CN101279243B - Mixing mode expanded adsorbent bed medium and method for producing the same - Google Patents

Abstract

The invention discloses a mixed model expanded adsorbent bed medium and a preparation method thereof. In the composition of medium, matrix is a composite microsphere with fibrin/inorganic weighting agent, and petunidin is sulfonyl group and mercapto benzimidazole. The composite microsphere with fibrin/inorganic weighting agent is acquired by inverse suspension thermal regeneration and is taken asthe matrix to mix divinyl sulfone, soda buffer solution and DMSO for activation, then swab-off activation matrix, the mercapto benzimidazole and sodium hydroxide containing ammonium persulfate are mixed for coupling, thus acquiring the mixed model expanded adsorbent bed medium taking the mercapto benzimidazole and the sulfonyl group as the petunidin. The novel expanded bed adsorbing medium developed by the invention combines with a plurality of petunidin-protein interaction mechanisms such as hydrophobic interaction, charge induction action, thiophilic action, etc, to form mixed model adsorption, which has the advantages of large adsorption capacity, high selectivity, etc., thus being used for high efficient separation and purification of bioactivators such as antibody, etc.

Description

A mixed mode expanded adsorption bed medium and its preparation method

technical field

The invention relates to a mixed-mode expanded bed adsorption medium and a preparation method thereof, which uses mercaptobenzimidazole and sulfone group as ligands, and belongs to the expanded bed adsorption and separation protein technology in chromatographic separation.

Background technique

Antibodies are an important biotechnology product widely used in immunotherapy, disease prevention, and medical diagnosis. In recent years, the separation technology for antibodies has made some progress, but it still needs to be continuously supplemented and improved.

Conventional ion-exchange chromatography and hydrophobic interaction chromatography can bind antibodies, but their specificity and selectivity are poor, and there are many separation steps, which affect the activity yield of antibodies. Protein A affinity chromatography media can selectively adsorb antibodies, but the media is expensive, and the ligand is easily degraded by proteases in the feed solution, the number of reuses is low, and the operating cost is extremely high, which limits its large-scale application. Mixed-mode chromatography (Mixed-mode chromatography) is a new biochemical separation technology developed in recent years. Combining two or more ligand-protein interaction modes can significantly improve the adsorption capacity and adsorption selectivity. Thiophilic chromatography is an important direction. Since Porath (Porath et al. FEBS letters, 1985, 185: 306) first applied thiophilic chromatography (thiophilic chromatography) to separate antibodies from serum in 1985, thiophilic chromatography has been used in protein purification, especially antibody purification. There are many applications. The sulfur of the sulfone group in the thiol is an electron-deficient region, which can be used as an electron acceptor, while the sulfur in the adjacent thioether is an electron-rich region, which can be used as an electron donor. This special structure can have a specific effect on some electron donor-acceptor sites in the conservative region of the antibody molecule, and improve the selectivity of antibody adsorption. However, traditional thiophilic chromatography must be under a certain concentration of ammonium sulfate or sodium sulfate to effectively adsorb antibodies, which is not conducive to the separation of antibodies and limits its large-scale application. Finding new, salt-tolerant thiophilic mixed-mode ligands is an important development trend.

Expanded Bed Adsorption (EBA) technology was formed in the 1990s. It is a new protein separation and purification technology that integrates solid-liquid separation, concentration and initial purification in one operating unit. EBA can directly capture the target product from cell fluid, cell homogenate or tissue extract, which reduces the number of operating units, shortens the processing time, and saves production costs. It is a new unit operation that has emerged in the past ten years. Therefore, the present invention uses cellulose/inorganic weighting agent composite microspheres as the expanded bed adsorption matrix, and the sulfone group introduced after divinyl sulfone activation and the coupled mercaptobenzimidazole are used as its main functional ligands to form a A new type of thiophilic mixed-mode expanded adsorption bed medium can be used for large-scale expanded bed adsorption and separation of antibodies, and has become a new type of efficient and integrated antibody separation process. Related research has not been reported in the literature.

Contents of the invention

The object of the present invention is to provide a mixed-mode expanded bed adsorption medium with mercaptobenzimidazole and sulfone group as ligands and a preparation method thereof.

