Nhs-activated lysine-agarose gel microspheres, method for preparing the same and use thereof

Abstract

The application relates to the technical field of biomaterials, and provides NHS-activated lysine-agarose gel microspheres, a preparation method thereof and application thereof. The preparation method comprises the following steps: gradient solvent replacement is performed on agarose gel so that the agarose gel is transferred from an aqueous phase to an acetone phase; the agarose gel in the acetone phase is mixed with a carbonyl diimidazole acetone solution to perform an activation reaction, and is transferred to an aqueous phase; an L-lysine solution is reacted with 2-octenyl succinic anhydride to obtain a carboxylated lysine aqueous solution; the activated agarose gel and the carboxylated lysine aqueous solution are reacted; dimethyl sulfoxide solutions of EDC and NHS are sequentially added into the carboxylated lysine-agarose gel to perform a reaction, and NHS-activated lysine-agarose gel microspheres are obtained. By introducing a multi-functional lysine molecule and performing carboxylation with an anhydride, the density of active groups on the surface of agarose microspheres is significantly increased, and the defect of low site density in a traditional direct activation method is overcome.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and particularly relates to NHS-activated lysine-agarose gel microspheres, their preparation methods, and applications. Background Technology

[0002] NHS-activated agarose microspheres are an important class of functionalized chromatographic media and bioimmobilization carriers, widely used in biotechnology fields such as monoclonal antibody preparation, recombinant protein purification, immunoassay, nucleic acid extraction, and cell sorting. These microspheres use cross-linked agarose as a matrix, and their surface-modified highly active N-hydroxysuccinimide ester (NHS ester) groups can undergo efficient and specific amidation reactions with primary amino groups in biomolecules such as proteins, antibodies, and peptides, achieving covalent immobilization of target molecules. This immobilization method has significant advantages such as stable binding, high coupling efficiency, and low non-specific adsorption.

[0003] Ideal NHS-activated agarose microspheres should possess the following core characteristics: First, a suitable particle size distribution (typically 40-120 μm) to balance high specific surface area with good hydrodynamic performance, meeting the requirements of centrifugation, filtration, and column packing operations; second, a high density of active groups to ensure excellent ligand coupling capacity; third, excellent physicochemical stability, maintaining structural stability in a wide range of buffer systems (pH 3-10) and various organic solvents, and withstanding the operating pressures during chromatography; and fourth, good biocompatibility to ensure maximum preservation of biomolecule activity during coupling.

[0004] To endow agarose microspheres with the aforementioned functions, their activation and modification strategies are crucial. Traditional activation methods mainly include bromine cyanide (CNBr) activation and epoxy activation. However, these methods all have inherent drawbacks that limit their performance in high-precision applications: the bromine cyanide reagent used in CNBr activation is highly toxic and volatile, posing significant safety risks; furthermore, the isourea bonds formed after coupling with ligands are chemically and biologically unstable, prone to hydrolysis, and may introduce non-specific ion exchange reactions. Epoxy activation requires harsh reaction conditions (usually strong alkalinity and high temperature), is time-consuming, and the epoxy groups can react with thiol groups, hydroxyl groups, etc., in addition to primary amino groups, resulting in relatively poor specificity.

[0005] Furthermore, there are limitations: most existing methods directly activate the hydroxyl groups of agarose, often resulting in a difficulty in simultaneously achieving the desired density and steric accessibility of active sites in the microspheres. This is particularly problematic when immobilizing large molecular weight proteins (such as antibodies), where steric hindrance can lead to poor coupling efficiency. There are also defects in the preparation process: improper control of key process parameters such as solvent replacement efficiency, activator hydrolysis control, and intermediate washing can easily result in significant batch-to-batch variations, low active ester content, and poor stability in the final product. Moreover, conventional methods fail to effectively address the hydrophilicity-hydrophobicity balance on the microsphere surface, potentially leading to increased non-specific adsorption. Therefore, developing NHS-activated agarose microspheres with stable processing, high active site density, excellent coupling efficiency, and good batch-to-batch consistency is urgently needed and of great significance for technological advancement in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides NHS-activated lysine-agarose gel microspheres, their preparation method, and applications, aiming to solve the problems mentioned in the background art.

[0007] In a first aspect, the present invention provides a method for preparing NHS-activated lysine-agarose gel microspheres, comprising the following steps:

[0008] Step a: Perform gradient solvent replacement on the agarose gel to transfer it from the aqueous phase to the acetone phase, and measure the filling volume of the agarose gel;

[0009] Step b: Mix the agarose gel in the acetone phase with the carbonyl diimidazole acetone solution to carry out the activation reaction. After the reaction is complete, wash and transfer to the aqueous phase to obtain the activated agarose gel.

