A method for preparing a composite emulsion for delivery of a biologically active substance by high pressure homogenization

Abstract

The application discloses a high-pressure homogenization preparation method of a composite emulsion for bioactive substance delivery, and relates to the technical field of emulsion preparation, which comprises the following steps: preparing an oil phase, an aqueous phase and a bioactive substance solution respectively, wherein the aqueous phase contains a water-soluble emulsifier and a non-ionic water-soluble surfactant, and the mass ratio of the water-soluble emulsifier to the non-ionic water-soluble surfactant is 0.10:1 to 0.20:1; the oil phase is added into the aqueous phase to realize shearing emulsification, and then high-pressure homogenization is carried out to obtain a high-pressure homogenization emulsion; then, ultrafiltration is carried out to reduce the content of the non-ionic water-soluble surfactant in the continuous aqueous phase to 20% to 45% of that before the ultrafiltration; subsequently, the bioactive substance solution and a water-soluble emulsifier solution are added, and low-pressure homogenization is carried out under a pressure corresponding to 5% to 20% of the highest processing pressure in the high-pressure homogenization process, so as to obtain the composite emulsion.

Description

Technical Field

[0001] This invention relates to the field of emulsion preparation technology, specifically to a high-pressure homogenization method for preparing a composite emulsion for delivering bioactive substances. Background Technology

[0002] Emulsion systems for delivering bioactive substances are among the commonly used dispersion carriers in pharmaceutical formulations, vaccine adjuvants, and functional biological products. Among them, oil-in-water emulsions are widely used for delivering bioactive substances such as proteins, peptides, and antigens because they can provide a large oil-water interface, good dispersion stability, and adjustable interfacial composition.

[0003] Existing emulsion delivery systems typically involve post-mixing a base emulsion with bioactive substances, or having the bioactive substances participate in emulsification and homogenization along with the aqueous phase. The former is prone to interfacial competition, local aggregation, and increased particle size distribution during the post-addition stage; the latter subjectes proteins or antigens to shear, pressure fluctuations, and temperature rises, resulting in a decrease in activity retention. In addition, excessive residual levels of some emulsifying agents in the continuous aqueous phase can also affect the stability of subsequent compounding. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the technical solution of this invention is as follows: A high-pressure homogenization method for preparing a composite emulsion for delivering bioactive substances includes the following steps: S1. Prepare oil phase, aqueous phase and bioactive substance solution respectively. The oil phase includes oil phase material and oil-soluble emulsifier. The aqueous phase includes aqueous medium, water-soluble emulsifier and nonionic water-soluble surfactant. The mass ratio of nonionic water-soluble surfactant to water-soluble emulsifier is 0.10:1 to 0.20:1. S2. The oil phase is added to the aqueous phase and shear emulsified to obtain a primary emulsion. The primary emulsion is then subjected to high-pressure homogenization to obtain a high-pressure homogenized emulsion. The high-pressure homogenization disperses the oil phase droplets in the continuous aqueous phase, and the nonionic water-soluble surfactant is retained in the continuous aqueous phase of the high-pressure homogenized emulsion. S3. The high-pressure homogenized emulsion is subjected to ultrafiltration liquid exchange treatment, and the nonionic water-soluble surfactant is discharged with the filtrate, so that the content of the nonionic water-soluble surfactant in the continuous aqueous phase is reduced to 20% to 45% before the ultrafiltration liquid exchange treatment, and a liquid exchange emulsion is obtained. S4. After adding the bioactive substance solution to the emulsion, add a water-soluble emulsifier to the resulting emulsion for replenishment, and then perform low-pressure homogenization treatment to obtain a composite emulsion. The pressure of the low-pressure homogenization treatment is 5% to 20% of the highest treatment pressure in the high-pressure homogenization treatment.

[0005] Furthermore, in S1, the nonionic water-soluble surfactant is a block copolymer containing polyoxyethylene segments and polyoxypropylene segments, and the nonionic water-soluble surfactant forms a clear aqueous phase in the aqueous medium.

[0006] Furthermore, in S1, the aqueous phase is prepared by first dissolving the nonionic water-soluble surfactant in the aqueous medium, then adding the water-soluble emulsifier and mixing to form a homogeneous aqueous phase.

[0007] Furthermore, in S1, the oil phase material is squalene; The oil-soluble emulsifier is sorbitan trioleate; The water-soluble emulsifier is polysorbate 80.

[0008] Furthermore, in S2, the high-pressure homogenization process includes a first high-pressure homogenization process and a second high-pressure homogenization process. The pressure of the first high-pressure homogenization process is 80 MPa to 120 MPa; The pressure of the second high-pressure homogenization process is 35 MPa to 70 MPa; The pressure of the second high-pressure homogenization process is 40% to 70% of the pressure of the first high-pressure homogenization process.

[0009] Furthermore, in S3, the ultrafiltration liquid exchange process is a tangential flow ultrafiltration liquid exchange process; The high-pressure homogenized emulsion circulates along the surface of the ultrafiltration membrane on the retention side, and the oil phase droplets are retained on the retention side; The liquid permeated through the ultrafiltration membrane is discharged as filtrate, while a replacement medium is added to the retention liquid side to perform a liquid exchange treatment on the high-pressure homogenized emulsion, so that the nonionic water-soluble surfactant is discharged with the filtrate.

[0010] Furthermore, in S3, the liquid exchange medium is a buffer solution with the same composition as the aqueous phase medium; The replacement medium is added to the retention liquid side in an equal volume manner, and the cumulative volume of the replacement medium added is 2 to 6 times the volume of the high-pressure homogenized emulsion.

