Strong cation exchange chromatography medium as well as preparation method and application thereof
The preparation of strong cation exchange chromatography media by single-step seed swelling technology and epoxy-thiol click reaction solves the problems of complex preparation and high cost in existing technologies, and achieves high loading capacity and low non-specific adsorption, which is suitable for biological separation and purification.
Patent Information
- Application Number
- CN202511661264.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2026-02-06
AI Technical Summary
The preparation process of existing strong cation exchange chromatography media is complex and costly, making it difficult to meet the needs of industrial production. Furthermore, traditional media suffer from non-specific adsorption problems during use.
A single-step seed swelling technique was used to synthesize epoxy porous polymer microspheres. Sulfonic acid groups were introduced through an epoxy-mercapto-click reaction, and the hydrophilicity of the medium was enhanced through a hydrolysis reaction, which simplifies the preparation process and reduces costs.
This invention enables the development of a strong cation exchange chromatography medium with high loading capacity and low non-specific adsorption, which simplifies the preparation process, reduces production costs, and improves the biocompatibility and industrial production efficiency of the medium.
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Figure CN121471403A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological separation and purification technology, and more specifically to a strong cation exchange chromatography medium, its preparation method, and its application. Background Technology
[0002] Ion exchange chromatography is a chromatographic technique that separates different biomolecules based on the differences in their charge properties and quantities. It is widely used for the large-scale separation and purification of various biomolecules such as proteins, vaccines, and antibodies. Ion exchange chromatography is a branch of high-performance liquid chromatography (HPLC) and a crucial foundation of it. Thanks to the high performance of its chromatographic packing materials, HPLC has become an indispensable tool in bioanalysis.
[0003] Ion exchange chromatography media consist of a support and surface functional groups, and can be classified into strong anion exchange chromatography media, strong cation exchange chromatography media, weak anion exchange chromatography media, and weak cation exchange chromatography media. The support matrix is usually a polymer or a sugar, and different functional groups are modified on its surface depending on the application. Commonly used polymer matrices include polystyrene and polymethacrylate, while agarose is a commonly used sugar matrix.
[0004] However, polymer microspheres are usually highly hydrophobic and cannot be used directly as chromatographic packing materials. They need to be modified to be hydrophilic on the surface first. The process is complex and difficult to control. In addition, the raw material costs of both polyethyleneimine and dextran are high, which is not conducive to industrial production.
[0005] Therefore, how to develop an ion exchange chromatography medium that is simple, efficient, low-cost, and conducive to industrial production is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the purpose of the present invention is to provide a strong cation exchange chromatography medium, its preparation method and application, so as to overcome the shortcomings of the prior art.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] A strong cation exchange chromatography medium, with the following structural formula: ,in, It is polyglycidyl methacrylate (PGMA).
[0009] A method for preparing the above-mentioned strong cation exchange chromatography medium specifically includes the following steps:
[0010] (1) Epoxy porous polymer microspheres were synthesized using a single-step seed swelling technique;
[0011] (2) Epoxy porous polymer microspheres were reacted with mercapto sulfonates in an epoxy-mercapto-click reaction;
[0012] (3) Hydrolyze the residual epoxy groups on the surface of the epoxy porous polymer microspheres to obtain a strong cation exchange chromatography medium.
[0013] This invention first uses a single-step seed swelling technique to synthesize a uniform porous polymer microsphere with a surface rich in epoxy groups and a specific pore structure; then, it uses a click reaction between the surface epoxy and mercaptosulfonate to obtain a strong cation exchange chromatography medium with a surface rich in sulfonic acid groups; finally, it undergoes a hydrolysis reaction to further enhance the hydrophilicity of the matrix, thereby reducing the non-specific adsorption of the chromatography medium.
