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Bioconjugation method for IgG4 antibody

An antibody and conjugation technology, which is applied in the production of antibody-drug conjugates, can solve the problems of reduced utilization of antibody raw materials, increased separation and purification operations, and complicated production processes, so as to improve the utilization rate and simplify the production process , The effect of small requirements for reaction volume

Inactive Publication Date: 2018-03-02
SHANGHAI WUXI BIOLOGIC TECH CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for IgG4 antibody, because its structure is different from that of IgG1, if the same partial reduction method is used for coupling, the obtained ADC with the same average number of connections is not ideal, and the unlinked antibody and ADC with high number of connections The content is very high and cannot be directly applied. The obtained IgG4-ADC must be further separated to remove unlinked antibodies and high-linked ADCs before it can be used in animals or clinically.
The increased separation and purification operation not only greatly reduces the utilization rate of antibody raw materials, but also increases the process steps, making the production process more complicated and increasing the cost

Method used

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  • Bioconjugation method for IgG4 antibody
  • Bioconjugation method for IgG4 antibody
  • Bioconjugation method for IgG4 antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0066] (1) Reduction of IgG4 antibody. Dissolve the antibody at a concentration of 8mg / ml in 20mM Histidine, 5% (w / v) sucrose, pH 5.5 buffer, add 20 equivalents of TCEP, place the reaction solution in a water bath at 37°C for 21 hours, PLRP -HPLC monitoring reaches the reaction end point (such as Figure 5 shown).

[0067] (2) Remove excess reducing agent. A desalting column with a molecular weight cut-off of 40KD was used to perform buffer exchange on the reduction reaction system. The antibody protein buffer was exchanged to 50mM NaPi, 50mM NaCl, 2mM EDTA, pH 6.5 while removing excess TCEP from the reaction.

[0068] (3) Partial oxidation of the reduced antibody. Add buffer, DMSO and DMSO stock solution of DHAA to the antibody purified by the desalting column, so that the antibody concentration is 5 mg / ml, the DMSO content is 5%, the equivalent ratio of DHAA to antibody is 6, and the reaction solution is placed at 4 ℃ for 3 hours, PLRP-HPLC monitoring reached the end of...

Embodiment 2

[0074] (1) Reduction of IgG4 antibody. Dissolve the antibody at a concentration of 10 mg / ml in PBS, pH 6.0 buffer, add 40 equivalents of DTT, place the reaction solution in a water bath at 37°C for 21 hours, and monitor by PLRP-HPLC to reach the end of the reaction (such as Figure 6 shown).

[0075] (2) Remove excess reducing agent. A desalting column with a molecular weight cut-off of 40KD was used to perform buffer exchange on the reduction reaction system. Buffer exchange the antibody protein to 50mM NaPi, 50mM NaCl, 2mM EDTA, pH 6.5 while removing excess DTT from the reaction.

[0076] (3) Partial oxidation of the reduced antibody. Add buffer, DMSO and DMSO stock solution of NAD to the antibody purified by the desalting column, so that the antibody concentration is 5 mg / ml, the DMSO content is 23%, and the equivalent ratio of NAD to antibody is 65. Place it at five degrees for 23 hours to complete the partial oxidation, so that the free sulfhydryl group reaches the ta...

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Abstract

The invention discloses a bioconjugation method for an IgG4 antibody. The bioconjugation method for the IgG4 antibody comprises the following steps of: step 1, reduction: the IgG4 is reduced, so fourpairs of interchain disulfide bonds in the IgG4 antibody are all made open; step 2, partial oxidation: the four pairs of open interchain disulfide bonds in the IgG4 antibody are partially oxidized, and the number of the free mercapto groups of the obtained IgG4 antibody reach the targeted average conjugation number of 4+ / -1; step 3, a coupling reaction: bioconjugation is conducted by adopting thefree mercapto groups of the IgG4 antibody and a connexon-conjugate complex; and step 4, purification of bioconjugation products: excess conjugates in the bioconjugation products which are obtained inthe step 3 are removed so as to obtain the final product. The bioconjugation product mixture obtained by using the method has better product distribution, the content of the conjugation products witha linker number of 0 is less than 5%, and the content of the conjugation products with a linker number of 8 is less than 15%; after routine purification is conducted on the obtained bioconjugation product mixture, the final product can be applied to animals or clinical trials directly.

Description

technical field [0001] The present invention relates to the technical field of biological conjugation in the biopharmaceutical industry, in particular to a technique for selectively utilizing interchain disulfide bonds in IgG4 antibodies and coupling them with drugs, and its application in antibody-drug coupling. Applications in ADC production. Background technique [0002] Bioconjugates such as ADCs (Antibody-Drug Conjugates) are a class of biological agents with great development potential, especially for tumor-targeted therapy. Structurally, ADCs can be divided into three parts: antibody, linker and toxin molecules. Antibodies are responsible for targeting and recognizing tumor molecules, toxin molecules are responsible for killing tumor cells, and linkers are the bridge between drugs and antibodies. It is necessary to ensure the stability of the ADC in vivo, and to enable toxin molecules to be effectively absorbed after reaching tumor cells. Play a role. Different fro...

Claims

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Application Information

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IPC IPC(8): C07K1/10
CPCC07K1/10
Inventor 金明志阴丽王俊周伟昌陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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