[0011] Furthermore, in S4, the bioactive substance solution is added to the liquid exchange emulsion and mixed to form a pre-composite emulsion; Based on the volume of the emulsion used for fluid exchange, the volume of the bioactive substance solution added is 5% to 15%. Based on the volume of the replacement emulsion, the volume of the water-soluble emulsifier replenishment is 2% to 8%, and the mass concentration of the water-soluble emulsifier in the water-soluble emulsifier replenishment is 0.05% to 0.30%. The water-soluble emulsifier is added to the pre-composite emulsion and mixed to form a replenishing emulsion; The replenishing emulsion is subjected to 2 to 5 cycles of low-pressure homogenization to form the composite emulsion.

[0012] The beneficial effects of this invention are as follows: 1. By adding a nonionic water-soluble surfactant to the aqueous phase and controlling its mass ratio with the water-soluble emulsifier, the interfacial state between the oil phase and the aqueous phase is improved, so that the oil phase can be fully broken and uniformly dispersed during shear emulsification and high-pressure homogenization. This helps to reduce the difference in emulsion droplet size and inhibit droplet aggregation, thereby improving the emulsification uniformity and dispersion stability of the oil and water phases.

[0013] 2. By performing ultrafiltration on the emulsion after high-pressure homogenization and controlling the content of nonionic water-soluble surfactants in the continuous aqueous phase to 20% to 45% of that before the liquid exchange, the composition of the continuous aqueous phase is adjusted while maintaining the dispersion state of the emulsion droplets. This reduces the influence of excessive surfactants on the mixing and dispersion process of different components and improves the mixing uniformity and dispersion stability of the emulsion system after the liquid exchange.

[0014] 3. By sequentially performing high-pressure homogenization emulsification, ultrafiltration liquid exchange, liquid replenishment, and low-pressure homogenization, the oil phase emulsification process is connected with the mixing and dispersion process of the subsequently added components. This allows the oil phase to first form a uniform emulsion through high-pressure homogenization, and then the subsequently added components are dispersed in the emulsion system under a lower homogenization pressure, thus taking into account both the uniformity of the oil phase emulsification and the mixing and dispersion stability of the subsequently added components. Attached Figure Description

[0015] Figure 1 This is a flowchart of the preparation method of the present invention; Figure 2 This is a schematic diagram of the tangential flow ultrafiltration fluid exchange of the present invention; Figure 3 This is a schematic diagram of the tangential flow ultrafiltration liquid exchange process of the present invention; Figure 4 This is a schematic diagram of the post-compounding treatment of bioactive substances according to the present invention. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. The raw materials, equipment, testing methods and data used in the following embodiments are used to illustrate the implementation of the present invention; without changing the steps and parameter ranges of this method, those skilled in the art can proportionally scale up or down the amount of materials used according to the production scale.

[0017] In the following examples, squalene is the oil phase material, sorbitan trioleate is the oil-soluble emulsifier, polysorbate 80 is the water-soluble emulsifier, and the nonionic water-soluble surfactant is a block copolymer containing polyoxyethylene segments and polyoxypropylene segments, specifically poloxamer 188.

[0018] In the following embodiments, the bioactive substance feed solution is a protein or antigen water-soluble feed solution. After the protein or antigen water-soluble feed solution is added to the change emulsion, it is at least partially distributed in the continuous aqueous phase and near the oil-water interface, and forms a post-composite dispersion state through replenishment with water-soluble emulsifier and cyclic low-pressure homogenization treatment.

[0019] In the following examples, the volume of bioactive substance added is determined based on the volume of the replacement emulsion and controlled between 5% and 15%.

[0020] In the following embodiments, the clarified aqueous phase refers to an aqueous phase in which no visible suspended matter, precipitate, or flocculent matter is observed, and the transmittance measured at 25°C and 600nm wavelength is not less than 95%; the equal volume replenishment method refers to the process in which the volume of filtrate discharged and the volume of replenishment medium are kept the same during the tangential flow ultrafiltration liquid exchange process, so that the volume of emulsion on the retained liquid side is maintained within the range of 95% to 105% of the initial volume; the circulating low-pressure homogenization treatment refers to the treatment method in which the replenished emulsion is recovered after passing through the low-pressure homogenizer once, and then enters the same low-pressure homogenizer for the next pass.

[0021] In the following embodiments, when the high-pressure homogenization process includes a first high-pressure homogenization process and a second high-pressure homogenization process, the low-pressure homogenization process pressure is calculated based on the first high-pressure homogenization process pressure, which is the highest processing pressure in the high-pressure homogenization process.

[0022] In the following embodiments, unless otherwise specified, shear emulsification is performed using a rotor-stator high-speed shearing machine, high-pressure homogenization and low-pressure homogenization are performed using a high-pressure homogenizer, and tangential flow ultrafiltration liquid exchange is performed using a tangential flow ultrafiltration device, with the processing temperature controlled within the range of 20°C to 25°C.

[0023] Example 1 Please refer to Figures 1 to 4 In this embodiment, influenza virus lysate antigen is used as the bioactive substance, and the resulting composite emulsion is used to evaluate the in vitro preparation stability and antigenicity retention of the water-soluble antigen solution in the post-composite emulsion.

[0024] S1. Prepare oil phase, aqueous phase and bioactive substance solution separately.

[0025] The oil phase was prepared as follows: 4.30 g of squalene and 0.50 g of sorbitan trioleate were weighed and added to a clean glass container. The mixture was stirred at 300 rpm for 10 min at 25 °C to ensure that the sorbitan trioleate was uniformly dispersed in the squalene, thus obtaining the oil phase. The oil phase appeared as a clear to slightly yellow transparent liquid, and no layering or visible suspended matter was observed after standing for 10 min.

