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Fc-binding protein, method for producing said protein, and antibody adsorbent using said protein, and methods for purifying and identifying antibody using said adsorbent

一种结合性、蛋白质的技术，应用在识别糖链有无附加于抗体,抗体吸附剂领域，能够解决难以控制抗体糖链、分析需要很多时间和劳力等问题

Active Publication Date: 2016-05-04
TOSOH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibody drugs are usually produced using genetic recombination technology using animal cells as hosts, and it is difficult to control the sugar chains attached to antibodies in the host
In addition, analysis of sugar chains of produced antibodies requires a lot of time and labor

Method used

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  • Fc-binding protein, method for producing said protein, and antibody adsorbent using said protein, and methods for purifying and identifying antibody using said adsorbent
  • Fc-binding protein, method for producing said protein, and antibody adsorbent using said protein, and methods for purifying and identifying antibody using said adsorbent
  • Fc-binding protein, method for producing said protein, and antibody adsorbent using said protein, and methods for purifying and identifying antibody using said adsorbent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1Fc

[0127] Example 1 Preparation of Fc-binding protein or human FcγRIIIa expression vector

[0128] (1) Based on the amino acid sequence from glycine (Gly) at position 17 to glutamine (Gln) at position 192 in the amino acid sequence of human FcγRIIIa described in SEQ ID NO: 1, DNAworks method (Nucleic Acids Res., 30, e43, 2002), designed a nucleotide sequence that converts codons from the human type to the E. coli type. The designed nucleotide sequence is shown in SEQ ID NO: 2.

[0129] (2) In order to prepare a polynucleotide comprising the sequence described in SEQ ID NO: 2, an oligonucleotide comprising the sequence described in SEQ ID NO: 3 to 20 was synthesized, and using the aforementioned oligonucleotide, the following procedures were performed: Two-stage PCR.

[0130] (2-1) PCR in the first stage: Prepare a reaction solution having the composition shown in Table 1, heat-treat the reaction solution at 98° C. for 5 minutes, then perform the first step at 98° C. for 10 seco...

Embodiment 2

[0140] Example 2 Introduction of mutations into Fc-binding protein and preparation of gene library

[0141] For the polynucleotide portion encoding the Fc-binding protein in the Fc-binding protein expression vector pER-eFcR prepared in Example 1, mutations were randomly introduced by error-prone PCR.

[0142] (1) Error-prone PCR was performed using pET-eFcR prepared in Example 1 as a template. Error-prone PCR was performed as follows: After preparing a reaction solution having the composition shown in Table 3, the reaction solution was heat-treated at 95°C for 2 minutes, the first step was performed at 95°C for 30 seconds, and the first step was performed at 60°C for 30 seconds. The second step of 1 second, the third step of 90 seconds at 72°C, these 3 steps were carried out as one cycle of reaction for 35 cycles, and finally heat treatment was performed at 72°C for 7 minutes. Mutations were favorably introduced into the polynucleotide encoding the Fc-binding protein by the a...

Embodiment 3

[0147] Example 3 Screening of thermostabilized Fc-binding proteins

[0148] (1) Inoculate the random mutant gene library (transformant) prepared in Example 2 into 2YT liquid medium (16 g / L of peptone, 10 g / L of yeast extract, chlorinated Sodium 5g / L) 200 μL, using a 96-well deep-well plate, shaking culture at 30°C for one night.

[0149] (2) After culturing, 5 μL of the culture solution was continuously implanted into 500 μL of IPTG (isopropyl-β-D-thiogalactopyranoside, isopropyl-β-D-thiogalactopyranoside) containing 0.05 mM, 0.3% glycine and In a 2YT liquid medium containing 50 μg / mL kanamycin, a 96-well deep-well plate was used, and cultured at 20° C. overnight with shaking.

[0150] (3) After culturing, the culture supernatant obtained by centrifugation was diluted 2-fold with 20 mM Tris hydrochloride buffer (pH 7.4) containing 150 mM sodium chloride. For the diluted solution, heat treatment was performed at 45°C for 10 minutes.

[0151] (4) The antibody-binding activity...

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Abstract

The present invention addresses the first problem of providing an Fc-binding protein having improved stability, especially stability to heat and acid, of the Fc-binding protein, a method for producing this protein, and an antibody adsorbent using this protein. The present invention also addresses the second problem of providing a method that makes it possible to identify the presence or absence of glycosylation of an antibody, and a material to be used in this method. The first problem is solved by an Fc-binding protein having improved stability to heat and acid obtained by substituting amino acid residues at specific positions in the extracellular domain within human Fc[gamma]RIIIa with other specific amino acids, a method for producing this protein, and an antibody adsorbent using this protein. The second problem is solved by using an adsorbent capable of specifically adsorbing an antibody having a sugar chain, the adsorbent being obtained by immobilizing human Fc[gamma]RIIIa on an insoluble carrier.

Description

technical field [0001] The present invention relates to an Fc-binding protein having an affinity for immunoglobulin, an antibody adsorbent using the protein, and a method for purifying and identifying an antibody using the adsorbent. More specifically, the present invention relates to an Fc-binding protein having higher heat and acid stability than wild-type, a method for producing the protein, and an antibody-adsorbent obtained by immobilizing the protein on an insoluble carrier. In particular, the present invention relates to an adsorbent capable of specifically adsorbing antibodies having sugar chains among antibodies, a method for purifying antibodies having sugar chains using the adsorbent, and a method for identifying whether sugar chains are attached to the antibody. Background technique [0002] Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Each molecule recognizes a single or the same group of immunoglobulin isotypes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/735C07K1/22C12N1/21C12N15/09C12P21/02
CPCC07H1/06C07K1/22C07K14/70535C07K17/00C07K16/283C07K2317/10C07K2317/732
Inventor 朝冈義晴田中亨井出辉彦
Owner TOSOH CORP
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