgcgaattct 60 a 61 <210> 7 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Primer EX3 <220>
<221> CDS
<222> (49)..(60) <400> 7 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg ggg gcc 57 Met Gly Ala
- 21 -cac ggtgagtact cgcgggct 78 His <210> 8 <211> 4 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence:Primer EX3 <400> 8 Met Gly Ala His <210> 9 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX4 <220>
<221> CDS
<222> (49)..(60) <400> 9 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg agc gcc 57 Met Ser Ala cac ggtgagtact cgcgggct 78 His <210> 10 <211> 4 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Primer EX4 <400> 10 Met Ser Ala His <210> 11 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX5
<213> Artificial Sequence <223> Description of Artificial Sequence:Primer EX3 <400> 8 Met Gly Ala His <210> 9 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX4 <220>
<221> CDS
<222> (49)..(60) <400> 9 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg agc gcc 57 Met Ser Ala cac ggtgagtact cgcgggct 78 His <210> 10 <211> 4 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Primer EX4 <400> 10 Met Ser Ala His <210> 11 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX5
- 22 -<220>
<221> CDS
<222> (49)..(60) <400> 11 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg ggg gtg 57 Met Gly Val ccc ggtgagtact cgcgggct 78 Pro <210> 12 <211> 4 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Primer EX5 <400> 12 Met Gly Val Pro <210> 13 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX6 <220>
<221> CDS
<222> (49)..(60) <400> 13 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg agc gtg 57 Met Ser Val cac ggtgagtact cgcgggct 78 His <210> 14 <211> 4 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Primer EX6 <400> 14 Met Ser Val His
<221> CDS
<222> (49)..(60) <400> 11 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg ggg gtg 57 Met Gly Val ccc ggtgagtact cgcgggct 78 Pro <210> 12 <211> 4 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Primer EX5 <400> 12 Met Gly Val Pro <210> 13 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX6 <220>
<221> CDS
<222> (49)..(60) <400> 13 tcacccggcg cgccccaggt cgctgaggga ccccggccag gcgcggag atg agc gtg 57 Met Ser Val cac ggtgagtact cgcgggct 78 His <210> 14 <211> 4 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Primer EX6 <400> 14 Met Ser Val His
- 23 <210> 15 <211> 59 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX
genome 1 <400> 15 ggacattcta gaacagatat ccaggctgag cgtcaggcgg ggagggagaa gggtggctg 59 <210> 16 <211> 48 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX13 <400> 16 gtgatggata tctctagaac agatagccag gctgagagtc aggcgggg 48 <210> 17 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EPO EX
<400> 17 atggatatca tcgattctag aacagatagc caggctgag 39
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX
genome 1 <400> 15 ggacattcta gaacagatat ccaggctgag cgtcaggcgg ggagggagaa gggtggctg 59 <210> 16 <211> 48 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EX13 <400> 16 gtgatggata tctctagaac agatagccag gctgagagtc aggcgggg 48 <210> 17 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer EPO EX
<400> 17 atggatatca tcgattctag aacagatagc caggctgag 39
- 24 -
Claims (47)
1. Human cell, characterized in that it contains a copy of an endogenous EPO gene in operative linkage with an heterologous promoter that is active in the human cell and is able to produce 200 - 7000 ng EPO/10 6 cells/24h, the distance between the heterologous promoter and the translation start of the EPO gene being less than 1100 bp, and said human cell is obtained by a process comprising the steps:
(a) providing human starting cells which contain at least one copy of an endogenous EPO gene, provided that the human starting cells are not germ line cells, (b) transfecting the cells with a DNA construct comprising:
two flanking DNA sequences which are homologous to regions of the human EPO gene locus in order to permit a homologous recombination, (ii) a positive selection marker gene and (iii) an heterologous expression control sequence which is active in the human cell, wherein the heterologous expression control sequence comprises a viral promoter and the distance between the heterologous promoter and the translation start of the EPO gene is less than 1100 bp, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selected according to step (c), and (e) identifying the EPO-producing cells.
