A kind of MPL triethylamine salt, its preparation process and application

Abstract

The present invention relates to a MPL triethylamine salt, a preparation process and an application thereof. The preparation method of the MPL triethylamine salt comprises the following steps: (1) dissolving a crude MPL product in chloroform and removing the solvent; (2) adding a TEA aqueous solution, and uniformly suspending the MPL in the solution by ultrasonication at room temperature; (3) transferring the above solution to pure water, and uniformly dispersing the solution by ultrasonication; (4) adding the TEA aqueous solution to the above system, ultrasonication, and freeze-drying. The present invention also relates to the application of the MPL triethylamine salt in the preparation of vaccine adjuvants and vaccines.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a MPL triethylamine salt, a preparation process and an application thereof. Background Art

[0002] MPL (monophosphoryl lipid A, also referred to as MPLA) is the innermost liposome (lipid A) part of endotoxin (LPS) in the cell wall of Gram-negative bacteria. The fat chain structure and phosphorylation degree will affect its activity and the effect of endotoxin. Lipid A from Salmonella minnesota contains a phosphate group at position 1 and position 4, which will over-activate the innate immune response, leading to clinical symptoms such as septic shock and strong side effects. Literature reports that after removing the phosphate group at position 1, its toxicity is reduced and the immune activity is retained, which is called monophosphoryl ester A (MPL).

[0003] As a new generation of vaccine adjuvants that have been marketed, MPL has a wide range of clinical uses. Common adjuvant systems include: AS01 (containing QS21 and MPL (liposome adjuvant), AS02 (MPL and QS21 added to water-in-oil emulsion containing squalene and α-tocopherol), AS04 (MPL adsorbed on aluminum hydroxide), AS15 (MPLA, QS21, CpG, liposomes), etc. Different formulations of adjuvant systems will affect the immune response effect and side effects of the adjuvant. Vaccine products containing adjuvants that have been marketed:

[0004] Fendrix TM: A hepatitis B vaccine developed by GSK in 2004, which consists of hepatitis B virus (HBV) surface antigen (HBsAg) and the AS04 adjuvant system.

[0005] Cervarix TM and Gardasil TM: The former is a bivalent cervical cancer vaccine developed by GSK in 2009, consisting of virus-like particles (VLPs) of the HPV L1 epitope and the AS04 system; the latter was developed by Merck and also contains the AS04 adjuvant.

[0006] Shingrix TM: A herpes zoster vaccine developed by GSK that uses the AS01 adjuvant system.

[0007] Since different formulations of adjuvant systems will affect the immune response and side effects of the adjuvant, MPL needs to go through different types of formulation processes to form an adjuvant preparation before it can be used. Therefore, there are high requirements for the solubility properties of the product.

[0008] At present, the source of MPL mainly relies on biological fermentation extraction, which contains a complex mixture of four or five long-chain acyl structural homologues with different chain lengths. The disadvantages are that the preparation and purification are difficult, the purity problems caused by the residual protein and sugar heterogeneity, and the cost is high. For example, a type of lipid A called SJ-19a is obtained by acid hydrolysis of Gram-negative bacteria Aspergillus liquefaciens, but chemical degradation may occur during the acid hydrolysis step, and the unstable phosphate bonds are broken, resulting in the heterogeneity of MPL.

[0009] The structure of synthetically prepared MPL is clear, and there is no purity problem caused by sugar heterogeneity, which is more suitable for industrial production. However, its water solubility is poor, which limits its application in preparations. The solubility of the currently reported MPL, base (non-salt type) and ammonium salt forms are relatively poor, which limits their use in liquid preparations; they are suspensions in water and cannot be filtered and sterilized.

[0010] The present invention is proposed in view of the above-mentioned technical problems in the prior art. Summary of the invention

[0011] The present invention firstly relates to a method for preparing MPL triethylamine salt (MPL-TEA), which comprises the following steps:

[0012] (1) MPL crude product was dissolved in chloroform and the solvent was removed;

[0013] (2) adding TEA aqueous solution and ultrasonically suspending MPL uniformly in the solution at room temperature;

[0014] (3) Transfer the above solution into pure water and disperse it evenly by ultrasonication;

[0015] (4) Add TEA aqueous solution to the above system, sonicate, and freeze-dry to obtain the product.

[0016] Specifically, the steps of the method are:

[0017] (1) The crude MPL is completely dissolved in chloroform (10%, wt%), filtered through a 0.45 μm microporous membrane and then the solvent is removed so that the MPL is evenly distributed at the bottom of a round-bottom flask; preferably, the solvent is removed by vacuum rotary evaporation;

[0018] (2) adding 1.0% TEA aqueous solution, and uniformly suspending MPL in the solution by ultrasound at room temperature. Preferably, the amount of the TEA aqueous solution added is 10 times that of the crude MPL (mass ratio);

[0019] (3) Transfer the above solution into pure water and disperse it evenly by ultrasonication; preferably, the amount of pure water added is 40 times (mass ratio) of the crude MPL;

[0020] (4) Add 0.2% TEA aqueous solution to the above system and dissolve it completely by ultrasonication; preferably, the amount of 0.2% TEA aqueous solution added is 50 times that of the crude MPL (mass ratio).