[0026] The aqueous medium is a phosphate buffer solution with a pH of 6.8, containing 10 mmol / L phosphate and 137 mmol / L sodium chloride, with an osmotic pressure of 286 mOsmol / kg to 304 mOsmol / kg. It is sterilized by filtration through a 0.22 μm filter membrane before use.

[0027] Add 90.00 mL of phosphate buffer to a mixing container, add 0.075 g of poloxamer 188, and stir at 400 rpm for 20 min at 25 °C until no undissolved particles are visible at the bottom of the container. After standing for 3 min, the liquid should not be visibly turbid, yielding the aqueous pre-phase solution. Poloxamer 188 is a block copolymer containing polyoxyethylene and polyoxypropylene segments.

[0028] Before taking the aqueous phase, the liquid was placed in a 1 cm cuvette, and the transmittance was measured at 25 °C with a wavelength of 600 nm. The transmittance was 97.4%. After the sample was left to stand for 10 min, no visible precipitate, flocculent matter or oily floating matter was observed. It was determined that poloxamer 188 formed a clear aqueous phase in the aqueous medium.

[0029] Add 0.50 g of polysorbate 80 to the aqueous phase precursor solution and continue stirring at 400 rpm for 15 min to obtain a homogeneous aqueous phase. The mass ratio of poloxamer 188 to polysorbate 80 is 0.15:1.

[0030] The bioactive substance was influenza virus lysis antigen solution. The influenza virus lysis antigen solution was diluted with phosphate buffer (pH 6.8) to a protein concentration of 0.20 mg / mL and stored at 2°C to 8°C for later use. Before use, it was equilibrated at 20°C to 25°C for 15 min. The protein concentration of the influenza virus lysis antigen solution was determined to be 0.20 mg / mL using the BCA method before addition, and the antigen binding signal measured by enzyme-linked immunosorbent assay (ELISA) was used as a 100% antigenicity reference.

[0031] S2. After adding the oil phase to the aqueous phase, shear emulsification and high-pressure homogenization are performed.

[0032] Under shearing conditions of 6000 rpm in the aqueous phase, the oil phase was added to the aqueous phase at a rate of approximately 1.0 mL / min. After the oil phase was added, the shearing speed was increased to 8000 rpm and shearing continued for 5 minutes to obtain the primary emulsion. During the shearing process, an ice-water bath was used to control the material temperature to not exceed 25°C. The primary emulsion appeared as a milky white, homogeneous liquid, and no visible oil layer was observed after standing for 5 minutes.

[0033] The colostrum is transferred to the feed tank of a high-pressure homogenizer and undergoes a first high-pressure homogenization process at a pressure of 100 MPa, repeated three times. Then, a second high-pressure homogenization process is performed at a pressure of 50 MPa, repeated twice, yielding a high-pressure homogenized emulsion. The pressure of the second high-pressure homogenization process is 50% of the pressure of the first process. After each cycle, the material is cooled to below 25°C using cooling coils before entering the next homogenization process.

[0034] The high-pressure homogenized emulsion was detected by dynamic light scattering method. The sample was diluted 50 times with phosphate buffer at pH 6.8 and the detection temperature was 25℃. The detection results were D50 of 136nm, D90 of 238nm and PDI of 0.110.

[0035] Take 2.0 mL of the high-pressure homogenized emulsion and centrifuge at 4℃ and 100,000 g for 60 min. Remove the upper layer of emulsion droplets and collect the middle and lower clear liquid as the continuous aqueous phase sample of the high-pressure homogenized emulsion. The mass concentration of poloxamer 188 in the continuous aqueous phase sample of the high-pressure homogenized emulsion was determined by high-performance liquid chromatography-evaporative light scattering detection. The result was 0.56 mg / mL, indicating that poloxamer 188 has a detectable retention in the continuous aqueous phase of the high-pressure homogenized emulsion.

[0036] S3. Perform tangential flow ultrafiltration liquid exchange treatment on the high-pressure homogenized emulsion.

[0037] The high-pressure homogenized emulsion was added to the retention tank of the tangential flow ultrafiltration device, with an initial retention volume of approximately 95 mL. The tangential flow ultrafiltration device used a polyethersulfone hollow fiber membrane with a filtration area of ​​0.05 m². 2 The nominal molecular weight cutoff is 300 kDa. The circulation pump is started to circulate the high-pressure homogenized emulsion along the surface of the ultrafiltration membrane on the retainer side. The circulation flow rate is controlled at 50 mL / min to 80 mL / min, the transmembrane pressure is controlled at 0.05 MPa to 0.12 MPa, and the processing temperature is controlled at 20℃ to 25℃. The liquid permeating through the ultrafiltration membrane is continuously discharged from the filtrate side as the filtrate.

[0038] The nominal molecular weight cutoff is used to retain oil phase droplets and allow free poloxamer 188 to be discharged with the filtrate. Under the condition that the oil phase droplets can be retained on the retention side and free poloxamer 188 in the continuous aqueous phase can pass through the ultrafiltration membrane, the nominal molecular weight cutoff can be selected in the range of 100 kDa to 500 kDa.

[0039] The replacement medium is a phosphate buffer solution with a pH of 6.8, which has the same composition as the aqueous phase medium. During the tangential flow ultrafiltration replacement process, when the liquid level in the retention tank decreases, replacement medium is added to the retention tank in a volume equal to the volume of filtrate discharged, so that the volume of the retention liquid is maintained within the range of 95% to 105% of the initial volume.

