A method for preparing a high-efficiency antibacterial microfiltration membrane for biopharmaceutical processes

Abstract

The present invention belongs to the field of membrane separation technology, and relates to a method for preparing a high-efficiency antibacterial microfiltration membrane for a biopharmaceutical process. A composite additive system is introduced into a casting solution, and a modifier is added. The modifier includes a composite alcohol porogen and an antibacterial agent. The casting solution composition and the membrane making process are coupled to obtain a high-throughput antibacterial membrane. The antibacterial agent can enhance the effect of the composite alcohol porogen, assist in membrane pore size regulation, and fix the membrane structure. The two act synergistically, so that the microfiltration membrane maintains high antibacterial performance while improving selective permeability, and realizes selective separation of microorganisms of different sizes. The membrane is expected to be a very potential candidate in the biopharmaceutical sterile filtration process.

Description

Technical Field

[0001] The invention belongs to the technical field of membrane separation and relates to a method for preparing a high-efficiency antibacterial microfiltration membrane used in a biopharmaceutical process. Background Art

[0002] In recent years, the biopharmaceutical industry has developed rapidly, and new biopharmaceuticals such as proteins, peptides, enzyme preparations, vaccines, etc. have continued to emerge. The production process of these biopharmaceuticals requires efficient separation and purification technology to ensure the quality and purity of the products. Membrane separation technology has attracted widespread attention because it can selectively screen according to molecular size and is very suitable for the separation and purification of biopharmaceuticals. Before the emergence of ultrafiltration membrane technology, commonly used separation technologies in the biopharmaceutical field, such as precipitation, centrifugation, chromatography, etc., all have problems such as low separation efficiency, complex operation, high cost, and easy inactivation of biomolecules. In contrast, ultrafiltration membrane technology has the advantages of simple operation, high separation efficiency, and little effect on the activity of biomolecules, which can better meet the separation needs in the biopharmaceutical field. However, organic microbial contaminants are an important problem faced in membrane separation applications. It will cause serious flux attenuation and thus affect the filtration efficiency. Therefore, it is of great significance to develop new high-throughput anti-microbial contamination composite membranes. Summary of the invention

[0003] The present invention provides a method for preparing a high-efficiency antibacterial microfiltration membrane for use in a biopharmaceutical process. By adding a composite alcohol porogen and an antibacterial agent into a membrane casting solution, the antibacterial agent can enhance the effect of the composite alcohol porogen, assist in membrane pore size regulation, and fix the membrane structure. The two act synergistically, so that the microfiltration membrane maintains high antibacterial performance while improving selective permeability, thereby achieving selective separation of microorganisms of different sizes.

[0004] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0005] The present invention provides a method for preparing a high-efficiency antibacterial microfiltration membrane for a biopharmaceutical process, comprising the following steps:

[0006] (1) Mixing a polymer material, a modifier and an organic solvent, stirring at a constant temperature to obtain a uniform casting solution, and then stopping the stirring and continuing to heat at 60°C, standing for 30 to 50 minutes to remove bubbles, to obtain a casting solution; the weight percentage of the polymer material in the casting solution is 6 to 10 wt%, the weight percentage of the organic solvent and the modifier in the casting solution is 90 to 94 wt%, and the total amount is 100 wt%; the polymer material is polyethersulfone (PES), and the modifier includes a composite alcohol pore-forming agent and an antibacterial agent; the composite alcohol pore-forming agent is polyvinyl pyrrolidone and polyethylene glycol in a mass ratio of 1 to 5:5 to 1, and the antibacterial agent is one or more of N-carboxymethyl chitosan, O-carboxymethyl chitosan, and N,O-carboxymethyl chitosan;

[0007] (2) The casting liquid is used to prepare a microfiltration membrane using a non-solvent induced phase inversion method: a clean and flat non-woven fabric is attached to a glass plate in advance, and then a scraper with a fixed thickness is used to evenly spread the casting liquid on the non-woven fabric at a uniform speed. The casting liquid is then quickly immersed in a deionized water coagulation bath at a fixed temperature. After the phase inversion process is completed and the membrane is formed, the membrane is stored in deionized water at room temperature and soaked to remove the residual organic solvent to obtain a microfiltration membrane.

[0008] Furthermore, the polyvinyl pyrrolidone model is selected from one of K12, K17, K25, K30, K60, and K90, and the molecular weight of the polyethylene glycol is 200-20000.

[0009] Furthermore, the composite alcohol porogen accounts for 1-6wt% of the total casting solution, and the antibacterial agent accounts for 0.05-0.2wt% of the total casting solution.

[0010] Furthermore, the organic solvent is one or more of dimethylformamide, dimethyl sulfoxide, dimethylacetamide and N-methylpyrrolidone (NMP).

