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Novel carbamylated EPO and method for its production

A technology for carbamylation and carbamylation of promoting erythrocytes, which is applied in the field of preparing the compound and can solve the problems of unsuitability of biopharmaceutical products and the like

Inactive Publication Date: 2007-07-11
H LUNDBECK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

and thus the presence of aggregates renders biopharmaceutical products unsuitable for use in humans

Method used

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  • Novel carbamylated EPO and method for its production

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0117] Preparation of carbamylated erythropoietin

[0118] In this example, the starting material of the method is purified recombinant human EPO.

[0119] First, the protein concentration is adjusted by ultrafiltration with the aim of keeping the processing volume low. The protein concentration was adjusted to 3 mg / ml. Ultrafiltration was performed by BioMax (Millipore) with a MWCO of 5 kDa.

[0120] After completing the concentration step, the EPO solution was mixed with a solution of 0.5M K-borate tetrahydrate 0.5M K-cyanate at pH 9.0. The solution was incubated at 32°C for 24 hours.

[0121] The reaction mixture of EPO and cyanate was desalted by gel filtration. Proteins were desalted into 25 mM Tris, 50 mM NaCl, pH 8.5 buffer. G-25 Fine (Amersham Biosciences) resin was used.

[0122] Using a flow rate of 90 cm / h on a column approximately 15 cm high, a sample loading of approximately 20% of the column volume was applied.

[0123] The desalted carbamylated EPO was co...

Embodiment 2

[0149] Total mass analysis

[0150] Using the method of Example 1, three carbamylated EPO samples were analyzed. After the carbamylation reaction, all 3 samples were purified using anion exchange as described in Example 1. One CEPO sample (named CEPO-CMC) was produced by CMC Biotech at 1 g scale (concentration 0.82 mg / ml; buffer: 20 mM sodium citrate, 0.3 mM citric acid, 0.1 M NaCl, pH 6.9-7.3) preparation. The remaining two samples, named CEPO-1 and CEPO-2, were prepared at 70 mg laboratory scale (concentration: 1.1 mg / ml; buffer: 25 mM Tris, 0.2M NaCl, pH 8.3-8.7). These CEPO samples were mixed with unmodified or initial EPO (concentration 0.82mg / ml; buffer: 2mM sodium citrate, 0.3mM citric acid, 0.1M NaCl, pH6.9-7.3) and simulated CEPO (after Add K-cyanate carbamylation process EPO) (concentration: 0.38mg / ml; buffer: 20mM sodium citrate, 0.3mM citric acid, 0.1M NaCI, pH6.9-7.3) for comparison .

[0151] ESI-mass spectrometry

[0152] Samples were enzymatically deglyc...

Embodiment 3

[0166] LysC peptide map

[0167] Peptide mapping of EPO and CEPO samples was performed using the endoprotease LysC and trypsin cleavage. All peptide mapping analyzes were performed on deglycosylated peptides. Enzymatic deglycosylation of the peptide is accomplished with simultaneous endoprotease digestion.

[0168] EPO and CEPO samples (about 150 μg each) were denatured and reduced by incubation with guanidine hydrochloride and DTT. Alkylation of free thiols with iodoacetic acid. Using a single gel filtration column, the alkylated samples were desalted and buffer exchanged to the appropriate buffer.

[0169] Endoprotease, N-glycosidase and neuraminidase were added simultaneously to the alkylated EPO and CEPO samples. Samples were incubated overnight at 37°C. After incubation, about 5 μg of each digest was used for RP-HPLC / MS analysis, using Jupiter, C18 RP-column of Phenomenix, combined with ESI-LCT of Waters. The UV signal at 22OnM and the total ion count (TIC) on the m...

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Abstract

The present invention discloses a method for production of novel carbamylated erythropoietin and compositions comprising the novel carbamylated erythropoietin and pharmaceutical compositions comprising this and uses thereof.

Description

[0001] introduction [0002] The present invention relates to a novel compound, and a process for preparing said compound. The novel compound, carbamylated erythropoietin (CEPO), is characterized by carbamylation on all or most of the primary amines of lysine and on the N-terminal amino acid of the molecule, and in addition, Primary amines of other amino acids in the compound molecule have low levels of carbamylation. In addition, the novel compound has no aggregated proteins and polymers, and is suitable for use in pharmaceutical compositions for disease treatment, such as the central and peripheral nervous systems, and other tissues expressing important EPO receptors. Another very surprising advantage of the present preparation method is that it provides a product that contains less aggregated protein than that obtained from other known methods of carbamylation of erythropoietin described and less polymer. Background of the invention [0003] Attenuation of the biohaemato...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/505A61K38/18
Inventor S·克里斯坦森L·福尔达杰J·瓦尔布乔恩M·H·图森A·H·佩德森M·芒克
Owner H LUNDBECK AS
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