Bovine serum albumin degreasing method
The problem of fatty acid removal from bovine serum albumin was solved by adsorption using pentyl-modified styrene-based hydrophobic resin. This method achieved efficient binding and stability of BSA on a magnetic microparticle platform, overcoming the shortcomings of traditional resins and enabling efficient utilization of BSA.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-03-27
AI Technical Summary
Existing technologies are insufficient to effectively remove fatty acids from bovine serum albumin, resulting in reduced binding of fatty acids to magnetic microparticles on the magnetic microparticle chemiluminescence platform, which affects colloidal stability and enzymatic reactions. Furthermore, BSA without fatty acid grade is mainly dependent on imports.
An adsorption method using pentyl-modified styrene-based hydrophobic resin was employed. This method combines ultrafiltration concentration, activated carbon adsorption, and freeze-drying with the preparation method of pentyl-modified styrene-based hydrophobic resin. By utilizing the hydrophobic chain matching and π-π interaction between the pentyl chain and fatty acids, efficient adsorption of fatty acids can be achieved while maintaining the flowability of BSA.
It achieves efficient removal of fatty acids, maintains the stability and activity of BSA, improves the binding ability of BSA with magnetic microparticles, solves the problems of large mass transfer resistance and insufficient specific surface area caused by the single pore size of traditional resins, and ensures the efficient utilization of BSA.
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Abstract
Description
TECHNICAL FIELD
[0001] The application relates to a bovine serum albumin degreasing method and belongs to the technical field of albumin production. BACKGROUND
[0002] Bovine serum albumin (BSA) is widely used in the diagnostic reagent industry due to its good stability, inertness and protein protection capability. With the continuous innovation and upgrading of the diagnostic reagent technology platform, the requirements for BSA are becoming higher and higher, especially on the magnetic microparticle chemiluminescence platform. Fatty acids can competitively occupy the hydrophobic binding sites of BSA, reduce the effective binding of BSA to the surface of magnetic microparticles, destroy the colloidal stability of magnetic microparticles, induce non-specific aggregation, and even directly bind to luminescent labels, interfere with enzyme reactions or luminescence processes. Therefore, the magnetic microparticle chemiluminescence platform needs to use fatty acid-free grade BSA. However, the fatty acid-free grade BSA currently mainly depends on import, which limits its application. SUMMARY
[0003] The purpose of the present application is to provide a bovine serum albumin degreasing method. The phenyl ring (pi system) of pentyl-modified styrene is closely connected with the pentyl chain (hydrophobic chain) in space, which can produce synergistic effects of hydrophobic effect and pi effect when adsorbing fatty acid molecules, and has stronger and more specific binding force, which can better adsorb fatty acids.
[0004] To achieve the above purpose, the technical scheme adopted by the present application is as follows:
[0005] A bovine serum albumin degreasing method, comprising the following steps:
[0006] ultrafiltration and concentration treatment of BSA stock solution;
[0007] active carbon adsorption and pentyl-modified styrene-based hydrophobic resin adsorption of the stock solution concentrate;
[0008] ultrafiltration and concentration treatment of flow-through liquid;
[0009] freeze-drying of the flow-through concentrate.
[0010] Preferably, before the ultrafiltration and concentration treatment of the BSA stock solution and the ultrafiltration and concentration treatment of the flow-through liquid, the pH of the BSA stock solution and the flow-through liquid is adjusted to 7.0-7.5 by using alkali solution.
[0011] The molecular weight cut-off of the membrane used for ultrafiltration and concentration is 10-30KD.
[0012] Preferably, before the active carbon adsorption, the pH of the stock solution concentrate is adjusted to 3-4 by using acid solution.
[0013] Preferably, the concentrate before activated carbon adsorption is prepared by adding 2-3% (w / v) of activated carbon powder to the concentrate after adjusting the pH, and then stirring for 1-2 hours for adsorption.
[0014] Preferably, before the hydrophobic resin adsorption, the liquid after activated carbon adsorption is filtered to remove the activated carbon powder from the liquid.
[0015] Preferably, the preparation method of pentyl-modified styrene-based hydrophobic resin includes the following steps:
[0016] S1. Under a nitrogen atmosphere, AlCl3 and THF were mixed and stirred in an ice-water bath. Then, a mixture of styrene and n-pentyl bromide was added dropwise to the system. After the addition was complete, the ice-water bath was removed, and the reaction was quenched after reacting at room temperature for a certain period of time. The mixture was then purified by extraction, washing, and drying to obtain pentyl-modified styrene.