[0014] Furthermore, in step (1) above, the method for synthesizing epoxy porous polymer microspheres is as follows: seed microspheres are added to an aqueous solution for dispersion to obtain a seed dispersion; epoxy monomers, crosslinking agents, pore-forming agents and initiators are added to an aqueous solution for homogenization and emulsification to obtain an emulsified oil phase; the emulsified oil phase is added to the seed dispersion for swelling, heating to initiate polymerization, and washing to obtain epoxy porous polymer microspheres.
[0015] The further beneficial effects of the above-mentioned method are that the present invention uses a single-step seed swelling technology to synthesize uniformly sized monodisperse porous polymer microspheres. The process is simple and controllable, and uniformly sized microspheres of the corresponding size can be obtained without a complex sieving process. Moreover, the pore properties of the porous microspheres are controllable.
[0016] Furthermore, the aforementioned seed microspheres are non-crosslinked linear polystyrene seed microspheres with a particle size of 3~10μm, preferably 4.5μm; the epoxy monomer is polyglycidyl methacrylate (PGMA); the crosslinking agent is triethylene glycol dimethacrylate (TEGDMA); the pore-forming agent is at least one of toluene and cyclohexanol; the initiator is benzoyl peroxide; and the aqueous phase solution is a mixture of 1% polyvinyl alcohol (PVA) and 0.25% sodium dodecyl sulfate (SDS).
[0017] The further beneficial effect of the above-mentioned method is that the present invention uses a hydrophilic crosslinking agent, triethylene glycol dimethacrylate, in the process of synthesizing porous polymer microspheres, thereby increasing the hydrophilicity of the microspheres.
[0018] Furthermore, the swelling temperature is 30-40°C, preferably 35°C; the time is 12-36 hours, preferably 24 hours.
[0019] Furthermore, the polymerization temperature is 65-80℃, preferably 70℃; the time is 12-24h, preferably 16h.
[0020] Furthermore, in step (2) above, the mercapto sulfonate is sodium 3-mercapto-1-propanesulfonate (MPS), sodium 2-mercaptoethanesulfonate (MESNA), or sodium 2,3-dimercaptopropanesulfonate (Na-DMPS).
[0021] Furthermore, in step (2) above, the epoxy-mercaptoclick reaction needs to be carried out under alkaline conditions. Even further, the alkaline reagent is potassium carbonate (K2CO3), sodium carbonate (Na2CO3), or lithium hydroxide (LiOH).
[0022] The further beneficial effect of the above-mentioned method is that the present invention uses an epoxy-mercapto-click reaction, which utilizes the mercapto group in mercaptosulfonate to react with the epoxy ring-opening reaction on the surface of polymer microspheres to introduce alcohol hydroxyl groups, and directly introduces long-chain alkane sulfonate groups on the surface of porous polymer.
[0023] Furthermore, in step (3) above, hydrolysis needs to be carried out under acidic conditions. Even further, the acidic reagent is 0.1-1M sulfuric acid, preferably 0.5M sulfuric acid.
[0024] The further beneficial effect of the above-mentioned method is that the residual epoxy is further hydrolyzed into a diol structure through a hydrolysis reaction, increasing the hydrophilicity of the medium, and the introduction of long-chain alkanes can reduce steric hindrance. Thus, the hydrophilicity of the medium is greatly improved, avoiding the complex hydrophilic modification process required in the production of traditional ion exchange chromatography media.
[0025] This invention also claims protection for the use of the above-described strong cation exchange chromatography medium or the strong cation exchange chromatography medium prepared by the above-described method in the separation and purification of biomacromolecules.
[0026] Furthermore, the aforementioned biological macromolecules include proteins, vaccines, and antibodies.
[0027] It is worth noting that the apparent properties of the strong cation exchange chromatography medium of the present invention can be characterized by optical microscopy and scanning electron microscopy, the performance can be characterized by the dynamic protein loading after it is packed into the column, the separation performance can be represented by the protein separation and purification effect after packing into the column, and the hydrophilicity can be characterized by the protein recovery rate after the column packing test.
