A hydroxyethyl chitosan gel microsphere and its application as a chromatography medium for antibody purification and separation.

Abstract

This invention discloses a hydroxyethyl chitosan gel microsphere and its application as a chromatography medium for antibody purification and separation. The preparation method of the hydroxyethyl chitosan gel microsphere of this invention is simple and inexpensive, and it has abundant internal pores while maintaining good mechanical strength and chemical stability. At the same time, the hydroxyethyl chitosan gel microsphere of this invention has more hydroxyl groups, making the cross-linking reaction more controllable, and providing more reaction sites for activating and coupling functional ligands. It meets the requirements for use as a chromatography medium and can replace traditional dextran gel and agarose gel for antibody purification. It can separate antibodies and small molecule proteins, has high economic value, and is suitable for large-scale production.

Description

(I) Technical Field

[0001] This invention belongs to the field of natural polymer materials technology, and more specifically, relates to a hydroxyethyl chitosan gel microsphere suitable for antibody purification and its application. (II) Background Technology

[0002] Chromatography, with its rapid, efficient, and highly specific characteristics, has become a favored method for protein separation and purification. Its mild operating conditions can better maintain the activity of protein molecules, making it suitable for the purification of most proteins. Currently, commonly used chromatographic methods include gel chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, short peptide biomimetic chromatography, and the emerging hydrophobic charge chromatography. Chromatographic techniques include ligands and media, among which the chromatographic media plays an important role. Choosing a suitable media is of great significance for the efficient purification of proteins.

[0003] Conventional chromatography processes mostly use soft matrices such as dextran gels or agarose gels as media. These media are mostly imported and expensive, and their preparation technologies are monopolized by a few large foreign biopharmaceutical companies. There is an urgent need to introduce high-performance and cost-effective biomaterials. Therefore, scholars at home and abroad have been searching for inexpensive, hydrophilic, and rigid media as alternatives. Chitosan is the second most abundant natural polysaccharide, mainly found in the shells of crustaceans and insects. Its structure and properties are similar to agarose and dextran, and it has excellent bioactivity, biodegradability, renewability, non-toxicity, and harmlessness. In particular, the chitosan molecular chain has a large number of reactive groups (amino and hydroxyl groups), providing active reaction sites for chemical modification and the introduction of various functional ligands. These characteristics make chitosan an ideal media material in chromatography.

[0004] Chitosan has a large relative molecular mass and strong intramolecular hydrogen bonding, resulting in poor water solubility; it dissolves only in dilute acids. The cross-linking process occurs under alkaline conditions, and chitosan easily precipitates out, forming solid microspheres, making it unsuitable for preparing gel microspheres. Hydroxyethyl chitosan (HECTS), an important derivative of chitosan, improves solubility while avoiding the loss of hydroxyl groups and retaining its ability to form cross-links between molecular chains. It can dissolve under alkaline conditions and form gel microspheres with a three-dimensional network structure under the action of cross-linking agents. These characteristics make it suitable as a raw material for synthesizing chromatographic media.

[0005] In recent years, there have been literature reports on the preparation of chitosan microspheres. Chitosan research is extensive, but most studies focus on its applications in drug delivery, bone repair, tissue engineering, and wound dressings. However, there are few reports on its use as a separation chromatography medium. Currently, there are no reports on the use of hydroxyethyl chitosan to prepare separation chromatography media. Using low-cost HECTS as a raw material to prepare a separation chromatography medium with good mechanical strength, chemical stability, and abundant pores can solve the problems of high price and technological monopoly of traditional chromatography media. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a hydroxyethyl chitosan gel microsphere and its application as a separation chromatography medium for antibody purification. The gel microspheres prepared by this method are inexpensive, have a distinct internal porous structure, and possess good mechanical strength and chemical stability. They can replace traditional dextran gels and agarose gels as separation and purification chromatography media for antibody purification.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides hydroxyethyl chitosan gel microspheres for antibody purification chromatography media, wherein the hydroxyethyl chitosan gel microspheres are prepared by the following method:

[0009] (1) Gel microspheres: Hydroxyethyl chitosan aqueous solution is dispersed in liquid paraffin containing crosslinking agent and Span 80 under stirring conditions of 40-60℃ and 100-500r / min. The reaction is carried out at 40-60℃ and 100-500r / min for 1-5h, then heated to 60-100℃ and reacted at 100-500r / min for 2-8h. The mixture is filtered, the filter cake is soaked in organic solvent, then washed with a 10-60% volume concentration organic solvent aqueous solution, and finally vacuum dried (preferably at 30℃) to obtain gel microspheres. The crosslinking agent includes one of glutaraldehyde, epichlorohydrin, and N,N-methylenebisacrylamide.

[0010] (2) Activation of gel microspheres: The gel microspheres from step (1) are added to a dimethyl sulfoxide (DMSO) aqueous solution, followed by the addition of allyl bromide and NaOH. The reaction is carried out at 20-40°C for 12-24 h (preferably at 30-40°C for 12-16 h). The reacted gel microspheres are then reacted with N-bromosuccinimide (NBS) and a 30-70% acetone aqueous solution at 20-40°C for 1-5 h (preferably at 30-40°C for 1-3 h). The mixture is then filtered, the filter cake is washed, and the activated gel microspheres are obtained.

[0011] (3) Connecting functional ligands: The activated gel microspheres in step (2) are reacted with 2-mercapto-1-methylimidazolium (MMI) in carbonate buffer (pH=10) at 20-40℃ for 10-20h (preferably 30-40℃ for 10-12h). After washing (preferably with deionized water), hydroxyethyl chitosan gel microspheres are obtained.

[0012] Further, in step (1), the mass concentration of the hydroxyethyl chitosan aqueous solution is 1-8% (preferably 4-8%), the volume ratio of the crosslinking agent to the hydroxyethyl chitosan aqueous solution is 0.1-1:1 (preferably 0.1-0.3:1), the volume ratio of the liquid paraffin to the hydroxyethyl chitosan aqueous solution is 1-10:1 (preferably 2-7:1), and the mass ratio of Span 80 to hydroxyethyl chitosan is 2-6:1 (preferably 3:1).

[0013] Furthermore, the organic solvent used for soaking in step (1) includes one of methanol, ethanol, petroleum ether, and acetone; the organic solvent in the 10-60% organic solvent aqueous solution used for rinsing includes one of methanol, ethanol, petroleum ether, and acetone.

[0014] Further, in step (2), the volume concentration of the dimethyl sulfoxide aqueous solution is 20-50% (preferably 40%), and the volume of the dimethyl sulfoxide aqueous solution is 0.5-2 mL / g (preferably 1 mL / g) based on the mass of the gel microspheres; the volume of allyl bromide is 0.1-1 mL / g (preferably 0.2-0.3 mL / g) based on the mass of the gel microspheres; the mass ratio of NaOH to gel microspheres is 0.1-1:1 (preferably 0.1-0.3:1); the mass ratio of N-bromosuccinimide to gel microspheres is 0.1-1:1 (preferably 0.1:1); and the volume of the 30-70% acetone aqueous solution is 0.5-2 mL / g (preferably 1 mL / g) based on the mass of the gel microspheres.

[0015] Furthermore, step (2) filter cake cleaning refers to cleaning with 50% acetone aqueous solution, 50% ethanol aqueous solution and deionized water respectively.

[0016] Furthermore, the volume of carbonate buffer used in step (3) is 1-5 mL / g (preferably 1.5 mL / g) based on the mass of the gel microspheres in step (2); the mass ratio of 2-mercapto-1-methylimidazole to the gel microspheres in step (2) is 0.01-1:1 (preferably 0.05-0.075:1).

