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Reorganizational hemopoietin and preparation method thereof

A technology of erythropoietin and synthetase, which is applied in the field of recombinant erythropoietin and its preparation, and can solve problems such as poor selectivity, incomplete reaction, and harsh reaction conditions

Inactive Publication Date: 2013-04-17
SUZHOU YUANJI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] So far, various types of polyethylene glycol chemical modification methods of recombinant erythropoietin have been disclosed. For example, WO94 / 28024 discloses a sugar-modified PEG conjugate with erythropoietin activity, wherein PEG is combined with The oxidized sugar chains of rEPO are connected. In the PEG chemical modification method, A) if only pure chemical methods are used to conjugate to PEG (for example, CERA of Roche Pharmaceuticals is PEG chemically modification), such methods usually have poor selectivity (cannot be oriented and / or specifically modified), and poor reaction conditions; The steps of glycosylation and glycosyl oxidation are involved before covalent modification. This method also has the defects of poor selectivity and incomplete reaction.

Method used

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  • Reorganizational hemopoietin and preparation method thereof
  • Reorganizational hemopoietin and preparation method thereof
  • Reorganizational hemopoietin and preparation method thereof

Examples

Experimental program
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Embodiment 1

[0040] Example 1: Recombinant erythropoietin with 4-acetylphenylalanyl residue at position 65 and / or position 152 chemically modified by polyethylene glycol

[0041] (1) Obtaining recombinant erythropoietin with 4-acetylphenylalanyl residues at position 65 and / or position 152

[0042] (1a) Construction of recombinant expression vector

[0043] a way:

[0044] see figure 1 , at first, construct the expression vector (pKeto) of 4-acetylphenylalanyl-tRNA synthetase gene in vitro, that is: amplify and synthesize the gene that contains coding 4-acetylphenylalanyl-tRNA synthetase (this gene sequence sees The DNA fragment of SEQ ID No.2), the obtained DNA fragment is introduced into a suitable vector by using a specific restriction site to obtain an expression vector containing a gene encoding 4-acetylphenylalanyl-tRNA synthetase. KetoRS (gene sequence is image 3 The DNA fragment of the shown SEQ ID No.2) was separated by agarose electrophoresis, and the gel was recovered to obt...

Embodiment 2

[0075] Example 2: Chemical modification of recombinant erythropoietin with 2-amino-8-oxononanoyl residue at position 65 and / or position 152 with polyethylene glycol

[0076] (1) Obtaining recombinant erythropoietin with 2-amino-8-oxononanoyl residues at position 65 and / or position 152

[0077] see image 3 , first, the double helix polynucleotide U6-pylT-BamHI-BglII (sequence is SEQ ID No.17) (ordered from Epoch Biolabs) is digested with endonucleases BamHI and BglII, and then inserted into the plasmid pcDNA3.1 / BglII site of hygro(+), the recombinant plasmid pcDNA3.1-pylT was obtained, and the sequence of PylT was SEQ ID No.5;

[0078] Then, with KetoKRS-XbaI-F (sequence is SEQ ID No.18), KetoKRS-ApaI-R (sequence is SEQ ID No.19) as primer pair 2-amino-8-oxononanoyl-tRNA synthetase ( KetoKRS) (sequence is SEQ ID No.6) to amplify, then digest with endonuclease XbaI, ApaI;

[0079] Then, insert the product digested with endonuclease XbaI and ApaI into the recombinant plasmid...

Embodiment 3

[0085] Embodiment three: the pharmacokinetic research and hematocrit determination of PEGylated EPO (AcF65), EPO (AcF152), EPO (KetoK65) and EPO (KetoK152)

[0086] PEGylated EPO(AcF65), EPO(AcF152), EPO(KetoK65) and EPO( KetoK152) samples with the same concentration. These PEGylated EPO compounds were used to compare pharmacokinetic profiles with native, unmodified, naturally functional EPO [from Amgen, Inc., Thousand Oaks, CA] as a benchmark since it is approved by the FDA. Approved product with long half-life due to protein hyperglycosylation. For the detection of proteins in blood, the chloramine T method, known in the art, was used with 125 I labeled five samples at tyrosine positions. Each molecule has about 1-2 125 I connect. Each sample (80ug) was labeled and desalted from residual unbound 125 I separated, detected the activity of the protein, and verified it with polyacrylamide gel electrophoresis and reversed-phase column. The following subpopulations were ass...

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Abstract

The invention relates to reorganizational hemopoietin with chemical modification and a preparation method thereof. The method adopts genetic engineering technology to reorganize hemopoietin, and introduces 4-acetophenone alanyl residue with chemical active ketone group into the hemopoietin; polyethylene glycol specifically reacts with the 4-acetophenone alanyl residue when polyethylene glycol carries out covalent modification on the reorganizational hemopoietin, the reaction is complete, reaction with other amino acid residues is avoided, and the required reaction condition is mild, therefore, the method solves the problems of bad selectivity and adverse reaction condition in the prior art. The pharmacokinetic study result and the haematocrit determination result of the reorganizational hemopoietin pegylation of pegylation are both superior to the natural erythropoietin.

Description

technical field [0001] The invention relates to recombinant erythropoietin and its preparation method, in particular to chemically modified recombinant erythropoietin and its preparation method. Background technique [0002] Erythropoietin (Erythropoietin), also known as erythropoietin, English abbreviation EPO, is a glycoprotein hormone, and its main physiological function is to stimulate the differentiation and proliferation of bone marrow red blood cell precursor cells, so EPO is considered to promote the production of red blood cells. It is also the main regulator that can regulate the total amount of red blood cells in the body at the optimum level. [0003] Natural human erythropoietin (hEPO) is a glycoprotein hormone mainly synthesized and secreted by the kidney. When the kidneys fail, EPO cannot be synthesized normally, which leads to a decrease in the production of red blood cells, which causes anemia. Anemia is one of the common symptoms of chronic kidney disease...

Claims

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Application Information

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IPC IPC(8): C07K14/505C07K1/107C12N15/85A61K38/18A61K47/48A61P7/06
Inventor 万为
Owner SUZHOU YUANJI BIOLOGICAL TECH
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