CN107812514B

CN107812514B - High-fluidity affinity chromatography medium

Info

Publication number: CN107812514B
Application number: CN201711054167.6A
Authority: CN
Inventors: 瞿欢欢; 朱至放
Original assignee: Suzhou Bogen Bioseparation Technology Co ltd
Current assignee: Suzhou Bogen Bioseparation Technology Co ltd
Priority date: 2017-10-31
Filing date: 2017-10-31
Publication date: 2020-09-29
Anticipated expiration: 2037-10-31
Also published as: CN107812514A

Abstract

The scheme relates to a high-circulation affinity chromatography medium, which takes macroporous silicon dioxide microspheres as an inner core, glycidyl methacrylate, polysaccharide and sodium metaaluminate are filled in pore channels of the macroporous silicon dioxide microspheres, one part of the surfaces of the medium silicon dioxide microspheres is bonded with oligosaccharide molecules, and the rest parts are connected with protein A ligand; the affinity chromatography medium provided by the invention has the advantages of firm skeleton, higher mechanical strength and pressure resistance, tight connection between the ligand and the inner core, high liquidity, good separation effect on target protein and high efficiency.

Description

High-fluidity affinity chromatography medium

Technical Field

The invention relates to a chromatography medium, in particular to a high-flow-through affinity chromatography medium.

Background

Proteins are present in all living bodies, are very important macromolecules, and as executives of biological functions, proteins are responsible for a variety of physiological functions such as biocatalysis, substance transport, exercise, defense, regulation, memory recognition and the like. However, both the intensive research on proteins and the preparation of functional proteins into biopharmaceuticals require the use of highly pure proteins, and therefore protein isolation and purification techniques are central techniques in the biological industry. Among them, chromatography is one of the important techniques for separating and purifying proteins, and includes affinity chromatography, adsorption chromatography, gel filtration chromatography, ion exchange chromatography, isoelectric focusing chromatography, and the like.

In recent years, the affinity chromatography technology is developed rapidly, shows great superiority in the separation and purification of functional drug protein, and realizes industrial application in the field of biological pharmacy. The solid phase matrix, the functional ligand and the spacer arm playing a connecting role are the most important three parts for forming the affinity chromatography medium, and the indexes of the particle size, the distribution, the flow performance, the pore structure, the loading capacity, the separation efficiency and the like are important basis for evaluating the performance of the affinity chromatography medium. The flow-through performance refers to the performance of a mobile phase passing through a medium, and the chromatography medium with high flow-through performance has firm skeleton, higher mechanical strength and pressure resistance and can improve the efficiency of protein separation and purification. The existing affinity chromatography medium has the problems of low circulation of the chromatography medium, poor separation efficiency and effect due to the problems of insecure connection between the ligand and the matrix, uneven structure of medium pore canals, more nonspecific adsorption and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a high pressure resistant affinity chromatography medium.

The technical scheme of the invention is summarized as follows:

taking macroporous silicon dioxide microspheres as an inner core; the pore canal of the macroporous silica microsphere is filled with glycidyl methacrylate, polysaccharide and sodium metaaluminate; oligosaccharide molecules are bonded on one part of the surface of the dielectric silica microsphere, and the rest part of the surface of the dielectric silica microsphere is connected with protein A ligand; the sodium metaaluminate is in the form of aluminum hydroxide dissolved in 0.1mol/L sodium hydroxide solution.

Preferably, the weight ratio of the glycidyl methacrylate to the polysaccharide to the sodium metaaluminate is as follows:

60-70 wt% of glycidyl methacrylate;

30-35 wt% of polysaccharide;

2-3 wt% of sodium metaaluminate;

the sum of the three dosage is 100 wt%.

Preferably, the particle size of the macroporous silicon dioxide microspheres is 30-50 μm, and the pore diameter is 200-300 nm.

Preferably, the polymerization degree of the polysaccharide is 20 to 30.