[0017] The present invention also provides an application of the hydroxyethyl chitosan microspheres as an antibody purification chromatography medium, wherein the antibody includes bovine immunoglobulin.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: the preparation method of the hydroxyethyl chitosan gel microspheres of the present invention is simple and inexpensive, and it has abundant internal pores while maintaining good mechanical strength and chemical stability; at the same time, the hydroxyethyl chitosan gel microspheres of the present invention have more hydroxyl groups, the cross-linking reaction is more controllable, and more reaction sites for activating and coupling functional ligands are provided, which meets the requirements as a chromatography medium, can replace traditional dextran gel and agarose gel for antibody purification, can separate antibodies and small molecule proteins, has high economic value, and is suitable for large-scale production. (iv) Description of the attached drawings

[0019] Figure 1 This is a schematic diagram of the molecular structure of hydroxyethyl chitosan gel microspheres.

[0020] Figure 2 This is a comparison image of hydroxyethyl chitosan gel microspheres (□) and commercially available dextran gel G75 (○) under an optical microscope.

[0021] Figure 3 The images are scanning electron microscope (SEM) images of the hydroxyethyl chitosan gel microspheres prepared in Example 1, magnified 60 times (a) and 8000 times (b).

[0022] Figure 4 The isothermal adsorption curve of bIgG on the hydroxyethyl chitosan gel microspheres prepared in Example 4 is shown.

[0023] Figure 5 These are the flow-through peak (lane 2) and elution peak (lane 3) of the mixed proteins in the hydrophobic charge chromatography column of Example 5, with lane 1 being the sample band.

[0024] Figure 6 This is the chromatogram of antibody and BSA separated by hydrophobic charge chromatography in Example 5.

[0025] Figure 7 These are the force / time curves for wet dextran gel G75 (a) and wet hydroxyethyl chitosan gel microspheres (b).

[0026] Figure 8 This is a chromatogram showing the separation of bovine antibody and tryptophan by hydroxyethyl chitosan gel microsphere chromatography. (V) Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: the room temperature described in the present invention is 25-30℃.

[0028] Example 1: Hydroxyethyl chitosan gel microspheres

[0029] (1) Preparation of gel microspheres: 20 mL of 4% HECTS aqueous solution was added to 140 mL of liquid paraffin containing 2.8 g Span 80 and 6 mL glutaraldehyde at 40 °C and 500 r / min. The reaction was carried out at 40 °C and 500 r / min for 1 h, and then the temperature was raised to 60 °C and the reaction was carried out at 500 r / min for 4 h. After the reaction was completed, the mixture was filtered, and the solid was soaked in acetone at room temperature for 10 min. Then it was washed with 60% petroleum ether aqueous solution until no oil film was formed. Finally, it was washed with deionized water until the solvent did not separate into layers and then dried under vacuum at 30 °C to obtain 0.6 g of HECTS gel microspheres.

[0030] (2) Activation of gel microspheres: Weigh 2g of HECTS gel microspheres and place them in 2mL of 40% DMSO aqueous solution, and add 0.6mL of allyl bromide and 0.25g of NaOH. React at 35℃ for 12h. After the reaction, the gel microspheres are reacted with 0.2g of NBS and 2mL of 30% acetone aqueous solution at 35℃ for 1h. After the reaction, wash with 50% acetone aqueous solution, 50% ethanol aqueous solution and deionized water respectively to obtain activated gel microspheres.

[0031] (3) Coupling ligands: The gel microspheres activated in step (2) were reacted with 0.15g MMI in 3mL of 1mol / L carbonate buffer (pH=10) at 35℃ for 10h. After filtration, the gel microspheres were washed with deionized water to obtain 1.2g of hydroxyethyl chitosan gel microspheres with a particle size distribution of 60-150μm and an MMI coupling density of up to 194μmol / g gel.

[0032] Hydroxyethyl chitosan gel microspheres and commercially available dextran gel G75 were examined using optical microscopy. The results are shown in [Figure number missing]. Figure 2 It can be seen that the particle size of hydroxyethyl chitosan gel microspheres is smaller than that of commercially available dextran gel G75.

[0033] Scanning electron microscopy images of hydroxyethyl chitosan gel microspheres at 60x and 8000x magnification. Figure 3 No pores were found in the microspheres.