[0017] S2. Prepare an aqueous phase containing gelatin and sodium chloride; prepare an oil phase containing pentyl-modified styrene, divinylbenzene, catalyst and pore-forming agent; under nitrogen protection, disperse the oil phase in the aqueous phase, heat and react, then cool naturally, and finally filter, wash, remove impurities, perform final washing, dry and sieve.
[0018] Preferably, in step S1, the ratio of AlCl3, THF, styrene and n-pentyl bromide is (15-20)g:(200-400)mL:(85-130)g:(60-85)g;
[0019] The temperature of the ice water bath is 0-5℃;
[0020] The addition time of styrene and n-pentyl bromide is 1-2 hours;
[0021] The reaction time at room temperature is 10-15 hours.
[0022] Preferably, in step S2, the concentration of gelatin in the aqueous phase is 0.3-0.7% (w / v), and the concentration of sodium chloride is 1.5-2.5% (w / v).
[0023] Preferably, in the oil phase, the pore-forming agent is toluene and n-heptane; and the ratio of pentyl-modified styrene, divinylbenzene, catalyst, toluene and n-heptane is (90-110)g:(50-75)g:(1-2)g:(90-100)mL:(55-65)mL.
[0024] Preferably, in step S2, the heating reaction is to first heat to 50-60°C and hold for 3-5 hours; then heat to 70-75°C and hold for 6-8 hours.
[0025] The beneficial effects of this invention are as follows:
[0026] By introducing pentyl-modified styrene into the resin, the pentyl chain length is similar to that of the alkyl chain of fatty acids, resulting in stronger van der Waals adsorption through the "hydrophobic chain matching effect." Simultaneously, the benzene ring structure provides the ability to engage in weak π-π or dipole interactions with the electron cloud near the carboxyl group of fatty acids, forming a synergistic adsorption of fatty acid molecules through multiple weak interactions, leading to higher selectivity.
[0027] By synergistically employing a dual-porogen (toluene / n-heptane) system with a segmented temperature polymerization process, the resin particles acquire a combined macroporous and microporous structure. The macropores (20-100 nm) ensure smooth passage of BSA protein and feed solution, preventing clogging and protein retention; the micropores (2-5 nm) provide a large specific surface area, rich in pentyl-modified styrene functional groups, serving as the primary sites for capturing fatty acid molecules. This structure overcomes the problems of high mass transfer resistance or insufficient specific surface area caused by the single pore size of traditional resins.
[0028] Because the molecular size of BSA is much larger than the micropore size of the resin, it is physically excluded from the micropore adsorption sites, and can only undergo very weak non-specific adsorption in the macropore channels. In contrast, smaller fatty acid molecules can freely enter the micropores and be specifically adsorbed. This ensures that while efficiently adsorbing fatty acids, the flow-through loss of BSA is extremely low.
[0029] The resin backbone is based on highly cross-linked divinylbenzene, exhibiting high mechanical strength and resistance to acids and alkalis. During regeneration with 1M NaOH, the NaOH neutralizes the adsorbed fatty acids, ionizing them and significantly reducing their hydrophobicity. This allows them to desorb from the hydrophobic sites of the resin and be discharged with the flushing solution. The resin's pore structure and chemical properties remain stable even after multiple regenerations. Detailed Implementation
[0030] In the following examples, the preparation method of the pentyl-modified styrene-based hydrophobic resin used in the hydrophobic resin chromatography column is as follows:
[0031] Under a continuous nitrogen flow, add 300 mL of anhydrous THF (tetrahydrofuran) and 16 g of anhydrous aluminum trichloride powder to the flask. Place the flask in an ice-water bath, start stirring, and allow the system to cool and maintain at 5°C.
[0032] Mix 104.1 g of styrene and 74.8 g of n-pentyl bromide thoroughly in an Erlenmeyer flask, and transfer the mixture to a constant-pressure dropping funnel. Slowly add this mixture dropwise (over 1.5 h) to a three-necked flask, ensuring the reaction temperature never exceeds 10°C.
[0033] After the addition is complete, remove the ice bath and allow the reaction solution to slowly heat to room temperature under stirring. Continue the reaction at this temperature for 12 hours (while maintaining a nitrogen atmosphere).
[0034] Slowly pour the reaction solution into a beaker containing 500 mL of ice-water mixture while stirring (operate slowly to avoid violent exothermic reactions). After quenching, transfer to a separatory funnel and extract the aqueous phase with 2 × 250 mL of dichloromethane. Combine the organic phases.