[0010] Step c: React L-lysine solution with 2-octenylsuccinic anhydride to obtain carboxylated lysine aqueous solution;

[0011] Step d: The activated agarose gel and carboxylysine aqueous solution are reacted. After the reaction is complete, the gel is washed to obtain carboxylysine-agarose gel.

[0012] Step e: Carboxylated lysine-agarose gel is reacted with EDC dimethyl sulfoxide solution and NHS dimethyl sulfoxide solution in sequence. After the reaction is complete, the gel is washed to obtain NHS-activated lysine-agarose gel microspheres.

[0013] Furthermore, in step a, the gradient solvent replacement of the agarose gel specifically involves:

[0014] S11: Filter and wash with 30% acetone aqueous solution;

[0015] S12: Filter and wash with 70% acetone aqueous solution;

[0016] S13: Filter and wash with pure acetone 3-5 times, soaking for 5 minutes after each wash to ensure that the agarose gel is completely replaced by acetone.

[0017] Further, in step b, the concentration of the carbonyl diimidazolium acetone solution is 0.1-0.5M, and the volume ratio of carbonyl diimidazolium acetone solution to agarose gel is 2-5:1. The activation reaction is specifically carried out by shaking at room temperature for 3-4 hours.

[0018] Further, step c specifically involves: using phosphate buffer as the reaction solvent, adding 2-octenyl succinic anhydride to the L-lysine solution, and reacting for 3-6 hours at pH 7.5-8.2 to obtain an aqueous solution of carboxylated lysine;

[0019] The concentration of the L-lysine solution was 0.5-1M, and the concentration ratio of L-lysine to 2-octenylsuccinic anhydride was 1:1.2.

[0020] Further, step d specifically involves: using phosphate buffer as the reaction solvent, adding carboxylysine aqueous solution to the activated agarose gel, reacting for 2 hours at pH=8.0-8.5, and then washing sequentially with phosphate buffer, glacial acetic acid / dimethyl sulfoxide solution, and dimethyl sulfoxide to obtain carboxylysine-agarose gel.

[0021] Further, in step e, the concentrations of the dimethyl sulfoxide solution of EDC and the dimethyl sulfoxide solution of NHS are both 0.5-2.0 mmol / mL, and the molar ratio of EDC:NHS is 1:1.

[0022] Secondly, the present invention provides NHS-activated lysine-agarose gel microspheres, which are prepared by the method for preparing NHS-activated lysine-agarose gel microspheres.

[0023] Furthermore, the application of NHS-activated lysine-agarose gel microspheres in coupling with ligands containing primary amino groups.

[0024] Furthermore, the ligands containing primary amino groups are proteins, polypeptides, antibodies, or amino-modified nucleic acids.

[0025] Thirdly, the present invention provides a separation medium for affinity chromatography comprising NHS-activated lysine-agarose gel microspheres.

[0026] The present invention has the following beneficial effects:

[0027] (1) The preparation method has high coupling efficiency. The mechanism is as follows: First, compared with glycine, which is a conventional amino and carboxyl donor material, the L-lysine used in this invention has a longer molecular chain, which can effectively reduce the steric hindrance effect of the active amino group, thereby simultaneously improving the reaction efficiency of the amino group with the acid anhydride and carbonyl diimidazole (CDI); Second, by introducing 2-octenyl succinic anhydride, this invention increases the number of carboxyl groups that can be activated by NHS on the agarose matrix: one carboxyl group comes from L-lysine, and the other comes from the carboxyl group generated after the acid anhydride in 2-octenyl succinic anhydride ring-opening, providing more carboxyl activation sites for 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), thereby increasing the density of N-hydroxysuccinimide (NHS) active ester, and finally greatly improving the coupling efficiency with the protein.

[0028] (2) The reaction conditions of the preparation method are mild, and the entire preparation process is carried out at room temperature, which avoids the damage of strong acid, strong alkali or high temperature to the agarose gel structure and maintains the good physical properties of the medium.

[0029] (3) The preparation method is controllable and reproducible. It adopts gradient solvent replacement, precise pH control and strict reagent dosage ratio to ensure the reproducibility of the preparation process and the stability of product quality.

[0030] (4) The obtained NHS-activated lysine-agarose gel microspheres have high density of NHS active ester groups introduced on the surface of the microspheres through lysine arms. They can be used to efficiently couple any ligand containing primary amines, such as antibodies, enzymes, protein A / G, etc., and have broad application prospects in the preparation of high-performance affinity chromatography media. Attached Figure Description

[0031] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0032] Figure 1 This is a schematic diagram of the process flow for preparing NHS-activated lysine-agarose gel microspheres according to the present invention.