(a) providing human starting cells which contain at least one copy of an endogenous EPO gene, provided that the human starting cells are not germ line cells, (b) transfecting the cells with a DNA construct comprising:
two flanking DNA sequences which are homologous to regions of the human EPO gene locus in order to permit a homologous recombination, (ii) a positive selection marker gene and (iii) an heterologous expression control sequence which is active in the human cell, wherein the heterologous expression control sequence comprises a viral promoter and the distance between the heterologous promoter and the translation start of the EPO gene is less than 1100 bp, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selected according to step (c), and (e) identifying the EPO-producing cells.
2. The human cell of claim 1, characterized in that it produces 200 - 3000 ng EPO/10 6 cells/24 h.
3. Human cell, characterized in that it contains copies of an endogenic EPO
gene obtained by gene amplification of the EPO gene contained in the cell according to claim 1 , each in operative linkage with a heterologous promoter active in the human cell and produces 1000 - 7000 ng EPO/10 6 cells/24 h.
gene obtained by gene amplification of the EPO gene contained in the cell according to claim 1 , each in operative linkage with a heterologous promoter active in the human cell and produces 1000 - 7000 ng EPO/10 6 cells/24 h.
4. The human cell of any one of claims 1 to 3, characterized in that it is an immortalized cell.
5. The human cell of any one of claims 1 to 4, characterized in that that it can be cultured in serum-free medium.
6. The human cell of any one of claims 1 to 5, characterized in that it is selected from the group consisting of an HT 1080 cell, an HeLa S3 cell and a Namalwa cell.
7. The human cell of any one of claims 1 to 6, wherein said viral promoter is a CMV promoter.
8. The human cell of claim 7, characterized in that the EPO gene, which is in operative linkage with the heterologous expression control sequence, has a signal peptide-coding sequence which codes for a signal peptide sequence modified in the region of the 4 first amino acids, which is:
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His.
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His.
9. The human cell of claim 8, characterized in that the first 4 amino acids are selected from the group consisting of:
(a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His, (c) Met-Gly-Val-Pro and (d) Met-Ser-Val-His.
(a) Met-Gly-Ala-His, (b) Met-Ser-Ala-His, (c) Met-Gly-Val-Pro and (d) Met-Ser-Val-His.
10. The human cell of claim 9, characterized in that the sequence of the first 4 amino acids of the signal peptide is Met-Ser-Ala-His.
11. Method for the preparation of a human EPO-producing cell according to any one of claims 1 to 10, comprising the steps of:
(a) providing human starting cells which contain one copy of an endogenic EPO gene, (b) transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous to regions of the human EPO gene locus, to permit a homologous recombination, (ii) a positive selection marker gene, and (iii) an heterologous expression control sequence which is active in the human cell, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selectable according to step (c), and (e) identifying of the EPO-producing cells.
(a) providing human starting cells which contain one copy of an endogenic EPO gene, (b) transfecting the cells with a DNA construct comprising:
(i) two flanking DNA sequences which are homologous to regions of the human EPO gene locus, to permit a homologous recombination, (ii) a positive selection marker gene, and (iii) an heterologous expression control sequence which is active in the human cell, (c) culturing the transfected cells under conditions in which a selection takes place for the presence of the positive selection marker gene, (d) analyzing the cells selectable according to step (c), and (e) identifying of the EPO-producing cells.
12. The method of claim 11, characterized in that the homologous DNA
sequences are selected from the regions of the 5'-untranslated sequences, exon 1 and intron 1 of the EPO gene.
sequences are selected from the regions of the 5'-untranslated sequences, exon 1 and intron 1 of the EPO gene.
13. The method of claim 12, characterized in that a sequence modified in the region of exon 1 is used.
14. The method of claim 12 or 13, characterized in that the neomycin phosphotransferase gene is used as selection marker gene.
15. The method of any one of claims 11 to 13, characterized in that the DNA
construct additionally comprises an amplification gene.
construct additionally comprises an amplification gene.
16. The method of claim 15, characterized in that a dihydrofolate reductase gene is used as amplification gene.
17. The method of claim 16, characterized in that the gene of a dihydrofolate reductase arginine-mutant is used as amplification gene.
18. The method of claim 16 or 17, furthermore comprising the steps:
(f) amplification of the EPO gene, and (g) obtaining of EPO-producing cells which contain several copies of an endogenic EPO gene each in operative linkage with a heterologous expression control sequence.