[0035] The organic phase was washed successively with 200 mL of saturated sodium bicarbonate solution and 2 × 200 mL of deionized water until neutral. The organic phase was then transferred to an Erlenmeyer flask and dried with sufficient anhydrous sodium sulfate for 2 hours.
[0036] The desiccant was removed by filtration, and most of the solvent was removed by rotary evaporation under reduced pressure in a 40°C water bath. The remaining crude product was purified by column chromatography (stationary phase: silica gel; mobile phase: petroleum ether / ethyl acetate = 20:1, v / v). The fraction containing the target product was collected, and the solvent was removed again by rotary evaporation to obtain a colorless and transparent pentyl-modified styrene liquid.
[0037] In a 2000 mL beaker, add 1500 mL of deionized water and heat to 55°C. While stirring, add 7.5 g of gelatin and 30 g of sodium chloride, continuing stirring until completely dissolved to obtain a homogeneous aqueous phase. Transfer the aqueous phase to a reaction vessel, turn on the circulating water bath, and stabilize the aqueous phase temperature at 40°C. Turn on the stirring and bubble with nitrogen gas for 30 minutes to remove dissolved oxygen.
[0038] In a 500 mL beaker, add 96 g of pentyl-modified styrene liquid, 60 g of divinylbenzene, 93.6 mL of toluene, and 62.4 mL of n-heptane sequentially, and stir until homogeneous. Then add 1.56 g of AIBN and stir until completely dissolved to obtain a transparent and homogeneous oil phase.
[0039] Adjust the stirring speed of the aqueous phase in the reactor to 450 rpm to form a stable vortex. Quickly pour the prepared oil phase into the reactor all at once. The oil phase will disperse into uniformly sized droplets under stirring. Maintain this stirring speed for 20 minutes to stabilize the droplet size within the range of 100-300 μm (observe by sampling under a microscope and adjust the stirring speed accordingly).
[0040] While maintaining a constant stirring speed and purging with nitrogen, increase the water bath temperature from 40°C to 55°C at a rate of 1°C / min. Once 55°C is reached, maintain this temperature and continue the reaction for 4 hours. Then, slowly increase the water bath temperature from 55°C to 70°C at a rate of 0.5°C / min. Once 70°C is reached, maintain this temperature and continue the reaction for 8 hours.
[0041] After polymerization, turn off the heating and allow the reaction system to cool naturally to room temperature with stirring. Turn off the stirring and pour all the material in the reactor into a Buchner funnel for filtration. Wash the resin particles repeatedly with 70°C hot water (3-5 L) until the filtrate is clear and foam-free to thoroughly remove gelatin and salt.
[0042] Transfer the wet resin particles into the filter paper cassette of a Soxhlet extractor. Add approximately 500 mL of analytical grade acetone to the extraction flask. Adjust the water bath temperature to maintain a stable reflux rate of 6-8 cycles per hour. Continue extraction for 48 hours to ensure complete removal of the toluene and n-heptane porogens.
[0043] After extraction, the resin particles were transferred to a beaker and washed once with 200 mL of ethanol, followed by washing with plenty of purified water until the eluent was neutral. The resin particles were then placed in a petri dish or tray and dried in a vacuum drying oven at 60 °C and -0.095 MPa for 24 h until constant weight was obtained. Dry, white, opaque, spherical resin particles were obtained.
[0044] Resin particles of 100-300 μm were sieved using a standard sieve as the product. The resulting resin particles have macropores of 20-100 nm and micropores of 2-5 nm.
[0045] Example 1: This example provides a method for defatting bovine serum albumin, specifically including the following steps:
[0046] 1. Raw material processing: 1200L of BSA stock solution with a concentration of 11mg / ml obtained by centrifugation from the heat shock process was adjusted to pH 7.0 with 1M NaOH.
[0047] 2. Ultrafiltration concentration: Using a 30kD ultrafiltration system, the feed solution from the previous step was replaced with purified water at a volume of 5 times and then concentrated to a BSA concentration of 50mg / ml.
[0048] 3. Activated carbon adsorption: Adjust the pH of the solution to 3.6 with 1M HCl, add 2% (w / v) of coconut shell activated carbon powder, and stir for 1 hour for adsorption.
