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Last Updated: October 22, 2019

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Claims for Patent: 7,186,529

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Summary for Patent: 7,186,529
Title:Preparation of erythropoietin by endogenous gene activation
Abstract: The invention relates to human cells which are capable, on the basis of an activation of the endogenous 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 endogenous EPO in human cells, and a method for the large technical production of EPO in human cells.
Inventor(s): Stern; Anne (Penzberg, DE), Brandt; Michael (Iffeldorf, DE), Honold; Konrad (Penzberg, DE), Auer; Johannes (Penzberg, DE), Koll; Hans (Weilheim, DE)
Assignee: Roche Diagnostics GmbH (Basel, CH)
Application Number:10/353,767
Patent Claims:1. DNA construct for activating an endogenous EPO gene in a human cell, comprising: (i) two flanking DNA sequences which are homologous to regions of the human 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 range of exon 1, coding for the amino acids: Met-X1-X2-X3 wherein X.sub.1 is Gly or Ser, X.sub.2 is Ala, Val, Leu, Ile, Ser or Pro, and X.sub.3 is Pro, Arg, Cys or His, provided that X.sub.1-X.sub.2-X.sub.3 is not the sequence Gly-Val-His, (ii) a positive selection marker gene, and (iii) a heterologous expression control sequence which is active in a human cell.

2. DNA construct according to claim 1, wherein the modified the amino acids is (a) Met-Gly-Ala-His, SEQ ID NO: 8, (b) Met-Ser-Ala-His, SEQ ID NO: 10, (c) Met-Gly-Val-Pro, SEQ ID NO: 12 or (d) Met-Ser-Val-His, SEQ ID NO: 14.

3. DNA construct for activating an endogenous EPO gene in a human cell, comprising: (i) two flanking DNA sequences which are homologous with regions of the human EPO gene locus selected from 5'-untranslated sequences, exon 1 and intron 1, in order 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 not greater than 1100 bp.

4. A nucleic acid molecule consisting of the nucleotide sequence of plasmid p189 (DSM 11661).

5. A method for preparing human EPO, comprising culturing a human cell in a suitable medium under conditions in which a production of EPO occurs and harvesting the EPO from the culture medium wherein said human cell contains a copy of an endogenous EPO gene in operable lineage with a heterologous promoter active in the human cell and the human cell is capable of the production of at least 200 ng EPO/10.sub.6 cells/24 h, wherein the endogenous EPO gene comprises a modified sequence being present in the range of exon 1, coding for the amino acids: Met-X.sub.1-X.sub.2-X.sub.3 wherein X.sub.1 is Gly or Ser, X.sub.2 is Ala, Val, Leu, Ile, Ser or Pro, and X.sub.3 is Pro, Arg, Cys or His, provided that X.sub.1-X.sub.2-X.sub.3 is not the sequence Gly-Val-His.

6. Method according to claim 5, wherein said medium is a serum-free medium.

7. Method according to claim 5 wherein the cells are cultured in suspension.

8. Method according to claim 5 the culturing is performed in a fermenter.

9. Method according to claim 8, wherein the volume of the fermenter is 10 l 50,000 l.

10. Method according to claim 5 wherein the harvesting of the EPO from the culture medium comprises the steps: (a) passing the cell supernatant 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 the EPO containing fractions or passing the EPO containing fractions over a reverse-phase HPLC medium, or concentrating and passing said EPO containing fractions over a reverse-phase HPLC column.

11. Method according to claim 10, wherein the affinity chromatography medium in step (a) is a blue sepharose medium.

12. Method according to claim 10 wherein the hydrophobic interaction chromatography medium in step (b) is a butyl sepharose medium.

13. Method according to claim 10 wherein the concentration is performed by exclusion chromatography.

14. Method according to claim 13, wherein an exclusion chromatography medium having an exclusion size of 10 kD is used.

15. Method according to claim 5 wherein said human EPO is obtained with a purity of at least 90%.

16. Method according to claim 5 wherein said human EPO is obtained with a specific activity in vivo of at least 100,000 IU/mg as determined by normocythemic mouse bioassay.

17. Method according to claim 16, wherein said human EPO is obtained with a specific activity in vivo (normocythemic mouse) of at least 175,000 IU/mg to 450,000 IU/mg as determined by normocythemic mouse bioassay.

18. Method according to claim 5 wherein said human EPO is obtained with a content of less than 0.2% N-glycolneuraminic acid with respect to the content of N-acetylneuraminic acid.

19. Method according to claim 5 wherein said human EPO comprises .alpha.-2,3-linked sialic acid residues.

20. Method according to claim 5 wherein said human EPO comprises .alpha.-2,3- and .alpha.-2,6-linked sialic acid residues.

21. Method according to claim 5 wherein said human EPO comprises a polypeptide with a length of 165 amino acids.

22. Method according to claim 5 wherein said human EPO comprises a polypeptide with a length of 166 amino acids.

23. Method according to claim 5 wherein said human EPO comprises a mixture of polypeptides with a length of 165 and 166 amino acids.

24. The DNA construct of claim 1 further comprising an amplification gene.

25. The DNA construct of claim 3 further comprising an amplification gene.

26. The method of claim 10 further comprising, after step (a), passing the fractions containing EPO over a hydrophobic interaction chromatography medium and harvesting the fractions containing EPO.

Summary for Patent:   Start Trial

Foriegn Application Priority Data
Foreign Country Foreign Patent Number Foreign Patent Date
97112640Jul 23, 1997
Germany197 53 681Dec 3, 1997

Details for Patent 7,186,529

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04   Start Trial Roche Diagnostics GmbH (Basel, CH) 2017-07-23 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04   Start Trial Roche Diagnostics GmbH (Basel, CH) 2017-07-23 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04   Start Trial Roche Diagnostics GmbH (Basel, CH) 2017-07-23 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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