[0034] Example 2: Hydroxyethyl chitosan gel microspheres

[0035] (1) Preparation of gel microspheres: 20 mL of 8% HECTS aqueous solution was added to 120 mL of liquid paraffin containing 6 g Span 80 and 4 mL epichlorohydrin at 60 °C and 400 r / min. The reaction was carried out at 60 °C and 400 r / min for 1 h, and then the temperature was raised to 80 °C and the reaction was carried out at 400 r / min for 4 h. After the reaction was completed, the mixture was filtered, and the solid was soaked in acetone at room temperature for 10 min. Then it was washed with 60% petroleum ether aqueous solution until no oil film was formed. Finally, it was washed with deionized water until the solvent did not separate into layers. The mixture was then dried under vacuum at 30 °C to obtain 1.3 g of HECTS gel microspheres.

[0036] (2) Activation of gel microspheres: Weigh 2g of HECTS gel microspheres prepared in step (1) and place them in 2mL of 40% DMSO aqueous solution. Add 0.4mL of allyl bromide and 0.48g of NaOH and react at 40℃ for 16h. After the reaction, react the gel microspheres with 0.2g of NBS and 2mL of 30% acetone aqueous solution at 40℃ for 3h. After the reaction, wash with 50% acetone aqueous solution, 50% ethanol aqueous solution and deionized water respectively to obtain activated gel microspheres.

[0037] (3) Coupling ligands: The gel microspheres that were fully activated in step (2) were reacted with 0.1g MMI in 3mL of 1 mol / L carbonate buffer (pH=10) at 40℃ for 10h. After filtration, the microspheres were washed with deionized water to obtain 1.4g of hydroxyethyl chitosan gel microspheres with a particle size distribution of 160-280μm and an MMI coupling density of up to 211.4μmol / g gel.

[0038] Optical microscopy results of hydroxyethyl chitosan gel microspheres showed that the particle size of the hydroxyethyl chitosan gel microspheres was similar to that of commercially available dextran gel G75, and 8000x scanning electron microscopy images showed obvious pores on the surface of the microspheres.

[0039] Example 3: Hydroxyethyl chitosan gel microspheres

[0040] (1) Preparation of gel microspheres: 15 mL of 8% HECTS aqueous solution was added to 40 mL of liquid paraffin containing 2.8 g Span 80 and 2 mL glutaraldehyde at 45 °C and 400 r / min. The reaction was carried out at 45 °C and 400 r / min for 5 h, and then the temperature was raised to 60 °C and the reaction was carried out at 400 r / min for 4 h. After the reaction was completed, the solid was filtered and soaked in acetone at room temperature for 10 min. Then it was washed with 60% petroleum ether aqueous solution until no oil film was formed. Finally, it was washed with deionized water until the solvent did not separate into layers. The solid was dried under vacuum at 30 °C to obtain 0.9 g of HECTS gel microspheres.

[0041] (2) Activation of gel microspheres: Weigh 2g of HECTS gel microspheres prepared in step (1) and place them in 2mL of 40% DMSO aqueous solution. Add 0.4mL of allyl bromide and 0.25g of NaOH and react at 30℃ for 12h. After the reaction, react the gel microspheres with 0.2g of NBS and 2mL of 30% acetone aqueous solution at 30℃ for 3h. After the reaction, wash with 50% acetone aqueous solution, 50% ethanol aqueous solution and deionized water respectively to obtain activated gel microspheres.

[0042] (3) Coupling ligands: The gel microspheres that were fully activated in step (2) were reacted with 0.1g MMI in 3mL of 1 mol / L carbonate buffer (pH=10) at 30℃ for 12h. After filtration, the microspheres were washed with deionized water to obtain 1.1g of hydroxyethyl chitosan gel microspheres with a particle size distribution of 300-600μm and an MMI coupling density of up to 176.9μmol / g gel.

[0043] Optical microscopy results of hydroxyethyl chitosan gel microspheres show that the particle size of the hydroxyethyl chitosan gel microspheres is slightly larger than that of commercially available dextran gel G75. 8000x scanning electron microscopy images show that the surface of the microspheres has tiny pores.

