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Hasylated polypeptides, especially hasylated erythropoietin

An erythropoietin and polypeptide technology, applied in the field of polypeptides, can solve the problems of lack of glycosylation, difficulty in producing polypeptides, polypeptides that do not have correct folding and natural conformation, etc., and achieve the effects of high biological activity and improved biological stability.

Inactive Publication Date: 2009-11-18
FRESENIUS KABI DEUT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, producing peptides in bacterial or insect cells is often difficult, as peptides are often produced without proper folding and native conformation, and lack proper glycosylation

Method used

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  • Hasylated polypeptides, especially hasylated erythropoietin
  • Hasylated polypeptides, especially hasylated erythropoietin
  • Hasylated polypeptides, especially hasylated erythropoietin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0514] Production of recombinant EPO

[0515] A) Production in mammalian cells

[0516] Recombinant EPO was produced in CHO cells as follows

[0517] A plasmid carrying human EPO cDNA was cloned into a eukaryotic expression vector (pCR3, hereinafter referred to as pCREPO). Site-directed mutagenesis was performed using standard methods as described (Grabenhorst, Nimtz, Costa et al., 1998, Invivospecificity of human alpha1, 3 / 4-fucosyltransferases III-VII in the biosynthesis of Lewis(x) and sialyl Lewis(x) motifs on complex- type N-glycans-Coexpression studies from BHK-21cell stogether with humanbeta-trace protein, J. Biol. Chem., 273(47), 30985-30994).

[0518] CHO cells stably expressing human EPO or its amino acid variants (e.g., Cys-29→Ser / Ala, or Cys-33→Ser / Ala, Ser-126→Ala, etc.) were generated by calcium phosphate precipitation as described (Grabenhorst et al. Human) was screened with sulfate G 418. Three days after transfection, the cells were subcultured 1:5 and s...

Embodiment 2

[0530] Formation of reactive HES derivatives

[0531] 1. SH-reactive HES

[0532] 1.1 Reaction of EMCH with oxo-HES12KD to form SH-reactive HES 12KD B

[0533]

[0534] 0.144 g (0.012 mmol) of oxo-HES12KD (Fresenius German Patent DE 196 28 705A1) was dissolved in 0.3 mL of anhydrous dimethylsulfoxide (DMSO), and added dropwise to 34 mg (0.15 mmol) of EMCH (Perbio Science , Deutschland GmbH, Bonn, Germany) in a mixture in 1.5 mL of DMSO. After stirring at 60 for 19 hours, the reaction mixture was added to 16 mL of a 1:1 mixture of ethanol and acetone. The precipitate was collected by centrifugation, redissolved in 3 mL DMSO, and reprecipitated as above. SH-reactive HES 12KDB was obtained by centrifugation and vacuum drying. The coupling reaction with thiol-EPO is described in Example 3, 2.2.

[0535] Options:

[0536] All crosslinkers with hydrazide and maleimide functionalities separated by a spacer can be used in this reaction. Table 2 lists some further examples ...

Embodiment 3

[0600] Coupling reaction with thiol-EPO

[0601] 1. Reaction of mercapto-EPO with haloacetamide-modified SH-reactive HES

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Abstract

The present invention relates to hydroxyalkylstarch (HAS)-polypeptide-conjugate (HAS-polypeptide) comprising one or more HAS moecules, wherein each HAS is conjugated to the polypeptide via a carbohydrate moiety or a thioether as well as to methods for the production thereof. In a preferred embodiment, the polypeptide is erythropoietin (EPO).

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 8, 2003, an application number of 03821464.4, and an invention title of "HASylated polypeptide, especially HASylated erythropoietin". technical field [0002] The present invention relates to polypeptides, especially erythropoietin, conjugated to hydroxyalkyl starch (HSS), especially to hydroxyethyl starch. Background technique [0003] The application of polypeptides, especially enzymes or cytokines, to the circulatory system in order to obtain a specific physiological effect is a well-known method in modern medicine. [0004] Erythropoietin (EPO) is a glycoprotein hormone necessary for the maturation of erythroid progenitor cells into red blood cells. Adults produce EPO in the kidneys. EPO is required to regulate the level of red blood cells in the circulation. Diseases marked by low tissue oxygen levels cause increased EPO biosynthesis, which subsequ...

Claims

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Application Information

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IPC IPC(8): C07K17/10A61K47/48A61K38/18A61P7/00A61P7/06A61K8/73A61K9/00A61K9/14A61K38/00C07K14/505C07K14/53C07K14/54C07K14/55C07K14/56C07K14/565C07K17/06C08B31/00C08B31/08C08B31/12C08B33/04C08B35/04C12N15/12
CPCC08B31/12C07K14/505A61K9/0021A61K47/4823C08B31/00C07K14/56C07K14/5412C07K17/10C07K14/565C08H1/00C07K14/53C07K14/55C08B31/006C08B31/08C08B31/185A61K38/00A61K47/61A61P7/00A61P7/06A61P43/00
Inventor 阿拉尔德·S·康拉德特埃卡尔特·格拉本赫斯特曼弗雷德·尼姆兹诺贝特·扎恩特尔罗纳德·弗兰克沃尔夫拉姆·艾希纳
Owner FRESENIUS KABI DEUT GMBH
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