[0049] 4. Hydrophobic resin adsorption: A stacked plate and frame filter is filled with a 10μm filter membrane and connected in series with a 3μm filter element for filtration. The supernatant obtained from filtration is passed through a hydrophobic resin chromatography column (30cm in diameter, 1m in packing height) at a rate of 10L / min. The flow-through solution is adjusted to pH 7.2 with 1M NaOH.
[0050] 5. Ultrafiltration concentration: The solution from the previous step was ultrafiltered and concentrated to 210 mg / ml using a 30 kD ultrafiltration system, and then sterilized by filtration with a 0.22 μm filter cartridge to obtain 58 L of solution, with a calculated yield of 92.2%; all the above operations were carried out at room temperature.
[0051] 6. Freeze-drying: After the above liquid is freeze-dried, BSA crystal powder is collected.
[0052] 7. Activated carbon and resin regeneration: The coconut shell activated carbon and hydrophobic resin packed chromatography column in the filter is rinsed with 1M NaOH for 1 hour, and then rinsed with purified water until neutral. After regeneration, it can be reused next time.
[0053] Example 2: This example provides a method for defatting bovine serum albumin, specifically including the following steps:
[0054] 1. Raw material processing: 1300L of BSA stock solution obtained by centrifugation from heat shock process, with a concentration of 10.5mg / ml, is adjusted to pH 7.2 with 1M NaOH.
[0055] 2. Ultrafiltration concentration: Using a 30kD ultrafiltration system, the feed solution from the previous step was replaced with purified water at a volume of 4 times and then concentrated to a BSA concentration of 45mg / ml.
[0056] 3. Activated carbon adsorption: Adjust the pH of the feed solution to 3.4 with 1M HCl, add 2.5% fruit shell activated carbon powder by mass and volume, and stir for adsorption for 1.5 hours.
[0057] 4. Hydrophobic resin adsorption: A stacked plate and frame filter is filled with a 10μm filter membrane and connected in series with a 3μm filter element for filtration. The supernatant obtained from filtration is passed through a hydrophobic resin chromatography column (30cm in diameter, 1m in packing height) at a rate of 10L / min. The flow-through solution is adjusted to pH 7.3 with 1M NaOH.
[0058] 5. Ultrafiltration concentration: The solution from the previous step was ultrafiltered and concentrated to 205 mg / ml using a 30 kD ultrafiltration system, and then sterilized by filtration with a 0.22 μm filter cartridge to obtain 61 L of solution, with a calculated yield of 91.6%; all the above operations were carried out at room temperature.
[0059] 6. Freeze-drying: After the above liquid is freeze-dried, BSA crystal powder is collected.
[0060] 7. Activated carbon and resin regeneration: The coconut shell activated carbon and hydrophobic resin packed chromatography column in the filter is rinsed with 1M NaOH for 1.5 hours, then rinsed with purified water until neutral. After regeneration, it can be reused next time.
[0061] Example 3: This example provides a method for defatting bovine serum albumin, specifically including the following steps:
[0062] 1. Raw material processing: 1150L of BSA stock solution obtained by centrifugation from the heat shock process, with a concentration of 11mg / ml, is adjusted to pH 7.5 with 1M NaOH.
[0063] 2. Ultrafiltration concentration: Using a 30kD ultrafiltration system, the solution from the previous step was replaced with purified water at a volume of 6 times and then concentrated to a BSA concentration of 60mg / ml.
[0064] 3. Activated carbon adsorption: Adjust the pH of the solution to 3.8 with 1M HCl, add 3% (w / v) of coconut shell activated carbon powder, and stir for 1 hour for adsorption;
[0065] 4. Hydrophobic resin adsorption: A stacked plate and frame filter is filled with a 10μm filter membrane and connected in series with a 3μm filter element for filtration. The supernatant obtained from filtration is passed through a hydrophobic resin chromatography column (30cm in diameter, 1m in packing height) at a rate of 10L / min. The flow-through solution is adjusted to pH 7.5 with 1M NaOH.
[0066] 5. Ultrafiltration concentration: The solution from the previous step was ultrafiltered and concentrated to 195 mg / ml using a 30 kD ultrafiltration system, and then sterilized by filtration with a 0.22 μm filter cartridge to obtain 60 L of solution, with a calculated yield of 92.5%; all the above operations were carried out at room temperature.
[0067] 6. Freeze-drying: After the above liquid is freeze-dried, BSA crystal powder is collected.
[0068] 7. Activated carbon and resin regeneration: The coconut shell activated carbon and hydrophobic resin packed chromatography column in the filter is rinsed with 1M NaOH for 2 hours, and then rinsed with purified water until neutral. After regeneration, it can be reused next time.