[0040] The equal volume replenishment method is as follows: for every 10 mL of filtrate collected, 10 mL of replacement medium is added to the retention liquid side; when the continuous collection volume of filtrate reaches 3 times the initial volume of the high-pressure homogenized emulsion, the replacement process is stopped, and the replacement emulsion is obtained.

[0041] Take 2.0 mL of the replacement emulsion and centrifuge at 4℃ and 100,000 g for 60 min to remove the upper layer of emulsion droplets. Collect the middle and lower clear liquid as the continuous aqueous phase sample of the replacement emulsion. The concentration of poloxamer 188 in the continuous aqueous phase sample of the replacement emulsion was determined by the same high performance liquid chromatography-evaporative light scattering detection method as S2, and the result was 0.178 mg / mL.

[0042] The percentage of poloxamer 188 content was calculated using the following formula: (Poloxamer 188 mass concentration in the continuous aqueous phase of the emulsion after liquid exchange ÷ Poloxamer 188 mass concentration in the continuous aqueous phase of the high-pressure homogenized emulsion) × 100%. The calculated result was 31.8%, meaning the poloxamer 188 content in the continuous aqueous phase of the emulsion after liquid exchange decreased to 31.8% of the level before the ultrafiltration liquid exchange treatment. Poloxamer 188 was detected in the filtrate collected during the tangential flow ultrafiltration liquid exchange treatment, indicating that poloxamer 188 was discharged with the filtrate.

[0043] The replacement emulsion was analyzed using dynamic light scattering (D50) at 142 nm, D90 at 247 nm, and PDI at 0.116. No visible oil layer, flocculent material, or sediment was observed after the replacement emulsion stood for 10 minutes.

[0044] S4. After adding the bioactive substance solution to the replacement emulsion, perform replenishment and low-pressure homogenization.

[0045] Take 90.00 mL of the exchange emulsion and add 9.00 mL of influenza virus lysis antigen solution. Stir at 300 rpm during the addition process, and continue stirring for 10 min after the addition is complete to form a pre-composite emulsion. The volume of influenza virus lysis antigen solution added is 10% of the volume of the exchange emulsion. This added volume is within the range of 5% to 15%.

[0046] Based on the volume of the replacement emulsion, add 5% (4.50 mL) of water-soluble emulsifier replenishment solution to the pre-composite emulsion. The water-soluble emulsifier replenishment solution is a phosphate buffer solution with a pH of 6.8 containing 0.15% polysorbate 80. Add the water-soluble emulsifier replenishment solution to the pre-composite emulsion at a rate of approximately 0.5 mL / min, and continue stirring at 300 rpm for 5 min after the addition is complete to form the replenishment emulsion.

[0047] The replenished emulsion was subjected to a circulating low-pressure homogenization process at a pressure of 10 MPa, repeated three times to obtain a composite emulsion. The low-pressure homogenization pressure was 10% of the initial high-pressure homogenization pressure. The circulating low-pressure homogenization process involved passing the replenished emulsion through the low-pressure homogenizer once, recovering it, and then re-entering the same homogenizer for the next processing. After each low-pressure homogenization process, the material temperature was controlled below 25°C.

[0048] The composite emulsion was detected by dynamic light scattering method. The sample was diluted 50 times with phosphate buffer at pH 6.8 and the detection temperature was 25℃. The detection results were D50 of 158nm, D90 of 272nm and PDI of 0.145.

[0049] The total detectable protein in the composite emulsion was determined using the BCA method, with the total protein before the addition of influenza virus lysate as a 100% reference. The protein recovery rate was calculated to be 92.4%.

[0050] The antigenicity retention rate of the composite emulsion was determined by enzyme-linked immunosorbent assay (ELISA). The influenza virus lysate that was not subjected to shear emulsification, high-pressure homogenization, and low-pressure homogenization was used as a 100% antigenicity reference. The antigenicity retention rate was calculated to be 90.6%.

[0051] The composite emulsion was stored at 2°C to 8°C in the dark for 30 days, and no visible layering, emulsification, flocculent matter, or precipitation was observed. After 30 days of storage, the D90 was measured by the same dynamic light scattering method and was 294 nm.

[0052] Example 2 This embodiment illustrates a preparation method under near-low-end conditions. The difference between this embodiment and Example 1 is that in S1, the amount of poloxamer 188 is 0.060 g, and the mass ratio of poloxamer 188 to polysorbate 80 is 0.12:1; in S2, the first high-pressure homogenization treatment pressure is 80 MPa, with 3 cycles, and the second high-pressure homogenization treatment pressure is 35 MPa, with 2 cycles; in S3, the cumulative volume of the replacement medium is twice the volume of the high-pressure homogenized emulsion; in S4, the volume of the influenza virus lysis antigen solution added is 5% of the replacement emulsion volume, the volume of the water-soluble emulsifier replenishment is 2% of the replacement emulsion volume, the mass concentration of polysorbate 80 in the water-soluble emulsifier replenishment is 0.05%, the low-pressure homogenization treatment pressure is 4 MPa, and the number of cycles is 2.

[0053] In this embodiment, the transmittance of the clarified aqueous phase at a wavelength of 600 nm was 96.8%; after tangential flow ultrafiltration liquid exchange treatment, the content of poloxamer 188 in the continuous aqueous phase of the liquid exchange emulsion was 41.2% of that before ultrafiltration liquid exchange treatment. The remaining preparation conditions were the same as in Example 1.