[0011] Furthermore, the constant temperature stirring temperature is controlled at 50°C to 70°C, and the stirring time is 5 to 8 h.

[0012] Furthermore, the preparation method of the modifier is: adding a composite alcohol porogen to an organic solvent, and then adding an antibacterial agent after mixing and dissolving.

[0013] Furthermore, the coagulation bath is deionized water, the coagulation bath temperature is controlled at 25±1° C., the film scraping rod thickness is 240-260 μm, and the coagulation bath immersion time is at least 24 hours.

[0014] Advantages and beneficial effects of the present invention:

[0015] 1. The present invention adds a composite alcohol porogen and an antibacterial agent to the casting solution to perform a co-modification on the microfiltration membrane, and the method adopted is the immersion precipitation phase transformation method. Since the added antibacterial agent molecules contain a large number of hydrophilic functional groups, and the bacterial cell membrane usually carries a negative charge, these hydrophilic groups can be protonated under certain conditions to form positively charged ions, destroying the integrity of the cell membrane, so that the bacteria cannot maintain normal life activities and die, thus showing a certain antibacterial property. The addition of an antibacterial agent can enhance the effect of the composite alcohol porogen, assist in the regulation of membrane pore size, and fix the membrane structure. The synergistic effect of the two allows the microfiltration membrane to maintain high antibacterial properties while improving the selective permeability, thereby achieving selective separation of microorganisms of different sizes.

[0016] 2. The separation system involved in the present invention is the separation of bacteria and proteins. The alcohol porogens and antibacterial agents used are low-priced, safe and non-toxic, and have certain application potential in the field of biopharmaceuticals.

[0017] 3. The method of the present invention is efficient and simple, and can improve the anti-pollution property of the PES microfiltration membrane while ensuring the permeation flux and separation performance, and significantly enhance the filtration efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison chart of the antibacterial performance of the microfiltration membranes prepared in Examples 1 to 5 against E. coli bacteria; the ordinate is the OD value of bacterial growth;

[0019] Figure 2 This is a comparison chart of the antibacterial performance of the microfiltration membranes prepared in Examples 1 to 5 against S. aureus bacteria;

[0020] Figure 3 The colony pictures of bacteria E. coli on the surface of the microfiltration membrane prepared in Examples 1 to 5;

[0021] Figure 4 These are colony pictures of bacteria S. aureus on the surface of the microfiltration membranes prepared in Examples 1 to 5. DETAILED DESCRIPTION

[0022] In order to better understand the structure and performance of the present invention, the preparation method of a highly selectively permeable antibacterial microfiltration membrane for biopharmaceutical process of the present invention is further described in detail below.

[0023] Embodiment 1~embodiment 5:

[0024] 5wt% PVP K30 and 1wt% PEG20000 were weighed and added to NMP. After stirring at 60℃ for 0.5 h, different contents of antibacterial agent CMCS (0-0.2wt%) were added. The total amount of polymer, modifier and organic solvent was 100wt%. After stirring for 1 h, 8wt% polymer membrane material PES was added. Stirring was continued at 60℃ for not less than 6 h to obtain a uniform casting solution. After stopping stirring, heating was continued at 60℃ and allowed to stand for 40 min to remove bubbles.

[0025] A clean and flat non-woven fabric was attached to the glass plate in advance, and then a 250 μm thick scraper was used to evenly spread the casting solution on the non-woven fabric, and then quickly immersed in a pre-prepared deionized water coagulation bath controlled at 25°C, and after waiting for the phase inversion process to form a membrane, it was transferred to another clean deionized water coagulation bath and soaked for at least 24 hours to remove the residual organic solvent, and a microfiltration membrane was obtained as a sample to be tested. The performance of the microfiltration membranes prepared in Examples 1 to 5 was tested, and the results are shown in Table 1.

[0026] Table 1 Effects of different antimicrobial content on the performance of polyethersulfone microfiltration membrane

[0027]