Mixed-mode expanded bed adsorption medium with mercaptobenzimidazole and sulfone groups as ligands: the matrix is composed of cellulose/inorganic weighting agent composite microspheres, and the ligands are sulfone groups introduced after activation of divinyl sulfone and The structure of the coupled mercaptobenzimidazole is:

The skeleton of the cellulose/inorganic weighting agent composite microsphere is cellulose, the weighting agent is titanium dioxide, stainless steel powder, nickel powder or tungsten carbide powder, and the mass percentage of the weighting agent in the wet bulb is 16%- 61%; the particle size of the cellulose/inorganic weighting agent composite microsphere is 50-250 μm. The ligand is 2-mercapto-1-R-benzimidazole, and R is a short-chain alkane with 0-2 carbon atoms. The ligand density is 28-58 μmol/ml medium.

The preparation method of the mixed-mode expanded bed adsorption medium: first fully mix the cellulose viscose and the inorganic weighting agent, the mass percentage of the inorganic weighting agent in the mixture is 16% to 61%, add 5 times of the cellulose viscose High-quality pump oil, control the speed of 500-800rpm, reverse-phase suspension thermal regeneration to make balls, wash with boiling water, and screen cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm as the matrix; secondly, cellulose/inorganic weighting agent composite microspheres Heavy agent composite microspheres, divinyl sulfone, 0.1-0.5M pH12 sodium carbonate buffer and dimethyl sulfoxide are mixed, and the volume of divinyl sulfone added is the volume of cellulose/inorganic weighting agent composite microspheres 18%～36%, activated in 180rpm shaker at 25°C for 4 hours, suction filtered, and washed with deionized water to obtain the activated matrix; then the activated matrix, mercaptoethylpyridine with 3 times the double Mix ammonium persulfate with 0.2-0.7M sodium hydroxide solution, and couple in a 180rpm shaker at 60°C for 8 hours; finally add the microspheres to a solution containing 1% H ₂ O ₂ and 1% glycerol for 2 hours, and remove After washing with ion water, a mixed-mode expanded bed adsorption medium with mercaptobenzimidazole and sulfone group as ligands is obtained. The schematic diagram of the reaction process is:

The invention fully combines various ligand-protein interaction principles such as hydrophobic interaction, charge induction effect and thiophilic effect to form a special mixed mode adsorption, and has the advantages of large adsorption capacity and high selectivity. At the same time, the new mixed-mode ligand is coupled to the cellulose/inorganic weighting agent composite expanded bed matrix to form a new expanded bed adsorption medium, which can be used for large-scale and efficient separation and purification of proteins such as antibodies.

Description of drawings

The static adsorption isotherm (different pH) of the mixed mode medium adsorption egg yolk antibody of Fig. 1;

Fig. 2 Static adsorption isotherms of egg yolk antibody adsorbed on mixed-mode media (different salt concentrations, pH=7).

Detailed ways

The present invention will be further described below by embodiment:

Example 1

Stir 50g of cellulose viscose and 40g of tungsten carbide powder in a 500ml round-bottomed flask at 300rpm, add 300g of pump oil, rotate at 700rpm, inversely suspend and regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, Screen cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm as the matrix; 10ml of drained composite microspheres, 3.6ml of divinyl sulfone, 9ml of 0.25M pH12 sodium carbonate buffer and 1.5ml of bismuth Methyl sulfoxide was added into the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercaptobenzimidazole with 3 times the molar amount of double bonds and 10ml of 0.5M sodium hydroxide solution containing 25mg/ml ammonium persulfate, perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours, and remove the mixed solution by suction filtration. Wash with deionized water and drain. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water, to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 1.81g/ml, The ligand density is 58 μmol/ml.

Example 2

Stir 50g of cellulose viscose and 40g of tungsten carbide powder in a 500ml round-bottomed flask at 300rpm, add 300g of pump oil, rotate at 700rpm, inversely suspend and regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, Screen cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm as the matrix; 10ml of drained composite microspheres, 0.9ml of divinyl sulfone, 9ml of 0.5M pH12 sodium carbonate buffer and 1.5ml of bismuth Methyl sulfoxide was added into the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercaptobenzimidazole with 3 times the molar amount of double bonds and 10ml of 0.7M sodium hydroxide solution containing 25mg/ml ammonium persulfate, perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours, and remove the mixed solution by suction filtration. Wash with deionized water and drain. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water, to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 1.81g/ml, The ligand density is 28 μmol/ml.