[0033] Figure 2 This is a microscope image of the process for preparing NHS-activated lysine-agarose gel microspheres according to the present invention.

[0034] Figure 3 This is a chemical schematic diagram illustrating the preparation of NHS-activated lysine-agarose gel microspheres according to the present invention, wherein:

[0035] Figure 3 In this context, 'b' represents the chemical principle behind step b.

[0036] Figure 3 In this context, 'c' represents the chemical principle behind step c.

[0037] Figure 3 In this context, d represents the chemical principle of step d.

[0038] Figure 3 In this context, 'e' represents the chemical principle of step 'e'.

[0039] Figure 4 This is a bar chart showing the coupling efficiency of different EDC / NHS dosages in Embodiment 1 of the present invention.

[0040] Figure 5 This is a bar chart showing the coupling efficiency of different BSA loading amounts in Embodiment 2 of the present invention.

[0041] Figure 6 This is a bar chart showing the coupling efficiency of different BSA loading amounts in Embodiment 3 of the present invention.

[0042] Figure 7 This is a bar chart showing the coupling efficiency of different BSA loading amounts in Comparative Example 1 and Example 4 of this invention. Detailed Implementation

[0043] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0045] This invention provides a method for preparing NHS-activated lysine-agarose gel microspheres, comprising the following steps:

[0046] Step a: Perform gradient solvent replacement on the agarose gel to transfer it from the aqueous phase to the acetone phase, and measure the filling volume of the agarose gel;

[0047] Step b: Mix the agarose gel in the acetone phase with the carbonyl diimidazole acetone solution to carry out the activation reaction. After the reaction is complete, wash and transfer to the aqueous phase to obtain the activated agarose gel.

[0048] Step c: React L-lysine solution with 2-octenylsuccinic anhydride to obtain carboxylated lysine aqueous solution;

[0049] Step d: The activated agarose gel and carboxylysine aqueous solution are reacted. After the reaction is complete, the gel is washed to obtain carboxylysine-agarose gel.

[0050] Step e: Carboxylated lysine-agarose gel is reacted with EDC dimethyl sulfoxide solution and NHS dimethyl sulfoxide solution in sequence. After the reaction is complete, the gel is washed to obtain NHS-activated lysine-agarose gel microspheres.

[0051] A schematic diagram illustrating the chemical principle for preparing NHS-activated lysine-agarose gel microspheres, as shown below. Figure 3 As shown.

[0052] In some embodiments, step a, the gradient solvent replacement of the agarose gel specifically involves:

[0053] S11: Filter and wash with 30% acetone aqueous solution;

[0054] S12: Filter and wash with 70% acetone aqueous solution;

[0055] S13: Filter and wash with pure acetone 3-5 times, soaking for 5 minutes after each wash to ensure that the agarose gel is completely replaced by acetone.

[0056] In some embodiments, in step b, the concentration of the carbonyl diimidazolium acetone solution is 0.1-0.5M, and the volume ratio of carbonyl diimidazolium acetone solution to agarose gel is 2-5:1. The activation reaction is specifically a shaking reaction at room temperature for 3-4 hours.

[0057] In some embodiments, step c specifically involves: using phosphate buffer as the reaction solvent, adding 2-octenyl succinic anhydride to the L-lysine solution, and reacting for 3-6 hours under conditions of pH=7.5-8.2 to obtain an aqueous solution of carboxylated lysine;

[0058] The concentration of the L-lysine solution was 0.5-1M, and the concentration ratio of L-lysine to 2-octenylsuccinic anhydride was 1:1.2.

[0059] In some embodiments, step d specifically involves: using phosphate buffer as the reaction solvent, adding an aqueous solution of carboxylysine to the activated agarose gel, reacting for 2 hours at pH 8.0-8.5, and then washing sequentially with phosphate buffer, glacial acetic acid / dimethyl sulfoxide solution, and dimethyl sulfoxide to obtain carboxylysine-agarose gel.

[0060] In some embodiments, in step e, the concentrations of the dimethyl sulfoxide solution of EDC and the dimethyl sulfoxide solution of NHS are both 0.5-2.0 mmol / mL, and the molar ratio of EDC:NHS is 1:1.

[0061] In some embodiments, the present invention provides NHS-activated lysine-agarose gel microspheres, which are prepared by the method for preparing NHS-activated lysine-agarose gel microspheres.

[0062] In some embodiments, the use of NHS-activated lysine-agarose gel microspheres in coupling with ligands containing primary amino groups.

[0063] In some embodiments, the ligand containing a primary amino group is a protein, polypeptide, antibody, or amino-modified nucleic acid.