[0028] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:
[0029] The strong cation exchange chromatography medium of this invention has advantages such as high protein loading capacity, low non-specific adsorption, and good mechanical properties. Moreover, the preparation process is simple and does not require special hydrophilic modification. Under the condition that conventional protein elution can be met, the process is simplified and the industrial production cycle is shortened. At the same time, the high efficiency of the click reaction can meet the characteristics of high loading capacity and good biocompatibility of the chromatography medium. Attached Figure Description
[0030] Figure 1 This is a process diagram of the preparation method of the strong cation exchange chromatography medium of the present invention;
[0031] Figure 2 SEM images of the strong cation exchange chromatography media in Examples 1-4;
[0032] Figure 3 The dynamic binding loading statistics of the strong cation exchange chromatography media in Examples 1-10 and Comparative Examples 1-2 are shown in the figure.
[0033] Figure 4 The graph shows the protein recovery rates of the strong cation exchange chromatography media in Examples 1-10 and Comparative Examples 1-2.
[0034] Figure 5 This is a dynamic binding loading spectrum of the strong cation exchange chromatography medium in Example 2;
[0035] Figure 6 This is a functional map of the target protein purified by strong cation exchange chromatography in Example 2. Detailed Implementation
[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0037] Example 1
[0038] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0039] (1) Synthesis of epoxy-rich polymer matrix
[0040] 20 g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5 μm were added to 900 g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5 L reactor, heated to 35 °C, and continuously stirred to obtain a seed dispersion. 200 g of PGMA, 500 g of TEGDMA, 260 g of toluene, and 3.15 g of benzoyl peroxide were weighed and added to 2400 g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was less than 1 μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24 h to allow swelling. 200 g of 1% PVA solution was added, and polymerization was initiated at 70 °C. The reaction was continued for 16 h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0041] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0042] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0043] (3) Hydrolysis of residual epoxy groups on the surface
[0044] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0045] Example 2
[0046] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0047] (1) Synthesis of epoxy-rich polymer matrix
[0048] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0049] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0050] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0051] (3) Hydrolysis of residual epoxy groups on the surface
[0052] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0053] Example 3
[0054] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0055] (1) Synthesis of epoxy-rich polymer matrix
[0056] 20 g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5 μm were added to 900 g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5 L reactor, heated to 35 °C, and continuously stirred to obtain a seed dispersion. 200 g of PGMA, 200 g of TEGDMA, 260 g of toluene, and 3.15 g of benzoyl peroxide were weighed and added to 2400 g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1 μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24 h to allow swelling. 200 g of 1% PVA solution was added, and polymerization was initiated at 70 °C. The reaction was continued for 16 h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0057] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0058] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0059] (3) Hydrolysis of residual epoxy groups on the surface
[0060] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0061] Example 4
[0062] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0063] (1) Synthesis of epoxy-rich polymer matrix
[0064] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 200g of TEGDMA, 160g of toluene, 100g of cyclohexanol, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0065] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0066] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0067] (3) Hydrolysis of residual epoxy groups on the surface
[0068] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0069] Example 5
[0070] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0071] (1) Synthesis of epoxy-rich polymer matrix
[0072] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0073] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0074] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of K2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0075] (3) Hydrolysis of residual epoxy groups on the surface
[0076] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0077] Example 6
[0078] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0079] (1) Synthesis of epoxy-rich polymer matrix
[0080] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0081] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0082] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of LiOH, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0083] (3) Hydrolysis of residual epoxy groups on the surface
[0084] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0085] Example 7
[0086] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0087] (1) Synthesis of epoxy-rich polymer matrix
[0088] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0089] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0090] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 100g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0091] (3) Hydrolysis of residual epoxy groups on the surface
[0092] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0093] Example 8
[0094] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0095] (1) Synthesis of epoxy-rich polymer matrix
[0096] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0097] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0098] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 200g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0099] (3) Hydrolysis of residual epoxy groups on the surface
[0100] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0101] Example 9
[0102] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0103] (1) Synthesis of epoxy-rich polymer matrix
[0104] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0105] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0106] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MESNA, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0107] (3) Hydrolysis of residual epoxy groups on the surface
[0108] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0109] Example 10
[0110] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0111] (1) Synthesis of epoxy-rich polymer matrix
[0112] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0113] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0114] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of Na-DMPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0115] (3) Hydrolysis of residual epoxy groups on the surface
[0116] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0117] Comparative Example 1
[0118] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0119] (1) Synthesis of epoxy-rich polymer matrix
[0120] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of TEGDMA, 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0121] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0122] Take 100g of porous PGMA microspheres, sonicate them in 1500mL of pure water, add 300g of MPS, and continue stirring for 30min until completely dissolved. Transfer the mixture to a 50℃ reactor, add 150g of Na2CO3, and continue stirring for 24h. After the reaction is complete, wash the mixture three times each with ethanol and water to obtain the strong cation exchange chromatography medium.