Example 3

Stir 50g of cellulose viscose and 80g of tungsten carbide powder in a 500ml round-bottomed flask at 300rpm, add 310g of pump oil, rotate at 800rpm, inversely suspend and thermally regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, Screen cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm as the matrix; 10ml of drained composite microspheres, 1.8ml of divinyl sulfone, 9ml of 0.1M pH12 sodium carbonate buffer and 1.5ml of bismuth Methyl sulfoxide was added into the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercaptobenzimidazole with 3 times the molar amount of double bonds and 10ml of 0.2M sodium hydroxide solution containing 25mg/ml ammonium persulfate, perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours, and remove the mixed solution by suction filtration. Wash with deionized water and drain. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 2.40g/ml, The ligand density is 41 μmol/ml.

Example 4

Stir 50g of cellulose viscose and 60g of tungsten carbide powder in a 500ml round-bottomed flask at 300rpm, add 300g of pump oil, rotate at 800rpm, inversely suspend and thermally regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, Screen cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm as the matrix; 10ml of drained composite microspheres, 3.6ml of divinyl sulfone, 9ml of 0.25M pH12 sodium carbonate buffer and 1.5ml of bismuth Methyl sulfoxide was added into the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercaptobenzimidazole with 3 times the molar amount of double bonds and 10ml of 0.5M sodium hydroxide solution containing 25mg/ml ammonium persulfate, perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours, and remove the mixed solution by suction filtration. Wash with deionized water and drain. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water, to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 2.11g/ml, The ligand density is 56 μmol/ml.

Example 5

Stir 50g of cellulose viscose and 12g of nickel powder in a 500ml round bottom flask at 300rpm, add 300g of pump oil, rotate at 700rpm, inversely suspend and thermally regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, and screen Cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm are used as the matrix; 10ml of drained composite microspheres, 3.6ml of divinyl sulfone, 9ml of 0.25M pH12 sodium carbonate buffer and 1.5ml of dimethyl The base sulfoxide was added to the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercaptobenzimidazole with 3 times the molar amount of double bonds and 10ml of 0.5M sodium hydroxide solution containing 25mg/ml ammonium persulfate, perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours, and remove the mixed solution by suction filtration. Wash with deionized water and drain. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water, to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 1.42g/ml, The ligand density is 57 μmol/ml.

Example 6

Stir 50g of cellulose viscose and 20g of stainless steel powder in a 500ml round bottom flask at 300rpm, add 300g of pump oil, rotate at 700rpm, inversely suspend and thermally regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, and screen Cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm are used as the matrix; 10ml of drained composite microspheres, 3.6ml of divinyl sulfone, 9ml of 0.25M pH12 sodium carbonate buffer and 1.5ml of dimethyl The base sulfoxide was added to the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercaptobenzimidazole with 3 times the molar amount of double bonds and 10ml of 0.5M sodium hydroxide solution containing 25mg/ml ammonium persulfate, perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours, and remove the mixed solution by suction filtration. Wash with deionized water and drain. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 1.71g/ml, The ligand density is 54 μmol/ml.

Example 7

Stir 50g of cellulose viscose and 15g of titanium dioxide in a 500ml round bottom flask at 300rpm, add 300g of pump oil, rotate at 700rpm, inversely suspend and thermally regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, and screen Cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm are used as the matrix; 10ml of drained composite microspheres, 3.6ml of divinyl sulfone, 9ml of 0.25M pH12 sodium carbonate buffer and 1.5ml of dimethyl The base sulfoxide was added to the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercaptobenzimidazole with 3 times the molar amount of double bonds and 10ml of 0.5M sodium hydroxide solution containing 25mg/ml ammonium persulfate, perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours, and remove the mixed solution by suction filtration. Wash with deionized water and drain. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water, to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 1.39g/ml, The ligand density is 58 μmol/ml.

Example 8

Stir 50g of cellulose viscose and 40g of tungsten carbide powder in a 500ml round-bottomed flask at 300rpm, add 300g of pump oil, rotate at 700rpm, inversely suspend and regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, Screen cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm as the matrix; 10ml of drained composite microspheres, 3.6ml of divinyl sulfone, 9ml of 0.25M pH12 sodium carbonate buffer and 1.5ml of bismuth Methyl sulfoxide was added into the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercapto-1-methyl-benzimidazole with 3 times the molar amount of double bonds and 10ml of 0.5M sodium hydroxide solution containing 25mg/ml ammonium persulfate, and perform coupling reaction in a shaker at 180rpm at 60°C for 8 hours. The mixture was removed by suction filtration, washed with deionized water, and dried. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water, to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 1.81g/ml, The ligand density is 51 μmol/ml.