[0064] In some embodiments, the present invention provides a separation medium for affinity chromatography comprising NHS-activated lysine-agarose gel microspheres.

[0065] Example 1:

[0066] (1) Solvent replacement of agarose gel microspheres:

[0067] Gently invert the container twice, take 5 mL of agarose gel microspheres CL-6B, centrifuge to remove the preservation solution, add to a glass G3 filter, and then filter and wash once with deionized water to remove surface impurities.

[0068] Perform gradient solvent replacement:

[0069] Wash once by filtration with 9 mL of 30% acetone aqueous solution;

[0070] Wash once by filtration with 9 mL of 70% acetone aqueous solution;

[0071] Wash five times with 15 mL of pure acetone, soaking for five minutes after each wash to ensure the agarose gel is completely replaced by acetone.

[0072] Transfer the agarose gel in acetone to a 50 mL centrifuge tube, centrifuge at 2600 rpm for 10 minutes, discard the supernatant, and record the filling volume of the agarose gel at the bottom of the tube as 3.0 mL.

[0073] (2) Carbonyl diimidazole (CDI) activated agarose

[0074] Prepare 10 mL of 0.25 M carbonyl diimidazolium acetone solution under dry conditions, add it to 3.0 mL of agarose gel obtained in step (1), and react with low-speed shaking at room temperature for 3 hours to obtain activated agarose gel.

[0075] Transfer the activated agarose gel to a glass G3 filter, filter and wash three times with 15 mL of pure acetone to remove unreacted carbonyl diimidazole, then filter and wash twice with 15 mL of deionized water. Transfer the activated agarose gel from the acetone phase to the aqueous phase, discard the washing solution, and set aside for later use.

[0076] (3) Lysine carboxylation

[0077] Using phosphate buffer as the reaction solvent and triethylamine to adjust the pH, 1.38 mL of 2-octenyl succinic anhydride was added to 10 mL of 0.5 M L-lysine aqueous solution, and the reaction was carried out at pH 7.5 for 3 hours to obtain carboxylated lysine aqueous solution.

[0078] (4) Lysine coupling

[0079] Using phosphate buffer as the reaction solvent and adjusting the pH with NaOH, 9 mL of carboxylysine aqueous solution obtained in step (3) was added to the activated agarose gel obtained in step (2). The reaction was carried out for 2 hours at pH=8.0. Then, the gel was washed 3 times with 9 mL of phosphate buffer, 2 times with 9 mL of 5% (v / v) glacial acetic acid / dimethyl sulfoxide (DMSO) solution, and finally 2 times with 9 mL of dimethyl sulfoxide to obtain carboxylysine-agarose gel. The carboxylysine-agarose gel was then transferred from the phosphate buffer phase to the dimethyl sulfoxide phase for later use.

[0080] (5) N-hydroxysuccinimide (NHS) activation

[0081] The carboxylysine-agarose gel obtained in step (4) was divided into three portions of 1 mL each. 4 mL of 0.5 mmol / mL, 1 mmol / mL, and 2 mmol / mL dimethyl sulfoxide solutions of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and NHS were prepared respectively. First, the dimethyl sulfoxide solution of EDC was added to the carboxylysine-agarose gel and the mixture was shaken at room temperature for 10 min. Then, the dimethyl sulfoxide solution of NHS was added and the mixture was shaken at room temperature for 2 hours to complete the conversion of the carboxyl group to NHS ester.

[0082] After the reaction was complete, the mixture was filtered and washed four times with 15 mL of dimethyl sulfoxide solution to completely remove free EDC, NHS and byproducts, thus obtaining NHS-activated lysine-agarose gel microspheres.

[0083] A schematic diagram of the process for preparing NHS-activated lysine-agarose gel microspheres is shown below. Figure 1 As shown.

[0084] Microscopy in the process of preparing NHS-activated lysine-agarose gel microspheres, such as Figure 2 As shown.

[0085] Test protein coupling efficiency: Take 1 mL of each of the 3 NHS activated lysine-agarose gels prepared in step (5), wash them 3 times with 5 mL of phosphate buffer (pH=7.4), and then transfer them into phosphate buffer.

[0086] For three NHS-activated lysine-agarose gels, the ratio of bovine serum albumin (BSA) to NHS-activated lysine-agarose gel was 5 mg: 1 mL. BSA was added to each gel, followed by 1.5 mL of phosphate buffer. The mixture was shaken at room temperature for 1 hour. After the reaction, the reaction solution was collected, and the concentration of uncoupled BSA in the supernatant was determined using the Bradford method. The coupling efficiency was calculated, and the results are shown in Table 1.

