Affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and preparation method thereof

A monoclonal antibody and chromatographic filler technology, applied in the field of biological separation and purification, can solve the problems of limiting affinity chromatographic separation and purification efficiency, and achieve the effect of increasing specific surface area and improving efficiency

Inactive Publication Date: 2011-09-14
WUXI GALAK CHROMATOGRAPHIC TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned solid phase supports are glass with controlled pore size of irregular shape, non-monodisperse (wider particle size distribution) microspheres or monodisperse non-porous microspheres, etc., which limit the further improvement of affinity chromatography separation and purification efficiency.

Method used

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  • Affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and preparation method thereof
  • Affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and preparation method thereof
  • Affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Preparation of affinity chromatography filler and affinity chromatography column with polymer polystyrene as solid phase carrier and protein A as ligand:

[0044] 1. Choose polystyrene (PS-DVB) porous polymer with uniform particle size and high cross-linking degree as the solid phase carrier. The particle size of the microsphere is about 30 μm, and the pore size of one part is 10-130nm, and the other part is 130-3000nm. It has good mechanical strength and chemical stability, and the surface is coated to enrich the primary alcohol functional group. The film covers the surface of all microspheres, will not be washed off by any organic solvent, and is guaranteed to be used within the pH range of 1-14. Take 10 g of the microspheres and place them in a 100 mL triangular ground-mouth round-bottom flask, which is equipped with a mechanical stirrer in the middle, a water condenser on one side, and a 50 mL dropping funnel on the other side. Mix 60mL of 3mol / L KOH solution and 1...

Embodiment 2

[0050] Isolation of human gamma globulin:

[0051] 1. Chromatographic retention behavior of human gamma globulin on blank column and affinity chromatography column

[0052] Connect the affinity chromatographic column or blank column to the high performance liquid chromatography, and the sample is human γ-globulin (Humanγ-Globulin). Mobile phase A was 20mM potassium phosphate buffer (pH 7.5) + 150mM NaCl, and mobile phase B was 0.1M glycine hydrochloride (pH 2.5). Dissolve human gamma globulin in mobile phase A to make a 1.0mg / mL gamma globulin solution, flow rate: 1mL / min; gradient conditions: 0-10min solution A, 10-20min solution B; 20-30min solution A, The injection volume is 20 μL, and the detection wavelength is 254 nm. like Figure 2A , indicating that human gamma globulin is not retained on the blank column, as Figure 2B As shown, it shows that human γ globulin can be retained on the affinity chromatography column and is eluted when the mobile phase is changed to B ...

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Abstract

The invention discloses an affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and a preparation method thereof. The affinity chromatographic packing consists of a monodisperse porous microsphere solid phase carrier and a protein A ligand. The preparation method comprises: bonding the protein A onto the surface of the solid phase carrier through amino acids containing a nitrogen group; and thus obtaining the affinity chromatographic packing. Concretely, the method comprises three steps, namely connecting an activated spatial arm, coupling the ligand and sealing residual active groups. Meanwhile the invention also discloses an affinity chromatographic column made of the affinity chromatographic packing. The affinity chromatographic packing and the affinity chromatographic column made of the affinity chromatographic packing can be used in the production process of monoclonal antibody medicines and the separation and purification of antibodyglobulin (IgG), and have high separation and purification efficiency.

Description

technical field [0001] The invention relates to the field of biological separation and purification, in particular to an affinity chromatography filler for separating and purifying monoclonal antibodies and antibody globulins and a preparation method thereof. Background technique [0002] Monoclonal antibodies (Monoclonal antibodies) are a class of biomolecules with a unique Y-shaped molecular structure, some with complex polysaccharide structures, and a molecular weight of about 150-160kD. Monoclonal antibodies can be produced by hybridoma or genetic engineering. Antibody globulin (IgG) exists in the body fluids of humans and animals and has important physiological functions. Strictly speaking, monoclonal antibody belongs to a special antibody globulin. In the past two decades, monoclonal antibody drugs have developed rapidly. It is widely used in heterogeneous diseases. [0003] At present, the most important method for separating and purifying monoclonal antibodies and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/282B01J20/286B01D15/08C07K1/22
Inventor 穆宁刘辉孙国威
Owner WUXI GALAK CHROMATOGRAPHIC TECH
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