[0054] Example 3 This embodiment illustrates a preparation method under near-high-end conditions. The difference between this embodiment and Example 1 is that in S1, the amount of poloxamer 188 is 0.090 g, and the mass ratio of poloxamer 188 to polysorbate 80 is 0.18:1; in S2, the first high-pressure homogenization treatment pressure is 120 MPa, with 4 cycles, and the second high-pressure homogenization treatment pressure is 70 MPa, with 2 cycles; in S3, the cumulative volume of the replacement medium is 6 times the volume of the high-pressure homogenized emulsion; in S4, the volume of the influenza virus lysis antigen solution added is 15% of the replacement emulsion volume, the volume of the water-soluble emulsifier replenishment is 8% of the replacement emulsion volume, the mass concentration of polysorbate 80 in the water-soluble emulsifier replenishment is 0.30%, the low-pressure homogenization treatment pressure is 18 MPa, and the number of cycles is 5.

[0055] In this embodiment, the transmittance of the clarified aqueous phase at a wavelength of 600 nm was 96.0%; after tangential flow ultrafiltration liquid exchange treatment, the content of poloxamer 188 in the continuous aqueous phase of the liquid exchange emulsion was 25.8% of that before ultrafiltration liquid exchange treatment. The remaining preparation conditions were the same as in Example 1.

[0056] Example 4 This embodiment discloses a method for preparing bioactive protein substances under different feed conditions. The difference between this embodiment and Example 1 is that the bioactive substance feed is an aqueous solution of recombinant protein antigen, prepared using a phosphate buffer solution with a pH of 7.2, and the protein concentration is 0.20 mg / mL; the aqueous phase medium in S1 is a phosphate buffer solution with a pH of 7.2; and the medium for changing the solution in S3 is a phosphate buffer solution with a pH of 7.2.

[0057] In this embodiment, after tangential flow ultrafiltration and fluid exchange treatment, the poloxamer 188 content in the continuous aqueous phase of the fluid exchange emulsion is 33.4% of that before ultrafiltration and fluid exchange treatment; in S4, after adding the recombinant protein antigen aqueous solution to the fluid exchange emulsion, the composite emulsion is prepared under the same fluid replenishment and circulating low-pressure homogenization conditions as in Example 1. The remaining preparation conditions are the same as in Example 1.

[0058] Example 5 This embodiment discloses a preparation method under scaled-up conditions. The feed amounts from Example 1 are scaled up by 5 times, with 21.50 g of squalene, 2.50 g of sorbitan trioleate, 450.00 mL of aqueous medium, 0.375 g of poloxamer 188, and 2.50 g of polysorbate 80.

[0059] In this embodiment, the shear emulsification speed is 8500 r / min, and the shearing time is 6 min; the first high-pressure homogenization treatment pressure is 100 MPa, and the cycle treatment is 4 times; the second high-pressure homogenization treatment pressure is 50 MPa, and the cycle treatment is 2 times; during the tangential flow ultrafiltration liquid exchange treatment, the cumulative volume of the liquid exchange medium added is 3 times the volume of the high-pressure homogenized emulsion; the proportion of influenza virus lysis antigen solution added, the proportion of water-soluble emulsifier replenishment added, the concentration of water-soluble emulsifier replenishment, and the cyclic low-pressure homogenization conditions in S4 are all the same as in Example 1.

[0060] In this embodiment, after tangential flow ultrafiltration liquid exchange treatment, the content of poloxamer 188 in the continuous aqueous phase of the liquid exchange emulsion is 33.7% of that before ultrafiltration liquid exchange treatment.

[0061] Table 1: Detection results of the composite emulsions obtained in Examples 1 to 5 Table 1-1: Emulsion Particle Size and Residual Ratio Table 1-2: Protein recovery, activity retention, and 30-day stability Note: The activity retention rate of Example 4 was determined by antibody binding ELISA corresponding to the recombinant protein antigen. The value is used to illustrate the retention of the recombinant protein antigen system under the same detection range and is not used for absolute value comparison with the influenza virus lysate antigen system.

[0062] Comparative Example 1 This comparative example illustrates the effect of using a nonionic water-soluble surfactant at a level lower than that in Example 1 on emulsion formation and subsequent compounding.

[0063] The difference between this comparative example and Example 1 is that the amount of poloxamer 188 used in S1 is 0.025g, the mass ratio of poloxamer 188 to polysorbate 80 is 0.05:1, and the other conditions are the same as in Example 1.

[0064] Comparative Example 2 This comparative example is used to illustrate the effect of using a nonionic water-soluble surfactant in a quantity higher than that in Example 1 on subsequent compounding.

[0065] The difference between this comparative example and Example 1 is that the amount of poloxamer 188 used in S1 is 0.150g, the mass ratio of poloxamer 188 to polysorbate 80 is 0.30:1, and the other conditions are the same as in Example 1.

[0066] Comparative Example 3 This comparative example illustrates the effect on composite emulsions when ultrafiltration fluid replacement treatment is not performed.

[0067] The difference between this comparative example and Example 1 is that ultrafiltration liquid exchange is not performed in S3, and the high-pressure homogenized emulsion obtained in S2 is directly used in S4. The other conditions are the same as in Example 1.

[0068] Comparative Example 4 This comparative example illustrates the effect on composite emulsions when the residual proportion of nonionic water-soluble surfactants is below a certain range after ultrafiltration fluid replacement treatment.

[0069] The difference between this comparative example and Example 1 is that the cumulative volume of the liquid exchange medium added in S3 is 8 times the volume of the high-pressure homogenized emulsion, which reduces the content of poloxamer 188 in the continuous aqueous phase of the liquid exchange emulsion to 12.6% before ultrafiltration liquid exchange treatment. The other conditions are the same as in Example 1.