[0087] Table 1. Coupling efficiency of NHS-activated lysine-agarose gel in Example 1

[0088]

[0089] Coupling efficiency (%) = [(protein loading amount - protein volume in supernatant) / protein loading amount] × 100%

[0090] The coupling efficiency test results are as follows Figure 4 As shown, the results indicate that under the conditions of Example 1, when the concentrations of EDC and NHS added were 1 mmol / mL and the amount of BSA loaded was 5 mg / mL, the coupling efficiency reached 93.38%.

[0091] Example 2:

[0092] (1) Solvent replacement of agarose gel microspheres:

[0093] Gently invert the container three times, take 7 mL of agarose gel microspheres CL-6B, centrifuge to remove the preservation solution, add to a glass G3 filter, and then filter and wash once with deionized water to remove surface impurities.

[0094] Perform gradient solvent replacement:

[0095] Wash once by filtration with 15 mL of 30% acetone aqueous solution;

[0096] Wash once by filtration with 15 mL of 70% acetone aqueous solution;

[0097] Wash five times with 25 mL of pure acetone, soaking for five minutes after each wash to ensure the agarose gel is completely replaced by acetone.

[0098] Transfer the agarose gel in acetone to a 50 mL centrifuge tube, centrifuge at 2600 rpm for 10 minutes, discard the supernatant, and record the filling volume of the agarose gel at the bottom of the tube as 5.0 mL.

[0099] (2) Carbonyl diimidazole (CDI) activated agarose

[0100] Prepare 15 mL of 0.25 M carbonyl diimidazolium acetone solution under dry conditions, add it to 5.0 mL of agarose gel obtained in step (1), and react with low-speed shaking at room temperature for 3 hours to obtain activated agarose gel.

[0101] Transfer the activated agarose gel to a glass G3 filter, filter and wash three times with 25 mL of pure acetone to remove unreacted carbonyl diimidazole, then filter and wash twice with 25 mL of deionized water. Transfer the activated agarose gel from the acetone phase to the aqueous phase, discard the washing solution, and set aside for later use.

[0102] (3) Lysine carboxylation

[0103] Using phosphate buffer as the reaction solvent and triethylamine to adjust the pH, 4.14 mL of 2-octenyl succinic anhydride was added to 15 mL of 1.0 M L-lysine aqueous solution, and the reaction was carried out at pH 8.2 for 6 hours to obtain carboxylated lysine aqueous solution.

[0104] (4) Lysine coupling

[0105] Using phosphate buffer as the reaction solvent and adjusting the pH with NaOH, 15 mL of carboxylysine aqueous solution obtained in step (3) was added to the activated agarose gel obtained in step (2). The reaction was carried out for 2 hours at pH=8.5. Then, the gel was washed 3 times with 15 mL of phosphate buffer, 2 times with 15 mL of 5% (v / v) glacial acetic acid / dimethyl sulfoxide solution, and finally 2 times with 15 mL of dimethyl sulfoxide to obtain carboxylysine-agarose gel. The carboxylysine-agarose gel was then transferred from the phosphate buffer phase to the dimethyl sulfoxide phase for later use.

[0106] (5) N-hydroxysuccinimide (NHS) activation

[0107] Prepare 10 mL of 0.5 mmol / mL EDC dimethyl sulfoxide solution and NHS dimethyl sulfoxide solution respectively. First, add EDC dimethyl sulfoxide solution to the carboxylysine-agarose gel obtained in step (4), shake at room temperature for 20 min, then add NHS dimethyl sulfoxide solution, shake at room temperature for 2 hours to complete the conversion of carboxyl group to NHS ester.

[0108] After the reaction was complete, the mixture was filtered and washed four times with 15 mL of dimethyl sulfoxide solution to completely remove free EDC, NHS and byproducts, thus obtaining NHS-activated lysine-agarose gel microspheres.

[0109] Test protein coupling efficiency: Take 2.5 mL of NHS activated lysine-agarose gel obtained in step (5), wash it 3 times with 15 mL of phosphate buffer (pH=7.4), and then transfer it into phosphate buffer.

[0110] The NHS-activated lysine-agarose gel was evenly divided into 5 equal parts. Bovine serum albumin (BSA):NHS-activated lysine-agarose gel was added in ratios of 1 mg:1 mL, 2 mg:1 mL, 3 mg:1 mL, 4 mg:1 mL, and 5 mg:1 mL, respectively. Then, 1 mL of phosphate buffer was added to each part, and the mixture was shaken at room temperature for 1 hour. After the reaction was completed, the reaction solution was collected, and the concentration of uncoupled BSA in the supernatant was determined by the Bradford method. The coupling efficiency was calculated, and the results are shown in Table 2.

