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Brush polymer modified graphene oxide-immobilized capture receptor compound and application thereof in enrichment of monoclonal antibodies

A polymer and graphene technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of small carrier specific surface area, limited application, large steric hindrance, etc., to save experimental costs, improve detection sensitivity, and reduce space. The effect of steric hindrance

Inactive Publication Date: 2021-06-25
BEIJING SHIJITAN HOSPITAL CAPITAL MEDICAL UNIVERSTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These carriers have small specific surface area and large steric hindrance in contact with drugs, which limits their application in drug pharmacokinetic research.

Method used

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  • Brush polymer modified graphene oxide-immobilized capture receptor compound and application thereof in enrichment of monoclonal antibodies
  • Brush polymer modified graphene oxide-immobilized capture receptor compound and application thereof in enrichment of monoclonal antibodies
  • Brush polymer modified graphene oxide-immobilized capture receptor compound and application thereof in enrichment of monoclonal antibodies

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the preparation of brush polymer modified GO

[0033] The reaction equation is as figure 1 shown.

[0034] Modification of ATRP initiator on GO surface: 100 mg of GO, 50 mL of freshly prepared tetrahydrofuran and 0.36 mL of triethylamine were placed in a 250 mL round bottom flask and vigorously stirred at 0-5 °C for 30 min under nitrogen atmosphere. Then 2-bromoisobutyryl bromide (0.32 mL, the mass ratio of graphene oxide to 2-bromoisobutyryl bromide is 1:0.006) was slowly added dropwise into the above reaction system. The reaction was stirred at this temperature for an additional 30 min before its temperature was raised to room temperature and stirring was continued for 4 h. The product was washed well with dichloromethane and dried in a vacuum oven at 50°C overnight.

[0035] Then SI-ATRP initiation was carried out on the surface of the above product: first, 50 mg of GO immobilized with the initiator was mixed with acrylic acid (2M), cupric chloride (0....

Embodiment 2

[0040] Example 2, GO-PAA immobilized capture receptor ErBb2

[0041] First, activate the terminal carboxyl group of GO-PAA: add 500 μL of NHS / EDC solution (400 mM EDC / 100 mM NHS), sonicate for 5 min, and mix at room temperature for 30 min. Subsequently, it was centrifuged at 10,000 rpm for 10 min to obtain the supernatant, and the material was washed twice with deionized water and PBS buffer respectively. Finally, 1 mL of trypsin solution (1 mg / mL) was added to the above GO-PAA polymer, and mixed overnight at 4°C. Calculated by BCA method, the loading capacity of GO-PAA on protein can reach 773.9mg / g.

Embodiment 3

[0042] Example 3, GO-PAA immobilized capture receptor ErBb2 (GO-ErBb2) enriched T-DM1

[0043] First determine the mass spectrometry detection conditions of T-DM1:

[0044] The theoretical values ​​of the MRM transitions of T-DM1 and its internal standard were calculated using Skyline software, as shown in Table 1 and Table 2, respectively. Under the selected transition conditions, use ESI ion source, positive ion mode, and multi-stage reaction monitoring (MRM) to optimize the chromatographic conditions as shown in Table 3: Waters ACQUITY UPLC BEH C 18 (50mm×2.1mm, 1.7μm) chromatographic column; mobile phase: A: water (0.1% formic acid) B: acetonitrile (0.1% formic acid); flow rate 0.3mL / min; injection volume 10μL. The temperature of the injector was controlled at 4°C. Obtain the spectrogram of T-DM1 under optimized chromatographic mass spectrometry conditions, such as Figure 5 shown.

[0045] Table 1 MRM conditions of T-DM1

[0046]

[0047] Table 2 MRM conditions of...

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Abstract

The invention relates to a brush polymer modified graphene oxide-immobilized capture receptor compound and application thereof in enrichment of monoclonal antibodies. The preparation method of the brush polymer modified graphene oxide compound comprises the following steps of: 1) modifying an initiator on the surface of graphene oxide; and (2) performing surface-initiated atom transfer radical polymerization on the product obtained in the step 1) and acrylic acid in the presence of CuCl, CuCl2 and N, N, N',N'',N''-pentamethyldiethylenetriamine to obtain the compound. According to the invention, specific enrichment of immobilized capture receptors and high-sensitivity detection of LC-MS are combined, so that accurate pharmacokinetic parameters and metabolic transformation process of monoclonal antibody drugs in vivo are expected to be obtained. According to the preparation method of the present invention, the efficient enrichment detection of different antibody drugs or protein drugs can be achieved by replacing a capture receptor, such that the efficient specific enrichment detection method can be popularized to the analysis detection and the pharmacokinetic research of other protein drugs so as to further guide the drug research and development, the mechanism research, the clinical medication and the like.

Description

technical field [0001] The invention relates to a brush polymer modified graphene oxide-immobilized capture receptor complex and its application in monoclonal antibody enrichment. Background technique [0002] Therapeutic monoclonal antibody is a drug prepared by antibody engineering technology based on cell engineering technology and genetic engineering technology. Since the first monoclonal antibody drug Muromonab-CD3 was approved by the US FDA in 1986, the research and development of antibody drugs has become a research hotspot in the field of biopharmaceuticals, and has occupied more and more market shares. Due to the advantages of such drugs as strong targeting, less toxic and side effects, and good therapeutic effects, they are widely used in the treatment of malignant tumors, infections, immune diseases, and organ transplant rejection. Compared with traditional small molecule drugs, the chemical nature of antibody drugs is glycoprotein, which also makes them have uni...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/08G01N30/72
CPCG01N30/02G01N30/08G01N30/72
Inventor 白海红王兴河程小强
Owner BEIJING SHIJITAN HOSPITAL CAPITAL MEDICAL UNIVERSTY
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