[0123] Comparative Example 2
[0124] Methods for preparing strong cation exchange chromatography media, such as Figure 1 As shown, the specific steps include:
[0125] (1) Synthesis of epoxy-rich polymer matrix
[0126] 20g of non-crosslinked linear polystyrene seed microspheres with a particle size of 4.5μm were added to 900g of an aqueous solution containing 2% PVA and 0.5% SDS, and ultrasonically dispersed for 30 min. The solution was then transferred to a 5L reactor, heated to 35℃, and continuously stirred to obtain a seed dispersion. 200g of PGMA, 350g of divinylbenzene (DVB), 260g of toluene, and 3.15g of benzoyl peroxide were weighed and added to 2400g of an aqueous solution. The solution was ultrasonically emulsified for 30 min until the emulsion droplet size was below 1μm to obtain an emulsified oil phase. The emulsified oil phase was slowly added dropwise to the reactor, and the mixture was continuously stirred for 24h to allow swelling. 200g of 1% PVA solution was added, and polymerization was initiated at 70℃ and continued for 16h. After the reaction, the mixture was filtered and washed to obtain uniformly sized monodisperse porous PGMA microspheres with a surface rich in epoxy groups.
[0127] (2) Epoxy-mercapto click reaction grafting sulfonic acid group
[0128] Take 100g of porous PGMA microspheres, ultrasonically disperse them in 1500mL of pure water, add 300g of MPS, continue stirring for 30min until completely dissolved, transfer to a 50℃ reactor, add 150g of Na2CO3, continue stirring for 24h, after the reaction is completed, wash with ethanol and water 3 times each.
[0129] (3) Hydrolysis of residual epoxy groups on the surface
[0130] The porous PGMA microspheres grafted with sulfonic acid groups were added to 1500 mL of 0.5 M sulfuric acid solution, heated to 60 °C and stirred continuously for 15 h. After the reaction, the microspheres were washed with water until neutral to obtain a strong cation exchange chromatography medium.
[0131] Performance testing
[0132] 1. SEM characterization
[0133] Scanning electron microscope (SEM) images of strong cation exchange chromatography media in Examples 1-4 are shown below. Figure 2 As shown.
[0134] Depend on Figure 2 It is known that the pore size of porous microspheres can be controlled by adjusting the amount of crosslinking agent triethylene glycol dimethacrylate. Under certain conditions of monomer and porogen dosage, the pore size of microspheres is inversely proportional to the amount of crosslinking agent within a certain range. On the other hand, the pore size of microspheres is related to the properties and type of porogen. By keeping the amount of porogen constant and using toluene and cyclohexanol as porogens, polymer microspheres with larger pore sizes can be obtained.