(f) amplification of the EPO gene, and (g) obtaining of EPO-producing cells which contain several copies of an endogenic EPO gene each in operative linkage with a heterologous expression control sequence.
19. The method of any one of claims 11 to 18, characterized in that a CMV
promoter/enhancer is used as expression control sequence.
promoter/enhancer is used as expression control sequence.
20. The method of any one of claims 11 to 18, characterized in that a plasmid corresponding to deposited accession number DSM 11661 is used in linearized form as DNA construct.
21. DNA construct for activating an endogenic EPO gene in a human cell, comprising:
(i) two flanking DNA sequences which are homologous to regions of the human EPO gene locus selected from 5'-untranslated sequences, exon 1 and intron 1, in order to permit a homologous recombination, a modified sequence being present in the region of exon 1, coding for the amino acids:
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His, (ii) a positive selection marker gene, and (iii) an heterologous expression control sequence that is active in a human cell.
(i) two flanking DNA sequences which are homologous to regions of the human EPO gene locus selected from 5'-untranslated sequences, exon 1 and intron 1, in order to permit a homologous recombination, a modified sequence being present in the region of exon 1, coding for the amino acids:
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, provided that X1-X2-X3 is not the sequence Gly-Val-His, (ii) a positive selection marker gene, and (iii) an heterologous expression control sequence that is active in a human cell.
22. The DNA construct of claim 21, further comprising an amplification gene.
23. The DNA construct of claim 21, characterized in that that the amino acids are selected from the group consisting of:
(a) Met-Gly-Ala-His.
(b) Met-Ser-Ala-His, (c) Met-Gly-Val-Pro and (d) Met-Ser-Val-His.
(a) Met-Gly-Ala-His.
(b) Met-Ser-Ala-His, (c) Met-Gly-Val-Pro and (d) Met-Ser-Val-His.
24. Plasmid p189 corresponding to deposited accession number DSM 11661.
25. Method for preparing human EPO, characterized in that a human cell according to any one of claims 1 to 10 is cultured in a suitable medium under conditions in which a production of EPO occurs and the EPO is harvested from the culture medium.
26. The method of claim 25, characterized in that a serum-free medium is used.
27. The method of claim 16 or 25, characterized in that the cells are cultured in suspension.
28. The method of any one of claims 25 to 27, characterized in that the culturing is performed in a fermenter.
29. The method of claim 28, characterized in that the volume of the fermenter is 10 1 - 50,000 1.
30. The method of any one of claims 25 to 29, characterized in that the harvesting of the EPO from the culture medium comprises the steps:
(a) passing the cell supernate over an affinity chromatography medium and harvesting the fractions containing EPO, (b) passing the fractions containing EPO over hydroxyapatite and harvesting the fractions containing EPO, and (c) concentrating or passing over a reverse-phase HPLC medium.
(a) passing the cell supernate over an affinity chromatography medium and harvesting the fractions containing EPO, (b) passing the fractions containing EPO over hydroxyapatite and harvesting the fractions containing EPO, and (c) concentrating or passing over a reverse-phase HPLC medium.
31. The method of claim 30, further comprising a step following step (a) of passing the fractions containing the EPO over a hydrophobic interaction chromatography medium and harvesting the fractions containing EPO.
32. The method of claim 30, characterized in that a blue sepharose .TM.
medium is used in step (a).
medium is used in step (a).
33. The method of claim 31, characterized in that a butyl sepharose .TM.
medium is used.
medium is used.
34. The method of any one of claims 30 to 33, characterized in that the concentration is performed by exclusion chromatography.
35. The method of claim 34, characterized in that a medium with an exclusion size of kD is used.
36. The method of any one of claims 25 to 35, characterized in that a human EPO is obtained with a purity of 90% to 95%.
37. The method of any one of claims 25 to 36, characterized in that a human EPO is obtained with a specific activity in vivo of at least 100,000 IU/mg.
38. The method of claim 27, characterized in that a human EPO is obtained with a specific activity in vivo of at least 175,000 IU/mg.