Preferably, the oligosaccharide molecules are formed by condensation polymerization of 2-10 monosaccharide molecules of the same type or different types; the monosaccharide molecules include penta-and hexa-aldoses.

Preferably, the ratio of the oligosaccharide molecules to the ligands is 1: 0.04-0.05.

The invention has the beneficial effects that: according to the scheme, macroporous silica microspheres with large pore diameters are used as an inner core, so that the surface area of the inner core of the chromatography medium can be increased, the inner core can be activated by filling glycidyl methacrylate in pores, the inner core can be connected with a matrix, and simultaneously, the structure of activated molecules which are affinity media in the pores of the inner core is tighter and firmer; the polysaccharide can increase the biocompatibility of the silicon dioxide microspheres, so that the protein loading capacity is increased; the sodium metaaluminate solution can be filled into the pore channels of the silica microspheres, and then the pH value of the solution is adjusted, so that an aluminum hydroxide solid frame can be formed in the pore channels, the mechanical strength and the pressure resistance of the medium can be improved, and the circulation performance is improved; the ligand and the oligosaccharide molecules are mixed and then bonded on the surface of the chromatography medium matrix together, and the proper proportion of the ligand and the oligosaccharide molecules is selected, so that the connection strength of the ligand and the matrix can be increased; the affinity chromatography medium provided by the invention has the advantages of firm skeleton, higher mechanical strength and pressure resistance, firm connection between the ligand and the matrix, high liquidity, good separation effect on target protein and high efficiency.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description. The present invention provides a high flow-through affinity chromatography medium, as illustrated in the following examples and comparative examples.

Example 1

The preparation process comprises the following steps:

1) 20g of macroporous silica microspheres with the particle size of 30-50 mu m and the pore diameter of 200-300 nm are soaked in 0.5mol/L sodium hydroxide solution, stirred at 50-55 ℃ for 1-1.5 hours to activate the macroporous silica microspheres, and dried in vacuum for 12 hours;

2) taking the macroporous silicon dioxide microspheres out of the vacuum drying furnace, quickly placing the microspheres into 0.1mol/L sodium hydroxide solution containing 30g of glycidyl methacrylate, 15g of polysaccharide and 1g of aluminum hydroxide, stirring the mixture at the temperature of between 35 and 40 ℃ for 2 to 3 hours, then adjusting the pH of the solution to be 7.0, continuously stirring the mixture for 3 to 4 hours, washing the mixture with deionized water and drying the mixture;

3) adding 0.2mol of oligosaccharide molecules, 0.01mol of protein A ligand, 1L of ethanol and 10g of sodium hydroxide into the microspheres obtained in the step 2), stirring and reacting at 55-60 ℃ for 12 hours, filtering to remove the solvent after the reaction is finished, washing for three times, and drying to obtain the final affinity chromatography medium.

Comparative example 1

The same procedure as in example 1 was repeated except that no glycidyl methacrylate was added in step 2) of example 1.

Comparative example 2

The procedure of example 1 was the same as that of example 1 except that no polysaccharide was added in step 2).

Comparative example 3

In step 2) of example 1, no sodium metaaluminate was added, and the remaining preparation process was the same as in example 1.

Comparative example 4

The preparation process of example 1 was the same as that of example 1 except that no oligosaccharide molecule was added in step 3).

Comparative example 5

In step 3) of example 1, 0.1mol of oligosaccharide molecule, 0.01mol of protein A ligand, 1L of ethanol and 10g of sodium hydroxide were added, and the rest of the preparation process was the same as in example 1.

Comparative example 6

Commercially available affinity chromatography media using protein A as the ligand.