[0135] 2. Protein loading test of chromatographic packing material
[0136] Ten mL of the strong cationic chromatography media prepared in Examples 1-10 and Comparative Examples 1-2 were loaded into chromatographic columns using a homogenization method and connected to a protein purification instrument. A 100 mg / mL lysozyme solution was injected via the pump head at a flow rate of 2 mL / min. Buffer A was pH 7.0, 0.02 M phosphate buffer, and Buffer B was pH 7.0, 0.02 M phosphate + 1 M NaCl. The sample was injected until 100% flow-through of the protein solution. Buffer B was then used for elution, and the dynamic binding capacity was measured. The statistical results are shown below. Figure 3 As shown.
[0137] A 10 mg / mL lysozyme solution was prepared, and the above process was repeated. After elution, the same sample was injected, but no column chromatography was performed. The protein recovery rate was tested, and the statistical results are shown below. Figure 4 As shown.
[0138] The dynamic binding loading spectrum of the strong cation exchange chromatography medium in Example 2 is shown below. Figure 5As shown in the figure, the functional map results of the purified target protein are as follows: Figure 6 As shown.
[0139] Table 1. Dynamic test results of strong cation chromatography media in Examples 1-10 and Comparative Examples 1-2
[0140] Depend on Figure 3-4 As shown in Table 1, Examples 1-4 controlled the pore size of the medium. The results showed that the dynamic binding capacity of the strong cation exchange chromatography medium first increased and then decreased with the increase of the medium pore size. This is because when the pore size is very small, the adsorption capacity of the medium is very weak, and the amount of protein that can be adsorbed is limited, resulting in a low loading capacity. As the pore size increases, the adsorption capacity of the medium is enhanced. However, if the pore size is too large, the specific surface area of the medium will decrease, the adsorption and carrying capacity of the medium for proteins will weaken, and the loading capacity will decrease. Therefore, a suitable pore size is needed to obtain a higher loading capacity. At the same time, due to the hydrophilic properties of the medium itself, the recovery rate of lysozyme is at a high level.
[0141] Examples 2 and 5-6 changed the type of alkali solution used in the epoxide-thiol click reaction grafting sulfonic acid group process. The results showed that Na2CO3, K2CO3 and LiOH alkali solutions are all suitable for the epoxide-thiol click reaction and have high efficiency. The final lysozyme recovery rate of the product is at a relatively ideal level.
[0142] Examples 2 and 7-8 show that the amount of mercaptosulfonate was adjusted during the grafting of sulfonic acid groups in the epoxy-mercapto-click reaction. The results show that the dynamic binding loading of the medium is positively proportional to the amount of mercaptosulfonate within a certain range. The increase in the amount of sulfonate means that the density of functional groups on the surface of the medium increases, and the dynamic binding loading shows a positive increasing trend within a certain range.
[0143] Examples 2 and 9-10 changed the types of thiosulfonates used in the epoxy-thiosulfonate click reaction grafting process. The results showed that the three thiosulfonates, sodium 3-mercapto-1-propanesulfonate (MPS), sodium 2-mercaptoethanesulfonate (MESNA), and sodium 2,3-dimercaptopropanesulfonate (Na-DMPS), all had ideal grafting effects.
[0144] Comparative Example 1 did not undergo hydrolysis after MPS grafting, and some epoxy groups remained on the surface of the medium, which made the hydrophilicity of the medium not very ideal, and the recovery rate of lysozyme was only 81.6%.
[0145] In Comparative Example 2, divinylbenzene (DVB) was used as a cross-linking agent in the synthesis of porous PGMA microspheres. While maintaining consistent pore properties, DVB was modified with sulfonic acid groups before hydrolysis. The final results showed that the medium exhibited significantly lower hydrophilicity, and the recovery rate of lysozyme was only 52.6%. This is because divinylbenzene, as a cross-linking agent, introduces many strongly hydrophobic groups such as phenyl groups and double bonds during the synthesis of PGMA microspheres, leading to severe non-specific adsorption and irreversible protein adsorption.