39. The method of any one of claims 25 to 38, characterized in that a human EPO is obtained with a content of N-glycol neuraminic acid of less than 0.2% of the total N-acetyl neuraminic acid content.
40. The method of any one of claims 25 to 39, characterized in that a human EPO is obtained with .alpha.-2,3-linked sialic acid residues.
41. The method of any one of claims 25 to 40, characterized in that a human EPO is obtained with .alpha.-2,3- and .alpha.-2,6-linked sialic acid residues.
42. The method of any one of claims 25 to 41, characterized in that a human EPO is obtained which comprises a polypeptide with a length of 165 amino acids.
43. The method of any one of claims 25 to 41, characterized in that a human EPO is obtained which comprises a polypeptide with a length of 166 amino acids.
44. The method of any one of claims 25 to 41, characterized in that a human EPO is obtained which comprises a mixture of polypeptides with a length of 165 and amino acids.
45. Isolated DNA which codes for a human EPO with a sequence modified in the area of the first 4 amino acids of a signal peptide defined by:
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, on the condition that X1-X2-X3 is not the sequence Gly-Val-His.
Met-X1-X2-X3 wherein X1 is Gly or Ser, X2 is Ala, Val, Leu, Ile, Ser or Pro, and X3 is Pro, Arg, Cys or His, on the condition that X1-X2-X3 is not the sequence Gly-Val-His.
46. The isolated DNA of claim 45, characterized in that the amino acid sequence is selected from the group consisting of:
(b) Met-Gly-Ala-His, (c) Met-Ser-Ala-His (d) Met-Gly-Val-Pro and (e) Met-Ser-Val-His.
(b) Met-Gly-Ala-His, (c) Met-Ser-Ala-His (d) Met-Gly-Val-Pro and (e) Met-Ser-Val-His.
47. The isolated DNA of claim 45 or 46, characterized in that it is a cDNA.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP97112640 | 1997-07-23 | ||
| EP97112640.4 | 1997-07-23 | ||
| DE19753681.6 | 1997-12-03 | ||
| DE1997153681 DE19753681A1 (en) | 1997-12-03 | 1997-12-03 | Erythropoietin composition has a highly specific activity |
| US09/113,692 | 1998-07-10 | ||
| US09/113,692 US6548296B1 (en) | 1997-07-23 | 1998-07-10 | Methods for identifying human cell lines useful for endogenous gene activation, isolated human lines identified thereby, and uses thereof |
| PCT/EP1998/004590 WO1999005268A1 (en) | 1997-07-23 | 1998-07-22 | Production of erythropoietin by endogenous gene activation |
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| Publication Number | Publication Date |
|---|---|
| CA2298015A1 CA2298015A1 (en) | 1999-02-04 |
| CA2298015C true CA2298015C (en) | 2015-01-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2298015A Expired - Lifetime CA2298015C (en) | 1997-07-23 | 1998-07-22 | Production of erythropoietin by endogenous gene activation |
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| Country | Link |
|---|---|
| EP (1) | EP0986644B1 (en) |
| JP (1) | JP4541539B2 (en) |
| CN (1) | CN1151258C (en) |
| AT (1) | ATE341625T1 (en) |
| AU (1) | AU754619B2 (en) |
| BR (1) | BRPI9811031B8 (en) |
| CA (1) | CA2298015C (en) |
| DE (1) | DE59813758D1 (en) |
| DK (1) | DK0986644T3 (en) |
| ES (1) | ES2273434T3 (en) |
| TR (1) | TR200000175T2 (en) |
| TW (1) | TW574372B (en) |
| WO (1) | WO1999005268A1 (en) |
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| US6855544B1 (en) | 1999-04-15 | 2005-02-15 | Crucell Holland B.V. | Recombinant protein production in a human cell |
| AT409379B (en) | 1999-06-02 | 2002-07-25 | Baxter Ag | MEDIUM FOR PROTEIN- AND SERUM-FREE CELL CULTURE |
