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New application of multi-arm polyethylene glycol modifier and its application in modification of asparaginase

A technology of asparaginase and polyethylene glycol succinimidyl acetate, which is applied in the application field of modifying asparaginase, can solve the problem of not involving the depolymerization of multi-subunit protein subunits, the presence of immunogens It can achieve the effect of low immunogenicity, maintaining biological activity and prolonging half-life.

Active Publication Date: 2019-04-05
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the unmodified original protein, the immunogenicity of PEG-modified L-asparaginase in all these patents and articles has been reduced, but there are still immunogenic problems
Also, none of these patents and articles deal with solving the problem of subunit depolymerization of multi-subunit proteins

Method used

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  • New application of multi-arm polyethylene glycol modifier and its application in modification of asparaginase
  • New application of multi-arm polyethylene glycol modifier and its application in modification of asparaginase
  • New application of multi-arm polyethylene glycol modifier and its application in modification of asparaginase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Preparation and analysis of asparaginase PEG conjugate

preparation example 1

[0093] Preparation Example 1. The PEGylated asparaginase of the present invention was prepared, purified and identified by the following methods:

[0094] 1. PEG conjugate sample preparation

[0095] Use 50mM pH5.0 acetic acid-sodium acetate buffer (purchased from Sinopharm Group) to dissolve asparaginase (purchased from Qianhong Pharmaceutical, with the sequence of SEQ ID NO:1) to prepare an 8mg / mL solution, respectively 4PALD5K, 4PALD10K (purchased from Beijing Jiankai Technology Co., Ltd.) are modified as polyethylene glycol modifier, asparaginase: polyethylene glycol modifier: reducing agent (sodium cyanoborohydride, purchased from sigma) The reaction was carried out at a molar ratio of 1:50:2500. After 12 hours of reaction at 4°C, 1M glycine was added to terminate the reaction.

[0096] In addition, asparaginase was dissolved in 50mM pH7.5 PB buffer (prepared with disodium hydrogen phosphate and sodium dihydrogen phosphate, the reagents were purchased from Sinopharm Group) to p...

Embodiment 2

[0112] Example 2: Preparation and analysis of PEG conjugate of alkaline phosphatase and urease

[0113] Alkaline phosphatase (AKP) is widely distributed in various organs of the human body. Alkaline phosphatase is an enzyme that can dephosphorylate the corresponding substrate by hydrolyzing the phosphate monoester to remove the phosphate group on the substrate molecule. Groups are removed and generate phosphate ions and free hydroxyl groups. Such substrates include nucleic acids, proteins, alkaloids, etc. AKP is a homodimeric protein with a molecular weight of 28KDa per subunit.

[0114] The Hp urease monomer is composed of two subunits A and B, which are hexamers. The molecular weights of the two sub-monomers A and B are about 30KDa and 64KDa, respectively, and the ratio is 1:1, which is used in this example The urease is unable to form a 6-mer, but a monomeric protein formed by two subunits A and B.

[0115] Dissolve alkaline phosphatase (purchased from SIGMA) and urease (purchas...

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Abstract

The invention discloses a new application of a multi-arm polyethylene glycol modifier and its application in modifying asparaginase. The multi-arm PEG modifier provided by the present invention can be used to strengthen the inter-subunit interaction force of multi-subunit proteins, keep multi-subunit proteins in the form of aggregation, thereby increasing the stability of multi-subunit proteins, enhancing or maintaining multi-subunit proteins biological activity, while reducing the chance of exposure of antigenic sites after depolymerization of each subunit, reducing immunogenicity. In addition, compared with the commercially available PEGylated asparaginase product "pegaspargase", the PEG-modified asparaginase prepared in the present invention has low immunogenicity, high activity and high stability, and PEG does not It has the advantages of falling off and the like, and is more suitable for the preparation of antitumor drugs.

Description

Technical field [0001] The invention relates to a new use of a multi-arm polyethylene glycol modifier and its application in modifying asparaginase. Background technique [0002] Polyethylene glycol (PEG) is a linear, uncharged polymer that can curl freely in solution. It has non-toxic, weak antigenicity and good biocompatibility. Using it to covalently modify proteins can increase the in vivo circulating half-life of the protein and reduce its antigenicity, increase the solubility of the protein and change the biological distribution of the protein in the human body. Since Abuchowski, Davis (J.Biol.Chem.1977,252:3578-3581.) et al. first reported the modification of proteins with PEG in 1977, PEG modification technology has been widely used in the fields of biomedicine and biotechnology. PEG has It is widely used in the modification of protein and peptide drugs. At present, protein PEGylation technology has become one of the most effective methods to reduce the immunogenicity o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N9/82C12N9/06A61K38/50A61K47/60A61P35/00A61P35/02
CPCA61K47/60A61K38/50C12N9/16C12N9/80C12N9/82C12N9/96C12Y301/03001C12Y305/01001C12Y305/01005A61K9/0019A61K9/19A61K38/44C12N9/0016C12Y104/01002A61P35/00A61P35/02
Inventor 马永王俊吴鼎龙邱晶徐春林陈晨王耀方
Owner ZONHON BIOPHARMA INST
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