[0070] Comparative Example 5 This comparative example illustrates the effect on composite emulsions when the residual proportion of nonionic water-soluble surfactants exceeds the specified range after ultrafiltration fluid replacement treatment.

[0071] The difference between this comparative example and Example 1 is that the cumulative volume of the replacement medium added in S3 is 1 times the volume of the high-pressure homogenized emulsion, so that the content of poloxamer 188 in the continuous aqueous phase of the replacement emulsion is reduced to 58.4% of that before the ultrafiltration replacement treatment. The other conditions are the same as in Example 1.

[0072] Comparative Example 6 This comparative example illustrates the effect of high-pressure homogenization on the retention of bioactive substances.

[0073] The difference between this comparative example and Example 1 is that the influenza virus lysate antigen solution was added to the aqueous phase before S2 shear emulsification and was subjected to the first high-pressure homogenization treatment and the second high-pressure homogenization treatment together with the colostrum. The other conditions were the same as in Example 1.

[0074] To differentiate the effects of the high-pressure homogenization stage and the subsequent ultrafiltration fluid exchange stage on activity retention, samples were taken from the high-pressure homogenized emulsion after the second high-pressure homogenization treatment and before entering S3, and tested using the same method. The protein recovery rate of the samples taken after S2 was 79.8%, and the antigenicity retention rate was 70.8%. The final test results after completing S3 and S4 are shown in Table 2-2.

[0075] Comparative Example 7 This comparative example illustrates the effect of not adding water-soluble emulsifiers to the compound emulsion.

[0076] The difference between this comparative example and Example 1 is that, in S4, after the influenza virus lysate antigen solution is added to the replacement emulsion, no water-soluble emulsifier is added for replenishment, and the cyclic low-pressure homogenization process is performed directly. The other conditions are the same as in Example 1.

[0077] Comparative Example 8 This comparative example illustrates the effect on composite emulsions when no cyclic low-pressure homogenization process is performed.

[0078] The difference between this comparative example and Example 1 is that after adding water-soluble emulsifier to replenish the liquid in S4, the mixture is stirred at 300 r / min for 15 min to form a composite emulsion, without undergoing cyclic low-pressure homogenization treatment. The other conditions are the same as in Example 1.

[0079] Table 2: Detection results of Comparative Examples 1 to 8 Table 2-1: Emulsion Particle Size and Residual Ratio Table 2-2: Protein recovery, activity retention, and 30-day stability Experimental Results and Beneficial Effects: As shown in Tables 1 and 2, the composite emulsions in Examples 1 to 5 were prepared according to the process route of this invention. The resulting composite emulsions had a D90 of 249 nm to 318 nm, a PDI of 0.114 to 0.162, a protein recovery rate of 91.6% to 93.4%, and an activity retention rate of 89.0% to 91.2%. No visible stratification was observed after standing at 2°C to 8°C for 30 days. These results indicate that when the mass ratio of nonionic water-soluble surfactant to water-soluble emulsifier, the residual proportion of ultrafiltration fluid, the replenishment of water-soluble emulsifier, and the low-pressure homogenization treatment are all within the specified ranges, the composite emulsion can maintain a narrow particle size distribution and good stability of bioactive substances.

[0080] Example 1 used a poloxamer 188 to polysorbate 80 mass ratio of 0.15:1. After changing the solution, the residual proportion of poloxamer 188 was 31.8%, and the resulting composite emulsion had a D90 of 272 nm, a PDI of 0.145, and an activity retention rate of 90.6%. Example 2 used a mass ratio close to the lower end of the range, 0.12:1. After changing the solution, the residual proportion of poloxamer 188 was 41.2%, and the resulting composite emulsion had a D90 of 318 nm, a PDI of 0.162, and an activity retention rate of 89.0%. Example 3 used a mass ratio close to the higher end of the range, 0.18:1. After changing the solution, the residual proportion of poloxamer 188 was 25.8%, and the resulting composite emulsion had a D90 of 249 nm, a PDI of 0.114, and an activity retention rate of 91.2%. The above results show that a mass ratio range of 0.10:1 to 0.20:1 and a residual ratio range of 20% to 45% can cover low-end, intermediate, and high-end conditions, and preparation results that meet the stability requirements of composite emulsions can be obtained under all conditions.

[0081] In Comparative Example 1, the mass ratio of poloxamer 188 to polysorbate 80 was 0.05:1, which is below the limit specified in this invention. The resulting composite emulsion had a D90 of 421 nm and a PDI of 0.268, and exhibited slight emulsification. This result indicates that when the amount of nonionic water-soluble surfactant is insufficient, the dispersion stability of the basic emulsion formed in the high-pressure homogenization stage decreases, and the particle size distribution becomes significantly wider after the subsequent addition of bioactive substance solution. In Comparative Example 2, the mass ratio of poloxamer 188 to polysorbate 80 was 0.30:1, which is above the limit specified in this invention. The resulting composite emulsion had a D90 of 356 nm, a PDI of 0.221, and an activity retention rate of 85.4%. This result indicates that when the amount of nonionic water-soluble surfactant is too high, both the emulsion dispersion state and the activity retention effect in the subsequent composite stage decrease.

