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PEGylated erythropoietin protein long-acting preparation

A technology of erythropoietin and PEGylation, which is applied in the fields of erythropoietin, extracellular fluid diseases, peptide/protein components, etc., can solve the problem of affecting the practical application value and significance of patents, affecting the secondary structure, biological activity and Product composition and other issues, to achieve the effects of prolonging plasma half-life, expanding clinical indications, and increasing activity in vivo

Inactive Publication Date: 2009-03-04
TIANJIN PAIGE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, different coupling reaction conditions, such as pH value and molar concentration ratio of PEG to protein, may also affect the secondary structure, biological activity and product composition of rhngEPO
Therefore, in the past, there were many patents (including US2007 / 0100133 A1) related to PEG-coupled recombinant proteins including rhEPO or rhngEPO. Because the scope of claims was too broad, the properties of PEG-coupled proteins were very different or even completely opposite, which greatly Affected the practical application value and significance of these patents

Method used

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  • PEGylated erythropoietin protein long-acting preparation
  • PEGylated erythropoietin protein long-acting preparation
  • PEGylated erythropoietin protein long-acting preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] Example 1 Expression and preparation of rhngEPO and rhngEPOm

[0164] Construct the prokaryotic expression system pET15b-rhngEPO to express rhngEPO efficiently:

[0165] 1. Strains and Plasmids

[0166] Escherichia coli DH5α (E.coli DH5α LacZΔM15 hsdR recA)

[0167] Escherichia coli BL21(DE3) (E.coli BL21(DE3))

[0168] Escherichia coli BL21(DE3) / pLysS(E.coli BL21(DE3) / pLys)

[0169] Plasmid pET15b

[0170] The above strains and plasmids were purchased from Novagen.

[0171] 2. Enzymes and reagents for molecular cloning

[0172] Restriction enzymes Nco I, Xho I; Pfu DNA polymerase were purchased from Shanghai Boya Bioreagent Company.

[0173] Agarose Gel DNA Purification Kit, T4 Ligase, DNA Fragment Purification Kit were purchased from OMEGA Ltd.

[0174] Nucleic acid molecular weight standard 1Kb Marker was purchased from BioLab.

[0175] Protein molecular weight standards (14.4-97.0kDa) were purchased from Shanghai Institute of Biochemistry.

[0176] 3. Metho...

Embodiment 2

[0244] Example 2 Preparation of single-site fixed PEG-rhngEPO and PEG-rhngEPOm

[0245] The above rhngEPO stock solution was concentrated by ultrafiltration (Satorius, Vivaflow / 200 tangential flow ultrafilter membrane bag), so that the protein concentration reached 2 mg / ml.

[0246] 750 mg of PEG propionaldehyde (PEG-ALD) with a molecular weight of 30 kDa (purchased from NOF Corporation, SUNBRIGHTME-300AL) was dissolved in 30 ml of 2 mg / ml rhngEPO stock solution (20 mM sodium acetate buffer, pH 6.0, 150 mM sodium chloride ), then add sodium borocyanide (NaCNBH 3 ) 1.4ml, the reaction solution was stirred at 4°C for 1 hour, then allowed to stand for 24 hours, and 300ml of 1mM dilute hydrochloric acid solution (pH3.5) was added. Use buffer, namely 20mM sodium acetate buffer (pH6.0), 150mM sodium chloride and 0.005% polysorbate 80 solution, equilibrate the cation exchange chromatography column (AKTA purifier, chromatography live INdEX 200 / 500, chromatography Medium Sepharose Fa...

Embodiment 3

[0247] Example 3 Physicochemical properties and in vitro activity of PEG-rhngEPO

[0248] 1) Solubility of PE-N-30

[0249] Experiments have shown that the protein concentration of PE-N-30 and rhEPO in 20mM sodium acetate buffer (pH6.0), 150mM sodium chloride and 0.005% polysorbate 80 solution can reach 2mg / ml, and at 4 ℃ Maintained for 3 months, determined by SEC: PE-N-30 did not find protein aggregation or degradation under this condition (such as Figure 10-A shown), a small amount of protein aggregation occurs in rhEPO under this condition, which is a soluble aggregate (such as Figure 10-B shown); and under the same buffer conditions as above, when the concentration of rhngEPO protein was 0.5mg / ml, put it at 4°C for 2 weeks, and it was found that protein aggregation was determined by SEC (such as Figure 10-C shown), indicating that PEGylated rhngEPO significantly increased the solubility or stability of the protein in water.

[0250] 2) In vitro thermal stability of P...

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Abstract

The invention discloses a type of polyethylene glycol erythropoietin, which is prepared from non-glycosylated rhEPO(rhngEPO) or point mutant thereof, and N-terminal amino of single and single chain PEG molecule having molecular weight greater than 20kDa or mercapto of cysteine mutational state by fixed point coupling, wherein the olyethylene glycol erythropoietin has the molecular structural formula of CH3-(CH2CH2-O)n-(CH2)r-NH-rhngEPO or CH3-(CH2CH2-O)n-X-S-rhngEPOm, wherein n is 100 to 2,200, r is 0 to 4, and X is S or C. The PEG-rhngEPO or PEG-rhEPOm molecule simultaneously has long-acting preparation characteristics such as long acting, high efficiency and low immunogenicity, and establishes foundation for preparing parent long-acting preparation for treating anemia caused by chronic renal insufficiency, tumor, anemia caused by AIDS chemotherapy, marrow brain nerve damage, and selective operation auto-transfusion.

Description

technical field [0001] The present invention relates to a class of pegylated (PEG) erythropoietin, in particular to a class of pegylated (PEG) erythropoietin conjugated compound molecules with pharmaceutically long-acting, high-efficiency, and low immunogenicity characteristics. It consists of a single, specific PEG molecule coupled to non-glycosylated recombinant erythropoietin protein or its point mutants. Compared with recombinant human erythropoietin, the compound molecule of the present invention has completely consistent or even better physicochemical and biological properties in vitro, such as solubility, thermal stability, anti-enzymolysis stability and specific activity, and has Pharmacological features such as prolonged half-life, increased activity and reduced immunogenicity. The present invention also relates to the production and application of such compound molecules, and as components of pharmaceutical preparations. Background technique [0002] Human Erythr...

Claims

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Application Information

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IPC IPC(8): C07K14/505A61K38/18A61P7/06
Inventor 张旋苏志国祁庆生
Owner TIANJIN PAIGE BIOTECHNOLOGY CO LTD
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