[0028] Table 1 shows the effect of different contents of antimicrobial agents on the performance of polyethersulfone microfiltration membranes. Among them, Example 1 is a control group, and the membrane number is Control. Examples 2 to 5 are polyethersulfone microfiltration membranes with different contents of antimicrobial agents added, and the membrane numbers are M-0.05C, M-0.1C, M-0.15C, and M-0.2C, respectively, as experimental groups. Compared with the control group Example 1, the water contact angle of the experimental group is reduced, indicating that the addition of antimicrobial agents improves the hydrophilicity of the polyethersulfone microfiltration membrane, and the retention rates of E. coli and S. aureus are both around 99%, indicating that the addition of antimicrobial agents makes the polyethersulfone microfiltration membrane have certain antibacterial properties, and the retention rate of BSA is significantly improved, proving that by introducing hydrophilic antimicrobial agents into the casting solution, the hydrophilicity and membrane structure of the microfiltration membrane can be improved, and it is ensured that the permeability of the microfiltration membrane is maintained at a high level when both the bacterial retention rate and the BSA retention rate are improved. Comparison of the antibacterial properties of the microfiltration membranes prepared in Examples 1 to 5 against E. coli bacteria Figure 1 As shown, the colony picture is as follows Figure 3 As shown; the antibacterial performance of the microfiltration membranes prepared in Examples 1 to 5 against S. aureus bacteria is compared. Figure 2 As shown, the colony picture is as follows Figure 4 as shown.

[0029] Embodiment 6 to Embodiment 9

[0030] 5wt% PVPK30 and 1wt% PEG20000 were weighed and added to NMP. After stirring at 60℃ for 0.5 h, three different antibacterial agents N-carboxymethyl chitosan (N-CMCS), O-carboxymethyl chitosan (O-CMCS), N,O-carboxymethyl chitosan (N,O-CMCS) (0.1wt%) were added respectively. The total amount of polymer, modifier and organic solvent was 100wt%. After stirring for 1 h, 8wt% polymer membrane material PES was added. Stirring was continued at a constant temperature for not less than 6 h to obtain a uniform casting solution. After stopping stirring, heating was continued and allowed to stand for 40 min to remove bubbles. A clean and flat non-woven fabric was attached to the glass plate in advance, and then a 250 μm thick scraper was used to evenly spread the casting solution on the non-woven fabric, and then quickly immersed in a pre-prepared deionized water coagulation bath controlled at 25°C, and after the phase inversion process formed a membrane, it was transferred to another clean deionized water coagulation bath and soaked for at least 24 hours to remove the residual organic solvent, and then it can be used as a sample to be tested. The performance of the microfiltration membranes prepared in Examples 6 to 9 was tested, and the test results are shown in Table 2.

[0031] Table 2 Effects of different antimicrobial agents on the performance of polyethersulfone microfiltration membrane

[0032]

[0033] Table 2 shows the effect of different antimicrobial agents on the performance of polyethersulfone microfiltration membrane. Example 6-Example 9 are respectively the control group and the experimental group with different antimicrobial agents added, and the prepared PES antimicrobial microfiltration membrane. By comparing Example 7-Example 9 with Example 6, it is found that the pure water flux, hydrophilicity and ability to intercept bacteria and proteins of the filtration membrane after adding the antimicrobial agent are significantly improved, among which the interception effect of the membrane of Example 8, especially the permeability, is the best, indicating that the antimicrobial agent can be well retained in the membrane through hydrogen bonding, and the antimicrobial agent can enhance the effect of the composite alcohol pore-forming agent, assist in the regulation of membrane pore size, and fix the membrane structure. The synergistic effect between the antimicrobial agent and the composite alcohol pore-forming agent makes the microfiltration membrane show good hydrophilicity and antimicrobial properties.

[0034] Embodiment 10~embodiment 14

[0035] Add 5wt% polyvinyl pyrrolidone and 1wt% polyethylene glycol to NMP, stir at 60℃ for 0.5h, then add 0.1wt% O-carboxymethyl chitosan, continue stirring for 1h, then add 8wt% polyethersulfone (polymer material), the total amount of polymer, modifier and organic solvent is 100wt%. Stir at different temperatures for 6h until completely dissolved, and after forming a uniform casting solution, turn off stirring and degas at 60℃ for 0.5h;

[0036] A clean and flat non-woven fabric was attached to the glass plate in advance, and then a 250 μm thick scraping rod was used to evenly spread the casting solution on the non-woven fabric, and then quickly immersed in a pre-prepared deionized water coagulation bath controlled at 25°C, and after waiting for the phase inversion process to form a membrane, it was transferred to another clean deionized water coagulation bath and soaked for at least 24 hours to remove the residual organic solvent, and a microfiltration membrane was obtained as a sample to be tested. The performance of the microfiltration membranes prepared in Examples 10 to 14 was tested, and the results are shown in Table 3.

[0037] Table 3 Effect of different reaction temperatures on the performance of polyethersulfone microfiltration membrane

[0038]

[0039] Table 3 shows the effect of different reaction temperatures on the performance of polyethersulfone microfiltration membrane. Examples 10 to 14 are to add a certain amount of modifier, prepare a casting solution at different temperatures, and prepare a microfiltration membrane by phase inversion. It can be seen from Examples 10 to 14 that, under a certain content of modifier, different reaction temperatures have little effect on the contact angle, but it is lower than that of Example 1. At different temperatures, the bacterial retention rate remains above 98%, and the pure water flux and BSA retention rate are significantly improved as the temperature rises to 60°C, indicating that the permeability of the microfiltration membrane can be improved by adjusting the reaction temperature without sacrificing hydrophilicity and BSA retention rate.