[0111] Table 2. Coupling efficiency of NHS-activated lysine-agarose gel in Example 2

[0112]

[0113] The coupling efficiency test results are as follows Figure 5 As shown, the results indicate that under the conditions of Example 2, when the amount of bovine serum albumin loaded was 1-5 mg / mL, the coupling efficiency of NHS-activated lysine-agarose gel was extremely high (>85%), and the stability was good, proving that it has high coupling capacity and efficiency.

[0114] Example 3:

[0115] (1) Solvent replacement of agarose gel microspheres:

[0116] Gently invert the container three times, take 3 mL of agarose gel microspheres CL-6B, centrifuge to remove the preservation solution, add to a glass G3 filter, and then filter and wash once with deionized water to remove surface impurities.

[0117] Perform gradient solvent replacement:

[0118] Wash once by filtration with 6 mL of 30% acetone aqueous solution;

[0119] Wash once by filtration with 6 mL of 70% acetone aqueous solution;

[0120] Wash five times with 10 mL of pure acetone, soaking for five minutes after each wash to ensure the agarose gel is completely replaced by acetone.

[0121] Transfer the agarose gel in acetone to a 50 mL centrifuge tube, centrifuge at 2600 rpm for 10 minutes, discard the supernatant, and record the filling volume of the agarose gel at the bottom of the tube as 2.0 mL.

[0122] (2) Carbonyl diimidazole (CDI) activated agarose

[0123] Prepare 6 mL of 0.25 M carbonyl diimidazolium acetone solution under dry conditions, add it to 2.0 mL of agarose gel obtained in step (1), and react with low-speed shaking at room temperature for 3 hours to obtain activated agarose gel.

[0124] Transfer the activated agarose gel to a glass G3 filter, filter and wash three times with 9 mL of pure acetone to remove unreacted carbonyl diimidazole, then filter and wash twice with 10 mL of deionized water. Transfer the activated agarose gel from the acetone phase to the aqueous phase, discard the washing solution, and set aside for later use.

[0125] (3) Lysine carboxylation

[0126] Using phosphate buffer as the reaction solvent and triethylamine to adjust the pH, 1.32 mL of 2-octenyl succinic anhydride was added to 6 mL of 0.8 M L-lysine aqueous solution, and the reaction was carried out at pH 8.0 for 4 hours to obtain carboxylated lysine aqueous solution.

[0127] (4) Lysine coupling

[0128] Using phosphate buffer as the reaction solvent and adjusting the pH with NaOH, 6 mL of carboxylysine aqueous solution obtained in step (3) was added to the activated agarose gel obtained in step (2). The reaction was carried out for 2 hours at pH=8.2. Then, the gel was washed 3 times with 6 mL of phosphate buffer, 2 times with 6 mL of 5% (v / v) glacial acetic acid / dimethyl sulfoxide solution, and finally 2 times with 6 mL of dimethyl sulfoxide to obtain carboxylysine-agarose gel. The carboxylysine-agarose gel was then transferred from the phosphate buffer phase to the dimethyl sulfoxide phase for later use.

[0129] (5) N-hydroxysuccinimide (NHS) activation

[0130] Prepare 4 mL of 1.0 mmol / mL EDC dimethyl sulfoxide solution and NHS dimethyl sulfoxide solution respectively. First, add EDC dimethyl sulfoxide solution to the carboxylysine-agarose gel obtained in step (4), shake at room temperature for 10 min, then add NHS dimethyl sulfoxide solution, shake at room temperature for 2 hours to complete the conversion of carboxyl group to NHS ester.

[0131] After the reaction was complete, the mixture was filtered and washed four times with 6 mL of dimethyl sulfoxide solution to completely remove free EDC, NHS and byproducts, thus obtaining NHS-activated lysine-agarose gel microspheres.

[0132] Test protein coupling efficiency: Take 2.0 mL of NHS activated lysine-agarose gel obtained in step (5), wash it 3 times with 6 mL of phosphate buffer (pH=7.4), and then transfer it into phosphate buffer.

[0133] The NHS-activated lysine-agarose gel was evenly divided into four equal parts. Bovine serum albumin (BSA) was added to the NHS-activated lysine-agarose gel at ratios of 4 mg:1 mL, 8 mg:1 mL, 16 mg:1 mL, and 32 mg:1 mL, respectively. Each part was then diluted with 2 mL of phosphate buffer and allowed to react at room temperature with shaking for 1 hour. After the reaction was completed, the reaction solution was collected, and the concentration of uncoupled BSA in the supernatant was determined using the Bradford method. The coupling efficiency was calculated, and the results are shown in Table 3.