[0146] Depend on Figure 5 It can be seen that the dynamic loading of the medium can be calculated by the ratio of the peak area of the flow-through peak to the elution peak.
[0147] Depend on Figure 6 It can be seen that when the strong cation exchange chromatography medium prepared in Example 2 is used for protein separation and purification, the target protein peak is sharp and there are few impurity peaks, indicating that the target protein with high purity can be obtained by using the medium of Example 2.
[0148] In summary, this invention first synthesizes uniformly sized, monodisperse, porous polyglycidyl methacrylate (PGMA) microspheres using a single-step seed swelling technique. The pore size and specific surface area of the microspheres are adjusted by regulating the amount of crosslinking agent. Sulfonic acid groups are then added by clicking the abundant epoxy groups on the microsphere surface with mercaptosulfonate. Finally, a diol is introduced through the hydrolysis of residual epoxy, resulting in a uniformly sized, strong cation exchange chromatography medium. This invention's strong cation exchange chromatography medium maximizes column packing stability and ensures high adsorption capacity. Simultaneously, the hydroxyl groups introduced after epoxy ring opening and the grafting of sulfonic acid groups minimize non-specific protein adsorption, while maintaining high loading capacity and high recovery rate.
[0149] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A strong cation exchange chromatography medium, characterized in that, The structural formula is: ,in, It is polyglycidyl methacrylate.
2. A method for preparing a strong cation exchange chromatography medium as described in claim 1, characterized in that, Specifically, the following steps are included: (1) Epoxy porous polymer microspheres were synthesized using a single-step seed swelling technique; (2) Epoxy porous polymer microspheres were reacted with mercapto sulfonates in an epoxy-mercapto-click reaction; (3) Hydrolyze the residual epoxy groups on the surface of the epoxy porous polymer microspheres to obtain the strong cation exchange chromatography medium.
3. The method for preparing a strong cation exchange chromatography medium according to claim 2, characterized in that, In step (1), the method for synthesizing the epoxy porous polymer microspheres is as follows: seed microspheres are added to an aqueous solution for dispersion to obtain a seed dispersion; epoxy monomers, crosslinking agents, pore-forming agents and initiators are added to an aqueous solution for homogenization and emulsification to obtain an emulsified oil phase; the emulsified oil phase is added to the seed dispersion for swelling, heating to initiate polymerization, and washing to obtain the epoxy porous polymer microspheres.
4. The method for preparing a strong cation exchange chromatography medium according to claim 3, characterized in that, The seed microspheres are non-crosslinked linear polystyrene seed microspheres; the epoxy monomer is polyglycidyl methacrylate; the crosslinking agent is triethylene glycol dimethacrylate; the pore-forming agent is at least one of toluene and cyclohexanol; the initiator is benzoyl peroxide; and the aqueous solution is a mixture of 1% polyvinyl alcohol and 0.25% sodium dodecyl sulfate.
5. The method for preparing a strong cation exchange chromatography medium according to claim 3, characterized in that, The swelling temperature is 30-40℃, and the time is 12-36h.
6. The method for preparing a strong cation exchange chromatography medium according to claim 3, characterized in that, The polymerization temperature is 65-80℃ and the time is 12-24h.
7. The method for preparing a strong cation exchange chromatography medium according to claim 2, characterized in that, In step (2), the mercapto sulfonate is sodium 3-mercapto-1-propanesulfonate, sodium 2-mercaptoethanesulfonate, or sodium 2,3-dimercaptopropanesulfonate.
8. The method for preparing a strong cation exchange chromatography medium according to claim 2, characterized in that, In step (3), the hydrolysis reagent is 0.1-1M sulfuric acid.
9. The application of a strong cation exchange chromatography medium as described in claim 1 or a strong cation exchange chromatography medium prepared by any one of claims 2-8 in the separation and purification of biomacromolecules.
10. The application according to claim 9, characterized in that, The biological macromolecules include proteins, vaccines, and antibodies.