[0040] Embodiment 15~embodiment 16

[0041] The difference from Example 12 is that 6 wt % of polyvinyl pyrrolidone or 6 wt % of polyethylene glycol is added to NMP. The performance of the microfiltration membranes prepared in Examples 15 and 16 was tested, and the results are shown in Table 4.

[0042] Table 4 Effect of adding different alcohol porogens on the performance of polyethersulfone microfiltration membrane

[0043]

[0044] Table 4 shows the effect of adding different alcohol porogens on the performance of polyethersulfone microfiltration membrane. It can be seen from Examples 12 and 15 to 16 that the addition of a two-component alcohol porogen significantly improves the pure water flux and greatly improves the interception efficiency of bacteria and proteins compared to a single-component alcohol porogen. This shows that the two alcohol porogens, PVP and PEG, have a synergistic effect on the permeability and separation efficiency of the membrane.

[0045] From Example 1 to Example 14, it can be seen that a high-performance ultrafiltration membrane can be prepared by adjusting the content, type and reaction temperature of the antibacterial agent. The method for preparing a high-efficiency antibacterial microfiltration membrane for a biopharmaceutical process disclosed in the present invention is simple to operate and does not require additional steps. It can play a certain role in regulating the membrane performance and structure, thereby preparing an antibacterial microfiltration membrane with high-efficiency filtration performance.

[0046] It can be seen from the above examples that the polyethersulfone microfiltration membrane prepared by the synergistic effect between the composite alcohol porogen and the antibacterial agent can achieve efficient separation of bacteria and proteins, and has excellent permeability and anti-fouling properties.

[0047] The above description is only a preferred implementation case of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a high-efficiency antibacterial microfiltration membrane for biopharmaceutical processes, characterized in that: The following steps are involved: (1) A polymer material, a modifier and an organic solvent are mixed, and stirred at a constant temperature to obtain a uniform mixed solution. After stopping the stirring, the mixture is heated and allowed to stand for 30 to 50 minutes to remove bubbles, thereby obtaining a casting solution. The weight percentage of the polymer material in the casting solution is 6 to 10 wt%, and the weight percentage of the organic solvent and the modifier in the casting solution is 90 to 94 wt%, with a total amount of 100 wt%. The polymer material is polyethersulfone, and the modifier includes a composite alcohol pore-forming agent and an antibacterial agent. The composite alcohol pore-forming agent is polyvinyl pyrrolidone and polyethylene glycol in a mass ratio of 1 to 5:5 to 1, and the polyvinyl pyrrolidone model is selected from one of K12, K17, K25, K30, K60 and K90. The molecular weight of the polyethylene glycol is 200 to 20,000, and the antibacterial agent is N-carboxymethyl chitosan, O-carboxymethyl chitosan , N, O-carboxymethyl chitosan; the total proportion of the composite alcohol porogen in the casting solution is 1-6wt%, and the total proportion of the antibacterial agent in the casting solution is 0.05-0.2wt%; the organic solvent is one or more of dimethylformamide, dimethyl sulfoxide, dimethylacetamide and N-methylpyrrolidone; the antibacterial agent enhances the effect of the composite alcohol porogen, assists in membrane pore size regulation, and fixes the membrane structure. The synergistic effect of the two allows the microfiltration membrane to maintain high antibacterial properties while improving selective permeability, thereby achieving selective separation of microorganisms of different sizes; (2) The casting liquid is used to prepare a microfiltration membrane using a non-solvent induced phase inversion method: a non-woven fabric is attached to a glass plate, and then a scraper is used to evenly spread the casting liquid on the non-woven fabric at a uniform speed, and then the casting liquid is quickly immersed in a constant temperature coagulation bath. After the phase inversion process is completed and the membrane is formed, the membrane is stored in deionized water at room temperature and immersed to obtain a microfiltration membrane.

2. The preparation method according to claim 1, characterized in that: The constant temperature stirring temperature is 50°C to 70°C, and the stirring time is 5 to 8 h.

3. The preparation method according to claim 1, characterized in that: The preparation method of the modifier is as follows: adding a composite alcohol porogen into an organic solvent, and then adding an antibacterial agent after mixing and dissolving.

4. The preparation method according to claim 1, characterized in that: The coagulation bath is deionized water, the coagulation bath temperature is controlled at 25±1° C., the film scraping rod thickness is 240-260 μm, and the coagulation bath immersion time is at least 24 hours.

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