[0044] Example 4: Adsorption capacity of antibody by hydroxyethyl chitosan gel microspheres as a chromatography medium

[0045] Determination of bovine immunoglobulin (bIgG) adsorption capacity: 0.04 g of hydroxyethyl chitosan gel microspheres prepared according to the method in Example 3 was placed in a 5 mL centrifuge tube, and 4 mL of bIgG solution (2 mol / L phosphate buffer, pH 7.4) at different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL) was added. The centrifuge tube was placed in a constant temperature shaker at 25°C and shaken at 180 r / min for 10 h. After centrifugation, the supernatant was collected and the absorbance was measured at 280 nm using a UV-Vis spectrophotometer. The adsorbed protein concentration was calculated based on the protein concentration and the A280 standard curve. The adsorption isotherm was fitted using the Langmuir equation. The results are shown in [Figure 1]. Figure 4 As shown, the maximum adsorption capacity Q of hydroxyethyl chitosan gel microspheres m The concentration was 61.35 ± 4.8 mg / g, and the adsorption equilibrium constant K was... d The value was 4.25 ± 0.49, indicating that the prepared hydroxyethyl chitosan gel microspheres have good antibody adsorption capacity as a chromatography medium for antibody purification.

[0046] Example 5: Adsorption and elution of antibodies using hydroxyethyl chitosan gel microspheres as a chromatography medium.

[0047] Hydrophobic charge-induced chromatography (HCIC) columns were prepared by loading hydroxyethyl chitosan gel microspheres (0.6 cm inner diameter, 4 cm packing height) into a chromatography column. The flow rate (equilibration, loading, and elution flow rates were the same) was adjusted to 0.2 mL / min. The column was equilibrated with 5 column volumes of 2 mol / L PBS buffer (pH = 7.4). 500 μL of 2 mol / L PBS buffer (pH = 7.4) containing 0.5 mg / mL bIgG and 0.5 mg / mL BSA was loaded. Elution was performed with 0.1 mol / L citrate-sodium citrate buffer (pH = 4). The sample peaks were detected using a UV detector, and collection began at the start of peak elution. The first peak was the flow-through peak. Figure 5 Lane 2), the second peak is the elution peak ( Figure 5 Lane 3), the chromatogram of the elution peak is shown in [reference needed]. Figure 6 As shown.

[0048] According to the above method, the SDS gel electrophoresis diagram shows that the MMI-HECTS chromatography medium has the ability to adsorb antibodies. It can adsorb antibodies under high salt and neutral conditions, and achieve antibody elution under low salt and pH=4 conditions.

[0049] Example 6: Mechanical strength, chemical stability, and antibody separation effect of hydroxyethyl chitosan gel microspheres

[0050] (1) Mechanical strength

[0051] Commercially available single wet dextran gel microspheres G75 and hydroxyethyl chitosan gel microspheres prepared by the method in Example 2 were subjected to pressure resistance tests in a physical property testing apparatus. The results are shown in [Figure 1]. Figure 7 As shown, Figure 7 The results show that the hydroxyethyl chitosan gel microspheres have a slightly higher compressive strength than the dextran gel G75 during compression deformation.

[0052] (2) Chemical stability

[0053] 0.2 g of hydroxyethyl chitosan gel microspheres prepared by the method in Example 2 were soaked in acetone, ethanol, acetonitrile of different concentrations (10%-50%) and HCl or NaOH aqueous solutions of different pH values ​​(pH=3-12) for 48 h at room temperature. After centrifugation, the supernatant was removed, and the microspheres were weighed to calculate the retention rate. The microspheres were then observed using an optical microscope. The optical microscope results showed that the shape of the microspheres did not change after soaking in different organic reagents, and the retention rate was above 98%, demonstrating that the hydroxyethyl chitosan microspheres have good chemical stability.

[0054] (3) Effect of antibody separation

[0055] The swollen hydroxyethyl chitosan gel prepared by the method in Example 2 was loaded into a chromatography column (24 cm high, 1.1 cm in diameter). 0.1 mol / L phosphate (PBS) buffer was used as the equilibration buffer. The column was driven by a peristaltic pump and the flow rate was set to 0.75 mL / min. 200 μL of 0.1 mol / L phosphate (PBS) buffer containing 10 mg / mL bovine antibody and 1 mg / mL tryptophan was loaded as the sample. The sample peaks were detected using a UV detector.