A flow-through performance experiment was performed on the chromatography media prepared in example 1 and comparative examples 1 to 6 under the same conditions, and 5mL of an affinity chromatography medium was loaded on a column, and connected to an AKTA Purifier 100 chromatography system, with ultrapure water as a mobile phase. After the column had stabilized, the flow rate was increased stepwise, and the column pressure at each flow rate was recorded, and when the flow rate was increased to a certain extent, the pressure was no longer stabilized at this flow rate point but was continuously increased, and the measurement was stopped at this point, which was called the flow rate stabilization point. And taking the flow rate before the flow rate stabilization point as a vertical coordinate, taking the column pressure corresponding to the flow rate as a horizontal coordinate, wherein the flow rate and the column pressure are in a linear relation, the slope of the flow rate can represent the circulation of the chromatographic medium, and the larger the slope is, the better the circulation performance is. Table 1 shows the flow rate (mL/h) and the slope of column pressure (Pa) of the chromatographic media prepared in example 1 and comparative examples 1 to 6, respectively, and the respective flow rates at 0.15KPa of the column pressure.

As can be easily seen from Table 1, when the flow-through performance experiment is carried out by using the affinity chromatography medium, namely embodiment 1, the affinity chromatography medium has obvious advantages compared with comparative examples 1-6, namely the affinity chromatography medium has better flow-through performance, regardless of the slope of the flow rate and the column pressure or the flow rate under the column pressure of 150 Pa; when the filler in the pore channel of the macroporous silica microsphere is not the condition limited by the invention, namely the comparative examples 1-3, the flow performance is obviously reduced; as can be known from the embodiment 1 and the comparative example 4, the addition of the oligosaccharide molecules into the ligand is helpful to improve the flow-through performance of the chromatography medium, the ligand can be better bonded with the matrix by mixing the ligand and the oligosaccharide, and the structure of the affinity chromatography medium is firmer; from example 1 and comparative example 6, it can be seen that the affinity chromatography media designed by the present invention has a significant improvement in flow-through performance compared to the prior art.

TABLE 1

Affinity chromatography media	Flow rate and column pressure slope (mL/h. Pa)	Flow Rate under column pressure of 150Pa (mL/h)
			Example 1	18.2	2752
Comparative example 1	13.4	2008
			Comparative example 2	15.9	2383
Comparative example 3	10.7	1614
			Comparative example 4	11.6	1755
Comparative example 5	16.7	2505
			Comparative example 6	8.3	1249

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.

Claims

1. A high flow-through affinity chromatography media, wherein the affinity chromatography media is prepared by:

(1) activating the macroporous silica microspheres: 20g of macroporous silica microspheres with the particle size of 30-50 mu m and the pore diameter of 200-300 nm are soaked in 0.5mol/L sodium hydroxide solution, stirred at 50-55 ℃ for 1-1.5 hours to activate the macroporous silica microspheres, and dried in vacuum for 12 hours;

(2) filling epoxypropyl methacrylate, polysaccharide and 0.1mol/L sodium hydroxide solution dissolved with aluminum hydroxide into the pore canal of the macroporous silica microsphere: taking the macroporous silicon dioxide microspheres out of the vacuum drying furnace, quickly placing the microspheres into 0.1mol/L sodium hydroxide solution containing 30g of glycidyl methacrylate, 15g of polysaccharide and 1g of aluminum hydroxide, stirring the mixture at the temperature of between 35 and 40 ℃ for 2 to 3 hours, then adjusting the pH of the solution to be 7.0, continuously stirring the mixture for 3 to 4 hours, washing the mixture with deionized water and drying the mixture;

(3) bonding oligosaccharide molecules to one part of the surface of the macroporous silicon dioxide microspheres, and connecting protein A ligand to the rest part: and (3) adding 0.2mol of oligosaccharide molecules, 0.01mol of protein A ligand, 1L of ethanol and 10g of sodium hydroxide into the microspheres obtained in the step (2), stirring and reacting at 55-60 ℃ for 12 hours, filtering to remove the solvent after the reaction is finished, washing for three times, and drying to obtain the final affinity chromatography medium.

2. The affinity chromatography media of claim 1, wherein the degree of polymerization of the polysaccharide is 20 to 30.

3. The affinity chromatography media of claim 1, wherein the oligosaccharide molecules are formed by the polycondensation of 2-10 monosaccharide molecules of the same or different types; the monosaccharide molecules include penta-and hexa-aldoses.