[0069] Comparative Example 1: It is basically the same as Example 1, except that the resin used in the chromatography column during the hydrophobic resin adsorption process is polystyrene-divinylbenzene resin.
[0070] The samples obtained in Examples 1-3 and Comparative Example 1 were compared with the imported control sample in terms of physicochemical properties and functional applications. The results are as follows:
[0071] 1. Physicochemical testing (prepared as a 5% aqueous solution)
[0072]
[0073] 2. Performance testing (on the magnetic microparticle chemiluminescence platform, CA724 project, and also as a component of enzyme diluent and blocking solution).
[0074]
[0075] As shown in Tables 1 and 2, the bovine serum albumin obtained in Examples 1, 2, and 3 showed highly consistent results with the control group in physicochemical detection and C724 performance testing, and its physicochemical properties were even superior to those of the control group. However, the bovine serum albumin obtained in Comparative Example 1 showed certain deviations from the control group in fatty acid content, background differences, and correlation.
Claims
1. A method for defatting bovine serum albumin, characterized in that, Includes the following steps: Ultrafiltration concentration treatment of BSA stock solution; Activated carbon adsorption and pentyl-modified styrene-based hydrophobic resin adsorption concentrate; Ultrafiltration concentration treatment of flow-through liquid; Freeze-dry the flow-through concentrate.
2. The method for defatting bovine serum albumin according to claim 1, characterized in that, Before ultrafiltration concentration of BSA stock solution and flow-through solution, adjust the pH of BSA stock solution and flow-through solution to 7.0-7.5 with alkaline solution. The molecular weight cutoff of the membrane used for ultrafiltration concentration is 10-30 kDa.
3. The method for defatting bovine serum albumin according to claim 1, characterized in that, Before activated carbon adsorption, the pH of the concentrate is adjusted to 3-4 with acid.
4. The method for defatting bovine serum albumin according to claim 3, characterized in that, Before activated carbon adsorption, the concentrate of the original solution is prepared by adding 2-3% (w / v) of activated carbon powder after adjusting the pH of the concentrate, and then stirring for 1-2 hours for adsorption.
5. The method for defatting bovine serum albumin according to claim 1, characterized in that, Before the hydrophobic resin adsorption, the liquid after activated carbon adsorption is filtered to remove the activated carbon powder from the liquid.
6. The method for defatting bovine serum albumin according to claim 1, characterized in that, The preparation method of pentyl-modified styrene-based hydrophobic resin includes the following steps: S1. Under a nitrogen atmosphere, AlCl3 and THF were mixed and stirred in an ice-water bath. Then, a mixture of styrene and n-pentyl bromide was added dropwise to the system. After the addition was complete, the ice-water bath was removed, and the reaction was quenched after reacting at room temperature for a certain period of time. The mixture was then purified by extraction, washing, and drying to obtain pentyl-modified styrene. S2. Prepare an aqueous phase containing gelatin and sodium chloride; prepare an oil phase containing pentyl-modified styrene, divinylbenzene, catalyst and pore-forming agent; under nitrogen protection, disperse the oil phase in the aqueous phase, heat and react, then cool naturally, and finally filter, wash, remove impurities, perform final washing, dry and sieve.
7. The method for defatting bovine serum albumin according to claim 6, characterized in that, In step S1, the ratio of AlCl3, THF, styrene and n-pentyl bromide is (15-20)g:(200-400)mL:(85-130)g:(60-85)g; The temperature of the ice water bath is 0-5℃; The dropping time for styrene and n-pentyl bromide is 1-2 hours; The reaction time at room temperature is 10-15 hours.
8. The method for defatting bovine serum albumin according to claim 6, characterized in that, In step S2, the concentration of gelatin in the aqueous phase is 0.3-0.7% (w / v), and the concentration of sodium chloride is 1.5-2.5% (w / v).
9. The method for defatting bovine serum albumin according to claim 6, characterized in that, In the oil phase, the pore-forming agents are toluene and n-heptane; and the ratio of pentyl-modified styrene, divinylbenzene, catalyst, toluene and n-heptane is (90-110)g:(50-75)g:(1-2)g:(90-100)mL:(55-65)mL.
10. The method for defatting bovine serum albumin according to claim 6, characterized in that, In step S2, the heating reaction is first raised to 50-60℃ and held for 3-5 hours; then the temperature is raised to 70-75℃ and held for 6-8 hours.