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Protein Modified Open-Tube Column and Its Application in the Separation of Monoclonal Antibody Charge Variant

A protein separation and open-column technology, which is applied in the field of instrumental analysis, can solve problems such as difficulties in the characterization of monoclonal antibodies, and achieve the effects of improving the separation effect, increasing the separation selectivity, and inhibiting the adsorption of proteins

Active Publication Date: 2019-01-25
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The complexity of monoclonal antibodies makes it difficult to characterize their heterogeneity

Method used

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  • Protein Modified Open-Tube Column and Its Application in the Separation of Monoclonal Antibody Charge Variant
  • Protein Modified Open-Tube Column and Its Application in the Separation of Monoclonal Antibody Charge Variant
  • Protein Modified Open-Tube Column and Its Application in the Separation of Monoclonal Antibody Charge Variant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The open-tube column modified by bovine serum albumin (BSA) is prepared by the following steps:

[0040] (1) Preparation of PDDA open-tube column: Dissolve PDDA and sodium chloride into 20mM Tris-HCl (pH 8.3) to obtain a PDDA solution with a PDDA concentration of 20mg / mL, wherein the salt ion concentration is 0.1M, and then Flush the capillary column for 1 h at a flow rate of 10 μL / min. Finally, remove excess PDDA solution with deionized water.

[0041] (2) Preparation of PDDA@BSA open-tubular column: After successful preparation of PDDA open-tubular column, take BSA and add 20mM Tris-HCl (pH 7.4) to make a 5mg / mL homogeneous solution, and then mix it with 10μL / min The flow rate was injected into the prepared PDDA open-tube column, and it was kept in an oven at 40° C. for 12 hours. Finally, the excess BSA was removed with deionized water, and dried under nitrogen protection at 40 °C to obtain a PDDA@BSA open-tube column.

Embodiment 2

[0043] The open-tube column modified by bovine serum albumin (BSA) is prepared by the following steps:

[0044] (1) Preparation of PDDA open-tube column: get PDDA and potassium chloride dissolved in 20mM Tris-HCl (pH value 8.3) to obtain a PDDA solution with a PDDA concentration of 10mg / mL, wherein the salt ion concentration is 1.0M, and then Flush the capillary column for 0.5 h at a flow rate of 10 μL / min. Finally, remove excess PDDA solution with deionized water.

[0045] (2) Preparation of PDDA@BSA open-tubular column: After successful preparation of PDDA open-tubular column, take BSA and add 20mM Tris-HCl (pH 7.4) to make a 10mg / mL homogeneous solution, and then prepare it at 10μL / min The flow rate was injected into the prepared PDDA open-tube column, and it was kept in an oven at 40° C. for 12 hours. Finally, the excess BSA was removed with deionized water, and dried under nitrogen protection at 40 °C to obtain a PDDA@BSA open-tube column.

Embodiment 3

[0047] The open-tube column modified by bovine hemoglobin (BHb) is prepared by the following steps:

[0048] (1) Preparation of PDDA open-tube column: Dissolve PDDA and sodium chloride into 20mM Tris-HCl (pH 8.3) to obtain a PDDA solution with a PDDA concentration of 20mg / mL, wherein the salt ion concentration is 2.0M, and then Flush the capillary column for 3 h at a flow rate of 10 μL / min. Finally, remove excess PDDA solution with deionized water.

[0049] (2) Preparation of PDDA@BHb open-tubular column: After successful preparation of PDDA open-tubular column, take BHb and add 20mM Tris-HCl (pH 7.4) to make a 5mg / mL homogeneous solution, and then prepare it at 10μL / min The flow rate was injected into the prepared PDDA open-tube column, and it was kept in an oven at 40° C. for 12 hours. Finally, excess BHb was removed with deionized water, and dried under nitrogen protection at 40 °C to obtain a PDDA@BHb open-tube column.

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Abstract

The invention discloses a protein stationary phase modification open tubular column and application of the protein stationary phase modification open tubular column to monoclonal antibody charge isomer separation. The open tubular column is prepared through the following steps that PDDA is fixed on the inner wall of a capillary tube by an electrostatic self-assembly method; then, protein is adsorbed on the PDDA surface through electrostatic self assembly; the protein stationary phase modification open tubular column is obtained. The open tubular column shows specific selectivity on monoclonal antibody charge isomers. Seven kinds of different-form charge isomers of cetuximab are successfully separated; two alkaline isomers and one acid isomer of the rituximab and two alkaline charge isomers and four acid isomers of trastuzumab are successfully separated from main peaks.

Description

technical field [0001] The invention belongs to the field of instrument analysis, and in particular relates to an open-tubular column modified by a protein stationary phase and its application in the separation of charge variants of monoclonal antibodies. Background technique [0002] Monoclonal antibody is a protein drug with broad application prospects in the field of biomedicine. Due to its good efficacy, high selectivity, and low side effects, it is widely used in the treatment of cancer. Currently, most clinically used monoclonal antibodies are of the immunoglobulin type. These monoclonal antibodies (~150kDa) are tetrameric glycoproteins composed of two identical heavy chains and two identical light chains, with a connected by several disulfide bonds. [0003] During production, extraction, formation, and storage, monoclonal antibodies undergo a series of post-translational modifications, including glycosylation, aggregation, fragmentation, and deamidation. Although t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 贾丽王文涛张亚敏
Owner SOUTH CHINA NORMAL UNIVERSITY
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