[0134] Table 3. Coupling efficiency of NHS-activated lysine-agarose gel in Example 3

[0135]

[0136] The coupling efficiency test results are as follows Figure 6 As shown, the results indicate that under the conditions of Example 3, when the bovine serum albumin loading amount was 4 or 8 mg / mL, the coupling efficiency of NHS-activated lysine-agarose gel microspheres was very high. However, when the bovine serum albumin loading amount was above 16 mg / mL, the coupling efficiency of NHS-activated lysine-agarose gel microspheres decreased significantly. This indicates that the maximum protein loading of NHS-activated lysine-agarose gel microspheres is approximately 16 mg / mL, which is much higher than the maximum protein loading of products prepared by traditional methods.

[0137] Example 4:

[0138] (1) Solvent replacement of agarose gel microspheres:

[0139] Gently invert the container twice, take 3 mL of agarose gel microspheres CL-6B, centrifuge to remove the preservation solution, add to a glass G3 filter, and then filter and wash once with deionized water to remove surface impurities.

[0140] Perform gradient solvent replacement:

[0141] Wash once by filtration with 9 mL of 30% acetone aqueous solution;

[0142] Wash once by filtration with 9 mL of 70% acetone aqueous solution;

[0143] Wash five times with 9 mL of pure acetone, soaking for five minutes after each wash to ensure the agarose gel is completely replaced by acetone.

[0144] Transfer the agarose gel in acetone to a 50 mL centrifuge tube, centrifuge at 2600 rpm for 10 minutes, discard the supernatant, and record the filling volume of the agarose gel at the bottom of the tube as 2.7 mL.

[0145] (2) Carbonyl diimidazole (CDI) activated agarose

[0146] Prepare 8.1 mL of 0.25 M carbonyl diimidazolium acetone solution under dry conditions, add it to 2.7 mL of agarose gel obtained in step (1), and react with low-speed shaking at room temperature for 3 hours to obtain activated agarose gel.

[0147] Transfer the activated agarose gel to a glass G3 filter, filter and wash twice with 9 mL of pure acetone to remove unreacted carbonyl diimidazole, then filter and wash twice with 10 mL of deionized water. Transfer the activated agarose gel from the acetone phase to the aqueous phase, discard the washing solution, and set aside for later use.

[0148] (3) Lysine carboxylation

[0149] Using phosphate buffer as the reaction solvent and triethylamine to adjust the pH, 2.07 mL of 2-octenyl succinic anhydride was added to 10 mL of 0.75 M L-lysine aqueous solution, and the reaction was carried out at pH 8.0 for 4 hours to obtain carboxylated lysine aqueous solution.

[0150] (4) Lysine coupling

[0151] Using phosphate buffer as the reaction solvent and adjusting the pH with NaOH, 8.1 mL of carboxylysine aqueous solution obtained in step (3) was added to the activated agarose gel obtained in step (2). The reaction was carried out for 2 hours at pH=8.0. Then, the gel was washed 3 times with 8.1 mL of phosphate buffer, 2 times with 8.1 mL of 5% (v / v) glacial acetic acid / dimethyl sulfoxide solution, and finally 2 times with 8.1 mL of dimethyl sulfoxide to obtain carboxylysine-agarose gel. The carboxylysine-agarose gel was then transferred from the phosphate buffer phase to the dimethyl sulfoxide phase for later use.

[0152] (5) N-hydroxysuccinimide (NHS) activation

[0153] Prepare 4 mL of 1.0 mmol / mL EDC dimethyl sulfoxide solution and NHS dimethyl sulfoxide solution respectively. First, add EDC dimethyl sulfoxide solution to the carboxylysine-agarose gel obtained in step (4), shake at room temperature for 10 min, then add NHS dimethyl sulfoxide solution, shake at room temperature for 2 hours to complete the conversion of carboxyl group to NHS ester.

[0154] After the reaction was complete, the mixture was filtered and washed four times with 8.1 mL of dimethyl sulfoxide solution to completely remove free EDC, NHS and byproducts, thus obtaining NHS-activated lysine-agarose gel microspheres.

[0155] Comparative Example 1:

[0156] Comparative Example 1 describes a method for introducing carboxyl groups into agarose gel microspheres using glycine. The entire preparation process was carried out under the conditions of Example 4, except that lysine in Example 4 was replaced with glycine, and the step of introducing 2-octenyl succinic anhydride was omitted. The coupling efficiency test results of the NHS-activated lysine-agarose gel microspheres prepared in Example 4 and the NHS-activated glycine-agarose gel microspheres prepared in Comparative Example 1 are shown in Table 4.

[0157] Table 4 Coupling efficiency of Comparative Example 1 and Example 4

[0158]

[0159] The coupling efficiency test results are as follows Figure 7 As shown, the results indicate that, compared to introducing carboxyl groups using glycine and then carboxylating with anhydride, the NHS-activated lysine-agarose gel microspheres prepared have a higher coupling efficiency with bovine serum albumin.