[0082] In Comparative Example 3, the ultrafiltration liquid replacement treatment was omitted, and the poloxamer 188 content in the continuous aqueous phase was not reduced by the liquid replacement. The resulting composite emulsion had a D90 of 374 nm, a PDI of 0.229, and an activity retention rate of 85.9%. In Comparative Example 4, after ultrafiltration liquid replacement, the residual proportion of poloxamer 188 was 12.6%, which is less than 20%. The resulting composite emulsion had a D90 of 392 nm and a small amount of flocculent matter appeared. In Comparative Example 5, after ultrafiltration liquid replacement, the residual proportion of poloxamer 188 was 58.4%, which is more than 45%. The resulting composite emulsion had a D90 of 416 nm and a PDI of 0.257. The above results indicate that ultrafiltration liquid replacement treatment has a regulatory effect on the content of nonionic water-soluble surfactants in the continuous aqueous phase. Both excessively low and excessively high residual proportions weaken the stability of the subsequent composite emulsion. Controlling the residual proportion within the range of 20% to 45% is beneficial for reducing dispersion disturbance when adding subsequent bioactive substances and maintaining the particle size stability of the emulsion system.

[0083] Comparative Example 6 involved shear emulsification and high-pressure homogenization of the influenza virus lysate antigen solution, resulting in an emulsion with a D90 of 279 nm and a PDI of 0.142. After S2, the protein recovery rate was 79.8%, and the activity retention rate was 70.8%. After S3 and S4, the final protein recovery rate was 78.6%, and the final activity retention rate was 69.4%, showing little difference between the two groups. This result indicates that the activity decrease in Comparative Example 6 mainly occurred during the shear emulsification and high-pressure homogenization stages, with subsequent ultrafiltration and low-pressure homogenization contributing little to this decrease. Adding the bioactive substance solution after solution exchange and using low-pressure homogenization for compounding helps reduce the adverse effects of high-pressure shear on protein-based bioactive substances.

[0084] Comparative Example 7, without the addition of a water-soluble emulsifier after the addition of the bioactive substance feed solution, resulted in a composite emulsion with a D90 of 362 nm and a PDI of 0.214, exhibiting slight emulsification. Comparative Example 8, after the addition of a water-soluble emulsifier feed solution, only involved stirring without cyclic low-pressure homogenization, resulting in a composite emulsion with a D90 of 346 nm and a PDI of 0.204. These results indicate that the addition of a water-soluble emulsifier after the addition of the bioactive substance feed solution improves the dispersion stability during the emulsion compounding process, while cyclic low-pressure homogenization further reduces local concentration differences and the risk of droplet aggregation. The combined use of these two methods results in a composite emulsion with a lower D90, a lower PDI, and better 30-day storage stability.

[0085] Example 4 used a recombinant protein antigen aqueous solution as the bioactive substance feed solution. The resulting composite emulsion had a D90 of 281 nm, a PDI of 0.136, a protein recovery rate of 92.3%, and an activity retention rate of 90.4%. These results indicate that the method of the present invention has certain applicability to protein-based bioactive substances. Example 5 prepared the composite emulsion under a 5 times feed rate. The resulting composite emulsion had a D90 of 286 nm, a PDI of 0.152, a protein recovery rate of 91.8%, and an activity retention rate of 89.6%, indicating that the method can maintain process stability even under small-scale amplification conditions.

[0086] In summary, this invention, by introducing a nonionic water-soluble surfactant during the high-pressure homogenization stage, performing ultrafiltration liquid exchange treatment before adding the bioactive substance feed solution, and adding a water-soluble emulsifier for replenishment and cyclic low-pressure homogenization treatment in the post-addition stage, enables the composite emulsion to simultaneously achieve a narrow particle size distribution, high protein recovery rate, high activity retention rate, and good low-temperature storage stability. This preparation method is suitable for the preparation and in vitro stability evaluation of composite emulsions containing water-soluble protein or antigenic substances such as influenza virus lysate antigens.

[0087] Detection method: Particle size and PDI were determined by dynamic light scattering. Before detection, 100 μL of the emulsion to be tested was taken, diluted to 5 mL with the corresponding aqueous medium, gently inverted to mix, placed in a disposable cuvette, and detected at 25℃. D50, D90, and PDI were recorded. Each sample was measured in triplicate, and the average value was taken.

[0088] The content of nonionic water-soluble surfactants was determined by high-performance liquid chromatography-evaporative light scattering (HPLC-ELISA). Before detection, high-pressure homogenized emulsions before and after ultrafiltration were centrifuged at 4°C and 100,000g for 60 min to remove the upper emulsion droplet enrichment layer. The middle and lower clear liquids were collected as continuous aqueous phase samples. A continuous aqueous phase without poloxamer 188 was used as a matrix blank for detection, and the poloxamer 188 content was the apparent mass concentration in the continuous aqueous phase sample.

[0089] The high-performance liquid chromatography (HPLC) conditions were as follows: C18 column, mobile phase of acetonitrile and water (65:35 v / v), flow rate of 1.0 mL / min, column temperature of 35 °C, and drift tube temperature of the evaporative light scattering detector (ELSD) of 60 °C. A standard curve was prepared using poloxamer 188 standard, and the mass concentration of poloxamer 188 in the continuous aqueous phase samples was calculated based on the standard curve.

[0090] The percentage of content after ultrafiltration fluid replacement treatment is calculated according to the following formula: Percentage of content after ultrafiltration fluid replacement treatment = Mass concentration of poloxamer 188 in the continuous aqueous phase sample of the fluid replacement emulsion ÷ Mass concentration of poloxamer 188 in the continuous aqueous phase sample of the high-pressure homogenized emulsion × 100%.

[0091] Protein recovery was determined using the BCA method. A blank emulsion of the same formulation without bioactive substances was used as background subtraction. The total protein content in the pre-added bioactive substance emulsion was taken as 100%, and the protein recovery rate was calculated based on the total detectable protein content in the composite emulsion.