| US7527961B2 (en) | 1999-11-26 | 2009-05-05 | Crucell Holland B.V. | Production of vaccines |
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| US7521220B2 (en) | 1999-11-26 | 2009-04-21 | Crucell Holland B.V. | Production of vaccines |
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| AU2002357019A1 (en) | 2001-11-28 | 2003-06-10 | Ortho-Mcneil Pharmaceutical, Inc. | Erythropoietin dosing regimen for treating anemia |
| EP1465987B1 (en) | 2001-12-07 | 2008-01-23 | Crucell Holland B.V. | Production of viruses, viral isolates and vaccines |
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| JPH01165393A (en) * | 1987-12-21 | 1989-06-29 | Sumitomo Chem Co Ltd | Method for purifying recombinant human erytheropoietin |
| AU635844B2 (en) * | 1989-11-06 | 1993-04-01 | Cell Genesys, Inc. | Production of proteins using homologous recombination |
| DK0505500T3 (en) * | 1989-12-22 | 1997-08-25 | Applied Research Systems | Modification of endogenous gene expression with regulatory element by homologous recombination |
| US5641670A (en) * | 1991-11-05 | 1997-06-24 | Transkaryotic Therapies, Inc. | Protein production and protein delivery |
| US6270989B1 (en) * | 1991-11-05 | 2001-08-07 | Transkaryotic Therapies, Inc. | Protein production and delivery |
| PT101031B (en) * | 1991-11-05 | 2002-07-31 | Transkaryotic Therapies Inc | PROCESS FOR THE SUPPLY OF PROTEINS BY GENETIC THERAPY |
| TW402639B (en) * | 1992-12-03 | 2000-08-21 | Transkaryotic Therapies Inc | Protein production and protein delivery |
| US5888774A (en) * | 1994-12-19 | 1999-03-30 | Cangene Corporation | Recombinant DNA molecules and expression vectors for erythropoietin |
| IL118201A (en) * | 1995-05-11 | 2004-12-15 | Roche Diagnostics Gmbh | Preparation comprising a protein with human erythropoietin activity which is free of serum and non-recombinant mammalian protein and process for the preparation thereof |
-
1998
- 1998-07-22 BR BRPI9811031 patent/BRPI9811031B8/en not_active IP Right Cessation
- 1998-07-22 TR TR2000/00175T patent/TR200000175T2/en unknown
- 1998-07-22 JP JP2000504243A patent/JP4541539B2/en not_active Expired - Lifetime
- 1998-07-22 DE DE59813758T patent/DE59813758D1/en not_active Expired - Lifetime
- 1998-07-22 EP EP98941401A patent/EP0986644B1/en not_active Expired - Lifetime
- 1998-07-22 ES ES98941401T patent/ES2273434T3/en not_active Expired - Lifetime
- 1998-07-22 AU AU89786/98A patent/AU754619B2/en not_active Expired
- 1998-07-22 CA CA2298015A patent/CA2298015C/en not_active Expired - Lifetime
- 1998-07-22 AT AT98941401T patent/ATE341625T1/en active
- 1998-07-22 WO PCT/EP1998/004590 patent/WO1999005268A1/en not_active Ceased
- 1998-07-22 CN CNB988074826A patent/CN1151258C/en not_active Expired - Lifetime
- 1998-07-22 DK DK98941401T patent/DK0986644T3/en active
- 1998-09-24 TW TW87116028A patent/TW574372B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| ATE341625T1 (en) | 2006-10-15 |
| AU8978698A (en) | 1999-02-16 |
| BRPI9811031B1 (en) | 2011-10-04 |
| DE59813758D1 (en) | 2006-11-16 |
| ES2273434T3 (en) | 2007-05-01 |
| BRPI9811031A (en) | 2000-08-08 |
| EP0986644B1 (en) | 2006-10-04 |
| TW574372B (en) | 2004-02-01 |
| CA2298015A1 (en) | 1999-02-04 |
| JP2001511343A (en) | 2001-08-14 |
| JP4541539B2 (en) | 2010-09-08 |
| DK0986644T3 (en) | 2007-01-29 |
| WO1999005268A1 (en) | 1999-02-04 |
| TR200000175T2 (en) | 2001-01-22 |
| CN1151258C (en) | 2004-05-26 |
| AU754619B2 (en) | 2002-11-21 |
| CN1265143A (en) | 2000-08-30 |
| EP0986644A1 (en) | 2000-03-22 |
| BRPI9811031B8 (en) | 2021-07-06 |
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