Example 9

Stir 50g of cellulose viscose and 40g of tungsten carbide powder in a 500ml round-bottomed flask at 300rpm, add 300g of pump oil, rotate at 700rpm, inversely suspend and regenerate to make balls, wash with boiling water and deionized water for 3 to 5 times, Screen cellulose/inorganic weighting agent composite microspheres with a particle size of 50-250 μm as the matrix; 10ml of drained composite microspheres, 3.6ml of divinyl sulfone, 9ml of 0.25M pH12 sodium carbonate buffer and 1.5ml of bismuth Methyl sulfoxide was added into the Erlenmeyer flask, activated in a shaker at 180 rpm at 25°C for 4 hours, the mixed solution was removed by suction filtration, washed with deionized water, and sucked dry. Add 2-mercapto-1-ethyl-benzimidazole with 3 times the molar amount of the double bond and 10ml of 0.5M sodium hydroxide solution containing 25mg/ml ammonium persulfate, and perform a coupling reaction in a shaker at 180rpm at 60°C for 8 hours. The mixture was removed by suction filtration, washed with deionized water, and dried. The medium was added to 100ml containing 1% H ₂ O ₂ and 1% glycerin solution, soaked for 2 hours, washed with deionized water, to obtain a hydrophobic charge-induced thiophilic expanded bed adsorption medium, and the wet true density of the medium was 1.81g/ml, The ligand density is 49 μmol/ml.

Example 10

Static adsorption experiment of mixed mode medium (ligand density: 58 μmol/ml) to adsorb egg yolk antibody. Firstly, the medium was equilibrated with citrate buffer solution of pH 5; after suction filtration, 0.4g of medium was accurately weighed into 25ml Erlenmeyer flasks with stoppers Add 8ml of buffer solutions with different yolk antibody (IgY) concentrations; place the Erlenmeyer flask in a water-bath shaker, shake at 180rpm at 25°C for 8h; take out the supernatant after equilibrium and measure the concentration of IgY; calculate it according to the material The amount of adsorbed protein and the remaining concentration of the solution were drawn to draw a static adsorption isotherm, and according to the Langmuir equation fitting, the saturated adsorption capacity of the medium was 167.3mg/ml. Using the same method, the static adsorption isotherms at pH 4 and pH 8 were measured, and the static saturated adsorption capacities of the medium for IgY at pH 4 and 8 were 101.9 and 114.9 mg/ml, respectively. The results showed that the medium had a high adsorption capacity for antibodies and was very sensitive to pH changes, showing typical mixed-mode adsorption characteristics.

Example 11

For the static adsorption experiment of mixed mode medium (ligand density: 58 μmol/ml) to adsorb egg yolk antibody, first prepare pH5 citric acid buffer solution containing 0.2M ammonium sulfate; balance the medium with buffer solution; Put 0.4g medium in a 25ml Erlenmeyer flask with a stopper, add 8ml of buffer solutions with different concentrations of egg yolk antibody (IgY); place the Erlenmeyer flask in a water bath shaker, shake at 180rpm at 25°C for 8h; take out the supernatant after equilibration Determine the concentration of IgY; obtain the amount of adsorbed protein and the remaining concentration of the solution according to the cross-calculation of the material, draw the static adsorption isotherm, and fit the saturated adsorption capacity of the medium according to the Langmuir equation to be 144.5 mg/ml. Using the same method, the static adsorption isotherm of IgY in pH 7 citrate buffer containing 0.6M ammonium sulfate was measured, and the static saturated adsorption capacity of IgY was 171.4 mg/ml. The results showed that the medium had a high adsorption capacity for antibodies and was insensitive to changes in salt concentration, reflecting typical mixed-mode adsorption characteristics.

Claims (1)

1. mixing mode expanded bed adsorbing medium, the composition mesostroma that it is characterized in that medium is cellulose/inorganic weighting agent complex microsphere, and aglucon is the sulfuryl introduced of divinylsulfone activation back and the mercaptobenzimidazole after the coupling, and structural group becomes:

The skeleton of described cellulose/inorganic weighting agent complex microsphere is a cellulose, and weighting agent is titanium dioxide, stainless steel powder, nickel powder or tungsten carbide powder.

Images