[0092] The activity retention rate of influenza virus lysate antigen solution was determined by enzyme-linked immunosorbent assay (ELISA). All samples were treated with the same dilution factor, and the influenza virus lysate antigen solution that did not undergo shearing emulsification and high-pressure homogenization was used as the 100% activity reference. The activity retention rate of recombinant protein antigen aqueous solution was determined by corresponding antibody binding ELISA.

[0093] The 30-day stability test method is as follows: the composite emulsion is stored in the dark at 2℃ to 8℃ for 30 days, and the presence of visible layering, emulsification, flocculent matter or precipitation is observed. The D90 is then tested according to the aforementioned dynamic light scattering method.

[0094] The above embodiments are used to illustrate possible implementations of the present invention and are not intended to limit the scope of protection of the present invention. Without changing the defined steps and key parameters, those skilled in the art can make routine adjustments according to the actual formulation scale and testing conditions.

Claims

1. A high-pressure homogenization method for preparing a composite emulsion for delivering bioactive substances, characterized in that, Includes the following steps: S1. Prepare oil phase, aqueous phase and bioactive substance solution respectively. The oil phase includes oil phase material and oil-soluble emulsifier. The aqueous phase includes aqueous medium, water-soluble emulsifier and nonionic water-soluble surfactant. The mass ratio of nonionic water-soluble surfactant to water-soluble emulsifier is 0.10:1 to 0.20:

1. S2. The oil phase is added to the aqueous phase and shear emulsified to obtain a primary emulsion. The primary emulsion is then subjected to high-pressure homogenization to obtain a high-pressure homogenized emulsion. The high-pressure homogenization disperses the oil phase droplets in the continuous aqueous phase, and the nonionic water-soluble surfactant is retained in the continuous aqueous phase of the high-pressure homogenized emulsion. S3. The high-pressure homogenized emulsion is subjected to ultrafiltration liquid exchange treatment, and the nonionic water-soluble surfactant is discharged with the filtrate, so that the content of the nonionic water-soluble surfactant in the continuous aqueous phase is reduced to 20% to 45% before the ultrafiltration liquid exchange treatment, and a liquid exchange emulsion is obtained. S4. After adding the bioactive substance solution to the emulsion, add a water-soluble emulsifier to the resulting emulsion for replenishment, and then perform low-pressure homogenization treatment to obtain a composite emulsion. The pressure of the low-pressure homogenization treatment is 5% to 20% of the highest treatment pressure in the high-pressure homogenization treatment.

2. The high-pressure homogenization preparation method of a composite emulsion for delivering bioactive substances according to claim 1, characterized in that, In S1, the nonionic water-soluble surfactant is a block copolymer containing polyoxyethylene segments and polyoxypropylene segments, and the nonionic water-soluble surfactant forms a clear aqueous phase in the aqueous medium.

3. The high-pressure homogenization preparation method of a composite emulsion for delivering bioactive substances according to claim 1, characterized in that, In S1, the aqueous phase is prepared by first dissolving the nonionic water-soluble surfactant in the aqueous medium, then adding the water-soluble emulsifier and mixing to form a homogeneous aqueous phase.

4. The high-pressure homogenization preparation method of a composite emulsion for delivering bioactive substances according to claim 1, characterized in that, In S1, the oil phase material is squalene; The oil-soluble emulsifier is sorbitan trioleate; The water-soluble emulsifier is polysorbate 80.

5. The high-pressure homogenization preparation method of a composite emulsion for delivering bioactive substances according to claim 1, characterized in that, In S2, the high-pressure homogenization process includes a first high-pressure homogenization process and a second high-pressure homogenization process; The pressure of the first high-pressure homogenization process is 80 MPa to 120 MPa; The pressure of the second high-pressure homogenization process is 35 MPa to 70 MPa; The pressure of the second high-pressure homogenization process is 40% to 70% of the pressure of the first high-pressure homogenization process.

6. The high-pressure homogenization preparation method of a composite emulsion for delivering bioactive substances according to claim 1, characterized in that, In S3, the ultrafiltration liquid exchange process is a tangential flow ultrafiltration liquid exchange process; The high-pressure homogenized emulsion circulates along the surface of the ultrafiltration membrane on the retention side, and the oil phase droplets are retained on the retention side; The liquid permeated through the ultrafiltration membrane is discharged as filtrate, while a replacement medium is added to the retention liquid side to perform a liquid exchange treatment on the high-pressure homogenized emulsion, so that the nonionic water-soluble surfactant is discharged with the filtrate.

7. The high-pressure homogenization preparation method of a composite emulsion for delivering bioactive substances according to claim 6, characterized in that, In S3, the fluid exchange medium is a buffer solution with the same composition as the aqueous phase medium; The replacement medium is added to the retention liquid side in an equal volume manner, and the cumulative volume of the replacement medium added is 2 to 6 times the volume of the high-pressure homogenized emulsion.

8. The high-pressure homogenization preparation method of a composite emulsion for delivering bioactive substances according to claim 1, characterized in that, In S4, the bioactive substance solution is added to the liquid exchange emulsion and mixed to form a pre-composite emulsion; Based on the volume of the emulsion used for fluid exchange, the volume of the bioactive substance solution added is 5% to 15%. Based on the volume of the replacement emulsion, the volume of the water-soluble emulsifier replenishment is 2% to 8%, and the mass concentration of the water-soluble emulsifier in the water-soluble emulsifier replenishment is 0.05% to 0.30%. The water-soluble emulsifier is added to the pre-composite emulsion and mixed to form a replenishing emulsion; The replenishing emulsion is subjected to 2 to 5 cycles of low-pressure homogenization to form the composite emulsion.

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