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3-hydroxybutyrylation modified protein medicine and preparation method and application thereof

A protein and drug technology, applied in the field of medicine, can solve the problems of protein-restricted medicine and commercial applications, antibody and receptor loss of antibody-targeted cells, Fc fragment fragmentation, etc.

Active Publication Date: 2021-02-09
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cleavage of the hinge region will lead to the fragmentation of the Fc fragment, so that the antibody cannot bind to the receptor and lose the function of the antibody to target cells and kill bacteria (Sun, H., et al., Plasminogens are a critical host pathogenicity factor for group A streptococcal infection. Science, 2004. 305(5688): p. 1283-6.)
Therefore, protein instability largely limits its medical and commercial applications

Method used

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  • 3-hydroxybutyrylation modified protein medicine and preparation method and application thereof
  • 3-hydroxybutyrylation modified protein medicine and preparation method and application thereof
  • 3-hydroxybutyrylation modified protein medicine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Recombinant expression of antibody (antibody preparation refers to "Wu, Y., et al., A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science, 2020. 368(6496): p. 1274-1278." Antibody B38 Preparation)

[0083] 1.1 Plasmid extraction

[0084] (1) Take 400mL of E. coli cultured overnight, centrifuge at 9000r / min for 10min, drain the supernatant as much as possible, and collect the bacteria;

[0085] (2) Resuspend the bacterial pellet with 25mL solution P1, and vortex until completely suspended;

[0086] (3) Add 25mL of solution P2, gently turn it up and down 4-7 times to fully lyse the bacteria, and place it at room temperature for 4 minutes;

[0087] (4) Add 25mL of solution P3, turn it up and down gently 4-7 times immediately, and mix well, at this time, a white flocculent precipitate will appear; let stand on ice for 3-5min, centrifuge at 13000r / min for 10min, and carefully take the supernatant to...

Embodiment 2

[0114] Example 2: Molecular weight detection after 3-hydroxybutyric acid modification of antibodies

[0115] Adjust the concentration of the antibody obtained in Example 1 to 1 mg / mL, add 1M 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1M 3-hydroxybutyric acid to a final concentration of 10 mM, React at room temperature for 2 hours, and then centrifuge 4 times with a 10kd ultrafiltration column to wash away unreacted compounds and perform mass spectrometry analysis.

[0116]The desalted antibody was made into a 0.2 mg / mL solution with 0.1% formic acid solution for flow injection analysis. Antibody protein samples for LC-MS analysis were directly prepared with ultrapure water, and 2 μL protein samples were injected directly with ultrapure water, and a micro syringe was used to draw protein samples, and the nanoACQUITY UPLC system was used to pass through the nanoACQUITY UPLC system at a flow rate of 1 μL / min for 30 minutes. Gradient elution separates the anal...

Embodiment 3

[0117] Example 3: Detection of modified sites after 3-hydroxybutyric acid modification of antibodies

[0118] The antibody obtained in Example 2 was detected by LC-MS / MS. Proteins were separated by SDS-PAGE, the gel band of interest was excised from the gel, reduced with 5 mM dithiothreitol, and alkylated with 11 mM iodoacetamide. Then, in-gel digestion was performed overnight at 37°C with sequencing-grade modified trypsin in 50 mM ammonium bicarbonate. Peptides were extracted twice with 0.1% trifluoroacetic acid in 50% acetonitrile in water for 30 minutes each. Tryptic peptides were redissolved in 20 μL 0.1% TFA and analyzed by LC-MS / MS. For LC-MS / MS analysis, peptides were separated by 85 min gradient elution at a flow rate of 0.30 μL / min using a Thermo-Dionex Ultimate 3000 HPLC system directly coupled to a Thermo Scientific QExactive mass spectrometer. The analytical column is a self-made fused silica capillary column (inner diameter 75 µm, length 150 mm, Upchurch) fille...

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Abstract

The invention relates to the technical field of medicine, discloses a 3-hydroxybutyrylation modified protein medicine (such as an antibody) and a preparation method and application thereof, and particularly relates to a 3-hydroxybutyrylation modified antibody and a preparation method and application thereof. By a large number of experiments, the inventor finds that after 3-hydroxybutyrate and an analogue thereof modify the protein medicine (such as the antibody), the thermal stability and protease hydrolysis resistance of the protein medicine can be remarkably improved, the isoelectric point of the protein medicine is reduced, and the half-life period of the protein medicine in the body of a subject is remarkably prolonged, so that the medicine effect of the protein medicine is further improved. The protein medicine obtained after modification has wide application prospect and high commercial value in the scientific research and clinical aspects.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a 3-hydroxybutyrylation-modified protein drug (such as an antibody) and a preparation method and application thereof. Background technique [0002] With the development of genetic recombination technology, the commercial production of protein drugs has become a reality, such as monoclonal antibodies and genetically engineered antibodies, recombinant vaccines, and so on. Compared with previous small-molecule drugs, protein drugs often have the characteristics of high activity, strong specificity, low toxicity, clear biological function, and favorable clinical application. They have become an important part of pharmaceutical products. [0003] Protein drug molecules are large, usually with a molecular weight of thousands or tens of thousands or even hundreds of thousands of daltons. In addition, proteins often have multiple functional groups and can undergo multiple reactions in t...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K1/107A61K39/44
CPCA61K2039/505C07K1/1075C07K16/10C07K2317/94
Inventor 陈国强李子华张雨点吴赴清
Owner TSINGHUA UNIV
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