[0160] The mechanism may be as follows: First, compared with glycine, L-lysine has a longer molecular chain, which can effectively reduce the steric hindrance effect of the active amino group, thereby simultaneously improving the reaction efficiency of the amino group with acid anhydride and carbonyl diimidazole (CDI); Second, by introducing 2-octenyl succinic anhydride, the number of carboxyl groups available for NHS activation on the agarose matrix is ​​increased: one carboxyl group comes from L-lysine, and the other comes from the carboxyl group generated after the anhydride in 2-octenyl succinic anhydride ring-opening, providing more carboxyl activation sites for EDC, thereby increasing the density of NHS active esters, and ultimately significantly improving the coupling efficiency with proteins.

[0161] In summary, this invention significantly increases the density of active groups on the surface of agarose microspheres by introducing multifunctional lysine molecules and carboxylating them with acid anhydrides, overcoming the low site density defect of traditional direct activation methods. The coupling efficiency between the prepared NHS-activated lysine-agarose gel microspheres and proteins reaches as high as 93.38%, which is significantly better than traditional methods. These NHS-activated lysine-agarose gel microspheres are used to prepare high-performance affinity chromatography media and have broad application prospects in the field of bioseparation and purification.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing NHS-activated lysine-agarose gel microspheres, characterized in that, Includes the following steps: Step a: Perform gradient solvent replacement on the agarose gel to transfer it from the aqueous phase to the acetone phase, and measure the filling volume of the agarose gel; Step b: Mix the agarose gel in the acetone phase with the carbonyl diimidazole acetone solution to carry out the activation reaction. After the reaction is complete, wash and transfer to the aqueous phase to obtain the activated agarose gel. Step c: React L-lysine solution with 2-octenylsuccinic anhydride to obtain carboxylated lysine aqueous solution; Step d: The activated agarose gel and carboxylysine aqueous solution are reacted. After the reaction is complete, the gel is washed to obtain carboxylysine-agarose gel. Step e: Carboxylated lysine-agarose gel is reacted with EDC dimethyl sulfoxide solution and NHS dimethyl sulfoxide solution in sequence. After the reaction is complete, the gel is washed to obtain NHS-activated lysine-agarose gel microspheres.

2. The preparation method according to claim 1, characterized in that: In step a, the gradient solvent replacement of the agarose gel is specifically performed as follows: S11: Filter and wash with 30% acetone aqueous solution; S12: Filter and wash with 70% acetone aqueous solution; S13: Filter and wash with pure acetone 3-5 times, soaking for 5 minutes after each wash.

3. The preparation method according to claim 2, characterized in that: In step b, the concentration of the carbonyl diimidazolium acetone solution is 0.1-0.5M, and the volume ratio of carbonyl diimidazolium acetone solution to agarose gel is 2-5:

1. The activation reaction is specifically carried out by shaking at room temperature for 3-4 hours.

4. The preparation method according to claim 3, characterized in that: Step c specifically involves adding 2-octenyl succinic anhydride to an L-lysine solution using phosphate buffer as the reaction solvent, and reacting for 3-6 hours at pH 7.5-8.2 to obtain an aqueous solution of carboxylated lysine. The concentration of the L-lysine solution was 0.5-1M, and the concentration ratio of L-lysine to 2-octenylsuccinic anhydride was 1:1.

2.

5. The preparation method according to claim 4, characterized in that: Step d specifically involves adding carboxylysine aqueous solution to the activated agarose gel using phosphate buffer as the reaction solvent, reacting for 2 hours at pH 8.0-8.5, and then washing sequentially with phosphate buffer, glacial acetic acid / dimethyl sulfoxide solution, and dimethyl sulfoxide to obtain carboxylysine-agarose gel.

6. The preparation method according to claim 5, characterized in that: In step e, the concentrations of both the dimethyl sulfoxide solution of EDC and the dimethyl sulfoxide solution of NHS are 0.5-2.0 mmol / mL, and the molar ratio of EDC:NHS is 1:

1.

7. NHS-activated lysine-agarose gel microspheres, characterized in that: It is prepared by any one of the preparation methods described in claims 1-6.

8. The application of the NHS-activated lysine-agarose gel microspheres as described in claim 7 in the coupling of ligands containing primary amino groups.

9. The application as described in claim 8, characterized in that: Ligands containing primary amino groups are proteins, polypeptides, antibodies, or amino-modified nucleic acids.

10. A separation medium for affinity chromatography, characterized in that, The product comprises NHS-activated lysine-agarose gel microspheres as described in claim 7.

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