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Long-acting anticoagulant polypeptide including immunoglobulin Fc segments as carriers and preparation method of same

An immunoglobulin, long-acting technology, used in carrier binding/immobilization of peptides, medical preparations containing active ingredients, peptide/protein components, etc., can solve problems such as loss of activity, improve usability, and prolong clinical administration. Interval time, the effect of increasing plasma half-life

Active Publication Date: 2012-01-11
BEIJING HANMI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for hirudin, the modification of the N-terminal amino acid can make it inactive

Method used

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  • Long-acting anticoagulant polypeptide including immunoglobulin Fc segments as carriers and preparation method of same
  • Long-acting anticoagulant polypeptide including immunoglobulin Fc segments as carriers and preparation method of same
  • Long-acting anticoagulant polypeptide including immunoglobulin Fc segments as carriers and preparation method of same

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preparation example Construction

[0038] According to another aspect of the present invention, the present invention provides a method for preparing the long-acting anticoagulant polypeptide, which includes the following steps:

[0039] a1) covalently linking an immunoglobulin Fc fragment or an anticoagulant polypeptide to one end of the activated PEG;

[0040] a2) isolating from the resulting reaction mixture a complex comprising an immunoglobulin Fc fragment or an anticoagulant polypeptide covalently linked to said PEG; and

[0041] a3) covalently linking the immunoglobulin Fc fragment or the anticoagulant polypeptide to the other end of the PEG in the separation complex to obtain a long-acting anticoagulant polypeptide containing the immunoglobulin Fc fragment and the anticoagulant polypeptide linked to each end of the PEG .

[0042] The preparation method may further comprise the following steps:

[0043] b) separating and purifying the long-acting anticoagulant polypeptide from the reaction solution.

...

Embodiment 1

[0072] Example 1 Preparation of bifunctional group polyethylene glycol (propionaldehyde-3.4KPEG-propionaldehyde, ALD-PEG-ALD) single modified hirudin HV2 (Lys47)

[0073] 1) PEGylation reaction and product separation:

[0074] ① Preparation of N-terminally modified PEGylated (N-PEGylated) hirudin

[0075] The hirudin used in the reaction is the variant 2 (HV2(Lys47)) in which the 47th asparagine is mutated into lysine, and the amino acid sequence is ITYTDCTESG QNLCLCEGSN VCGKGNKCILGSNGKGNQCV TGEGTPKPES WGDFEEIPEE YLT, which is expressed by recombinant Pichia pastoris. The purity was analyzed by high performance liquid chromatography, and the result was greater than 95%. Reaction system is: 16mg hirudin, concentration is 5mg / mL; Solvent is 20mM phosphate buffer saline (pH6.0); Add polyethylene glycol with the molar ratio of hirudin:polyethylene glycol=1:10; And add Sodium cyanoborohydride (SCB) to a final concentration of 20 mM; react at 4°C for 1 hour.

[0076] AKTAexplorer...

Embodiment 2

[0084] Embodiment 2 Preparation of Hirudin-PEG-Fc

[0085] Take PEG-hirudin with a hirudin content of 5.09 mg and 552 mg of immunoglobulin Fc fragment HMC001 (preparation method as shown in Chinese Patent 200480001775) (molar ratio is 1:12), and the buffer is 100 mM K-P containing 20 mM SCB ( pH 6.0), react at 4°C for 16 hours. The replacement buffer component was buffer 20 mM Tris (pH 7.5). First, a Source Q column (16mm I.D., 105mL, GE Healthcare) was used for preliminary purification, that is, the column was equilibrated with 20mM Tris (pH 7.5), and the sample was loaded and washed, and then eluted with a salt gradient. And mix the active ingredients. Then, use Source ISO chromatographic column (16mm I.D., 21mL, GE Healthcare) to further purify, that is, buffer 20mM Tris, pH7.5, 1.6M (NH 4 ) 2 SO 4 Equilibrate the chromatographic column, load the sample and buffer with 20mM Tris, pH7.5, 1.6M (NH 4 ) 2 SO 4 After washing, respectively, with 20mM Tris, pH7.5, 1.44M an...

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Abstract

The invention provides a long-acting anticoagulant polypeptide including immunoglobulin Fc segments as carriers and a preparation method of the same. The long-acting anticoagulant polypeptide is formed by covalently conjugating anticoagulant polypeptide molecules and the immunoglobulin Fc segments through polyethylene glycol. The invention also provides the application of the long-acting anticoagulant polypeptide in the preparation of drugs for treating and / or preventing thrombotic diseases, and a pharmaceutical composition including the long-acting anticoagulant polypeptide. When the long-acting anticoagulant polypeptide provided by the invention is adopted, the plasma half-life of the drugs is greatly prolonged, clinical dosing intervals are extended, and the applicability of anticoagulant drugs is improved on the basis that the original anticoagulant activity of the anticoagulant polypeptide is substantially kept unchanged.

Description

technical field [0001] The invention relates to a carrier-bound long-acting anticoagulant polypeptide and a preparation method thereof, in particular to a long-acting anticoagulant polypeptide containing an immunoglobulin Fc fragment as a carrier and a preparation method thereof (C07K17 / 00). Background technique [0002] Hirudin is a single chain cyclic peptide compound. The peptide chain consists of 65 amino acid residues with a relative molecular weight of 7000 Daltons. There are three disulfide bridges (-S-S-) at the N-terminal of the molecule, and the cysteine ​​residues at positions 6, 14, 16, 28, 22, and 39 (Cys6...Cys14, Cys16. ..Cys28, Cys22...Cys39), making the N-terminal peptide chain wrap around to form a dense core cyclic peptide structure. The C-terminus is rich in acidic amino acid residues, and the tyrosine at position 63 is sulfated. There are more than ten variants of hirudin, and the main three variants are referred to as HV1, HV2 and HV3 for short. Its...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K45/00A61K38/58C07K17/00A61P7/02
Inventor 薛剑锋杜伯雨张磊周小平杨依丽刘家望连忠辉李炳善文圣焕
Owner BEIJING HANMI PHARMA CO LTD
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