[0056] Figure 8 The results showed that hydroxyethyl chitosan gel microspheres could completely separate antibodies and small molecules, with a resolution of 1.82.

Claims

1. A hydroxyethyl chitosan gel microsphere for antibody purification chromatography, characterized in that, The hydroxyethyl chitosan gel microspheres were prepared according to the following method: (1) Gel microspheres: Hydroxyethyl chitosan aqueous solution was dispersed in liquid paraffin containing crosslinking agent and Span 80 under stirring conditions of 40-60℃ and 100-500r / min. The reaction was carried out at 40-60℃ and 100-500r / min for 1-5h, and then the temperature was raised to 60-100℃ and the reaction was carried out at 100-500r / min for 2-8h. The mixture was filtered, and the filter cake was soaked in organic solvent, then washed with a 10-60% volume concentration organic solvent aqueous solution, and finally vacuum dried to obtain gel microspheres. The crosslinking agent included one of glutaraldehyde, epichlorohydrin, and N,N-methylenebisacrylamide. (2) Activation of gel microspheres: Add the gel microspheres from step (1) to a dimethyl sulfoxide aqueous solution, then add allyl bromide and NaOH, and react at 20-40℃ for 12-24h. After the reaction, react the gel microspheres with N-bromosuccinimide and a 30-70% volume concentration acetone aqueous solution at 20-40℃ for 1-5h. Filter, wash the filter cake, and obtain the activated gel microspheres. (3) Connecting functional ligands: The gel microspheres activated in step (2) are reacted with 2-mercapto-1-methylimidazole in carbonate buffer at 20-40℃ for 10-20h. After washing, hydroxyethyl chitosan gel microspheres are obtained.

2. The hydroxyethyl chitosan gel microspheres as described in claim 1, characterized in that, In step (1), the mass concentration of the hydroxyethyl chitosan aqueous solution is 1-8%, the volume ratio of the crosslinking agent to the hydroxyethyl chitosan aqueous solution is 0.1-1:1, the volume ratio of the liquid paraffin to the hydroxyethyl chitosan aqueous solution is 1-10:1, and the mass ratio of Span 80 to hydroxyethyl chitosan is 2-6:

1.

3. The hydroxyethyl chitosan gel microspheres as described in claim 1, characterized in that, The organic solvent used for soaking in step (1) includes one of methanol, ethanol, petroleum ether, and acetone; the organic solvent used for rinsing in the 10-60% aqueous solution of organic solvent includes one of methanol, ethanol, petroleum ether, and acetone.

4. The hydroxyethyl chitosan gel microspheres as described in claim 1, characterized in that, Step (2) The volume concentration of the dimethyl sulfoxide aqueous solution is 20-50%, and the volume of the dimethyl sulfoxide aqueous solution is 0.5-2 mL / g based on the mass of the gel microspheres; the volume of allyl bromide is 0.1-1 mL / g based on the mass of the gel microspheres; the mass ratio of NaOH to gel microspheres is 0.1-1:1; the mass ratio of N-bromosuccinimide to gel microspheres is 0.1-1:1; and the volume of the 30-70% acetone aqueous solution is 0.5-2 mL / g based on the mass of the gel microspheres.

5. The hydroxyethyl chitosan gel microspheres as described in claim 1, characterized in that, Step (2) Filter cake cleaning refers to cleaning with 50% acetone aqueous solution, 50% ethanol aqueous solution and deionized water respectively.

6. The hydroxyethyl chitosan gel microspheres as described in claim 1, characterized in that, The volume of carbonate buffer used in step (3) is 1-5 mL / g based on the mass of the gel microspheres in step (2); the mass ratio of 2-mercapto-1-methylimidazole to the gel microspheres in step (2) is 0.01-1:

1.

7. The application of the hydroxyethyl chitosan microspheres of claim 1 as an antibody purification chromatography medium.

8. The application as described in claim 7, characterized in that, The antibodies include bovine immunoglobulins.

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