Fc-binding protein having improved antibody separation ability, and method for separating antibody using same

A separation method and protein technology, which can be applied to the preparation methods of peptides, immunoglobulins, chemical instruments and methods, etc., can solve the problems of insufficient improvement of the separation degree and adsorption capacity of antibody analysis, and achieve the effect of improving the separation ability.

Active Publication Date: 2019-10-08
TOSOH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such findings are insufficient to improve the resolution and adsorption capacity associated with antibody analysis
Therefore, in the industrial production of antibody drugs, it is difficult to apply FcγRIIIa-immobilized carriers to step analysis and product analysis

Method used

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  • Fc-binding protein having improved antibody separation ability, and method for separating antibody using same
  • Fc-binding protein having improved antibody separation ability, and method for separating antibody using same
  • Fc-binding protein having improved antibody separation ability, and method for separating antibody using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1F

[0067] Preparation of Example 1 FcR9 Amino Acid Substitution Product

[0068] The amino acid substitution introduced into the Fc-binding protein FcR9 (SEQ ID NO: 5) was prepared according to the method disclosed in the following WO2015 / 199154 ​​to confirm that the valine at position 192 (corresponding to that described in SEQ ID NO: 1) was replaced by a different amino acid. The usefulness of valine at position 176 in wild-type human FcγRIIIa composed of amino acid sequences). Specifically, amino acid substitutions were introduced by PCR into plasmid pET-FcR9 (WO2015 / 199154) containing a polynucleotide (SEQ ID NO: 6) encoding FcR9, thereby producing a 192nd FcR9 (SEQ ID NO: 5) having other amino acid substitutions. Valine-positioned Fc-binding protein. FcR9 (SEQ ID NO: 5) is an Fc-binding protein containing the wild-type FcγRIII extracellular domain described in SEQ ID NO: 4, wherein the Val amino acid at position 43 is replaced by Glu (corresponding to position 27 in SEQ ID ...

Embodiment 2F

[0083] Example 2 Evaluation of IgG1-binding ability of Fc-binding proteins

[0084] (1) Each transformant expressing the Fc-binding protein produced in Example 1 was cultured in 20 mL of 2YT liquid medium supplemented with 50 μg / mL kanamycin, and cultured with aerobic shaking at 37° C. overnight, Thereby pre-cultivation was carried out respectively.

[0085] (2) Cultivate 10 mL of each pre-culture solution in 1000 mL of 2YT liquid medium (peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) supplemented with 50 μg / mL kanamycin, And cultured with aerobic shaking at 37°C overnight.

[0086] (3) The culture temperature was changed to 20° C. 1.5 hours after the start of culture, followed by shaking culture for 30 minutes. Thereafter, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.01 mM, and aerobic shaking culture was continued overnight at 20°C.

[0087] (4) After the end of the culture, the cells were collected by centrifugation, susp...

Embodiment 3

[0097] Example 3 Preparation of Fc-binding protein (FcR9_F_Cys) of the present invention with cysteine ​​tag added

[0098] (1) PCR was performed using the expression vector pET-FcR9_F containing the polynucleotide described in SEQ ID NO: 10 encoding the protein composed of the amino acid sequence described in SEQ ID NO: 9 produced in Example 2 as a template. The primers used in PCR were oligonucleotides consisting of the sequences described in SEQ ID NO: 11 (5′-TAGCCATGGGCATGCGTACCGAAGATCTGCCGAAAGC-3′) and SEQ ID NO: 12 (5′-CCCAAGCTTATCCGCAGGTATCGTTGCGGCACCCTTGGGTAATGGTAATATTCACGGTCTCGCTGC-3′). A reaction solution having the composition described in Table 3 was prepared. Thereafter, the reaction solution was heat-treated at 98°C for 5 minutes, and the first step was performed at 98°C for 10 seconds, the second step was performed at 55°C for 5 seconds, and the second step was performed at 72°C for 1 minute. 3 steps, these 3 steps were repeated 30 cycles as one cycle of reacti...

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Abstract

The present invention addresses the problem of providing an Fc-binding protein having an improved antibody separation ability. The present invention also addresses the problem of providing a high-accuracy antibody separation method using an insoluble carrier having the protein immobilized thereon. The problems can be solved by: an Fc-binding protein in which at least an amino acid substitution ata specific position therein occurs and which has reduced affinity for an antibody; and an antibody separation method comprising a step of allowing an equilibration buffer solution to pass through a column in which an insoluble carrier having the protein immobilized thereon is filled to equilibrate the column, a step of adding a solution containing an antibody to cause the adsorption of the antibody onto the carrier, and a step of eluting the antibody adsorbed on the carrier using an elution solution.

Description

technical field [0001] The present invention relates to an Fc-binding protein having improved ability to separate antibodies (immunoglobulins), and a method for isolating antibodies using the same. More specifically, the present invention relates to an Fc-binding protein that has a reduced affinity for antibodies compared to conventionally known Fc-binding proteins, so that it has improved antibody (immunoglobulin) separation ability; and using A method for isolating antibodies on an insoluble carrier on which an Fc-binding protein is immobilized. Background technique [0002] The sugar chain structure of antibody drugs is closely related to drug efficacy and stability. Therefore, it is extremely important to control the sugar chain structure in the production of antibody drugs. Among Fc-binding proteins, FcγRIIIa is known to recognize the sugar chain structure of antibodies (immunoglobulins). Antibodies can be separated based on sugar chain structures by using an adsorbe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/735C07K1/22C07K16/00C12N15/09
CPCC07K1/22C07K16/00C12N15/09C07K14/00
Inventor 寺尾阳介朝冈义晴大岳辽子远藤谕山中直纪山本侑枝大江正刚
Owner TOSOH CORP
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