[0021] The present invention also includes the step of preparing the MPL triethylamine salt into a nano solution, and the specific steps are:

[0022] (5) homogenizing the reaction solution in step (4) by a high-pressure homogenizer and filtering it through a 0.45 μm aqueous microporous filter membrane;

[0023] The present invention also includes the step of further freeze-drying the MPL triethylamine salt nano solution, and the specific steps are:

[0024] (6) Transfer to a freeze dryer and perform freeze drying according to a freeze drying curve, wherein the freeze drying steps are as follows:

[0025] Pre-freezing: -30.0℃, 180min;

[0026] One-time drying:

[0027] Stage 1: -15.0℃, cooling for 30min, lasting for 480min;

[0028] Stage 2: -5.0℃, heating for 30min, lasting for 240min;

[0029] Stage 3: 0℃, heating for 15min, lasting for 240min;

[0030] Desorption drying: 20.0℃, heating for 15min, lasting for 480min;

[0031] After freeze-drying, flush with nitrogen, seal and store at 2-8°C.

[0032] Most preferably, the method comprises the steps of:

[0033] Take 1 g of crude MPL and dissolve it completely in 10 mL of chloroform, filter it with a 0.45 μm microporous filter membrane, add 5 mL of methanol and rotary evaporate to remove the solvent so that it is evenly distributed at the bottom of the round-bottom flask.

[0034] 10 mL of 1.0% TEA aqueous solution was added to the flask and ultrasonicated at 25-30° C. for 0.5 h to uniformly suspend MPL in the solution.

[0035] The above solution was transferred into a container containing 40 mL of pure water and dispersed evenly by ultrasonication for 10 min.

[0036] 50 mL of 0.2% TEA aqueous solution was added to the above system and ultrasonicated for 10 min.

[0037] The final liquid was 100 ml, in which MPL was 10 mg / ml and TEA concentration was 0.2%.

[0038] The above solution was homogenized twice by a high-pressure homogenizer, the D50 particle size was <100 nm, the solution was filtered through a 0.45 μm aqueous phase microporous filter membrane, transferred to a freeze dryer, and freeze-dried according to the freeze-drying curve. After the freeze-drying was completed, nitrogen was flushed into the freeze dryer, the bottle stopper was sealed, the bottle was taken out, and stored at 2-8°C.

[0039] The invention also relates to the MPL triethylamine salt prepared by the method.

[0040] The solubility of the MPL triethylamine salt in aqueous solution is 8 mg / mL;

[0041] The average particle size distribution data of the aqueous solution prepared by dissolving the MPL triethylamine salt in pure water is: the average particle size is about 900-950nm.

[0042] The average particle size distribution data of the aqueous solution prepared by solubilizing 10 mg of MPL triethylamine salt with 1 mL of DMSO and then dissolving it in 10 mL of deionized water is: the average particle size is about 110 to 130 nm.

[0043] The present invention also relates to the method for preparing the MPL triethylamine salt or the application of the MPL triethylamine salt in preparing a vaccine adjuvant.

[0044] The vaccines are preferably: hepatitis B vaccine, herpes zoster vaccine, and cervical cancer vaccine.

[0045] The vaccine adjuvant is preferably a liposome or micelle adjuvant, and more preferably, the adjuvant is: AS01, AS02, AS04, AS15.

[0046] The chemical synthesis process of the crude MPL product can be found in CN202010305764, CN202010306822, CN202010305752, CN202010306826, CN202010305744, and CN202010305754.

[0047] The beneficial effects of the present invention are:

[0048] (1) Overcoming the problem of poor solubility of existing MPL in water and conventional organic solvents. Through salt type screening, it was found that triethylamine salt has good solubility and stability. After high-pressure homogenization, it can form a nano solution. The resulting lyophilized powder has good solubility and is suitable for the formulation of vaccine adjuvants.

[0049] (2) The present invention has simple operation steps and complete salt formation. The triethylamine content is 5.12% as characterized by DSC, 4.7% as detected by gas phase, and 5.48% as theoretical triethylamine. The particle size is reduced and the solubility is significantly improved by freeze-drying according to the freeze-drying parameter process.

[0050] (3) Compared with MPL base, the lyophilized powder of triethylamine salt can be directly dissolved in water to form nanoparticles. It can be dispersed into a stable nano solution with a particle size of about 125 nm by solubilization with a small amount of organic solvent, which is suitable for the preparation of nanoformulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 , purity verification of synthetic MPL,

[0052] 1A, HPLC characterization results of synthesized MPL;

[0053] 1B, NMR characterization of synthesized MPL;

[0054] 1C. High-resolution mass spectrometry characterization results of synthesized MPL.

[0055] Figure 2 , purity verification of the synthesized MPL triethylamine salt,

[0056] 2A, the synthesized MPL triethylamine salt was characterized by TGA for triethylamine content;

[0057] 2B, DSC characterization of synthesized MPL triethylamine salt;

[0058] 2C. The triethylamine content in the synthesized MPL triethylamine salt was further characterized by gas phase analysis.

[0059] Figure 3 , appearance and detection of aqueous solutions of different MPL salts and MPL base,

[0060] 3A. Appearance of solubility of different MPL salts and MPL base in water (from left to right: MPL base solution, MPL triethylamine salt solution, MPL tert-butylamine salt solution and MPL ammonium salt solution);

[0061] 3B, DLS data of MPL triethylamine salt solution after dispersion in water.

[0062] Figure 4 Different MPL salts and MPL base were first dissolved in a small amount of DMSO and then dispersed in water for solution characterization.

[0063] 4A, solution appearance (from left to right: MPL base solution, MPL triethylamine salt solution, MPL tert-butylamine salt solution and MPL ammonium salt solution);

[0064] 4B, DLS data of MPL base solution;

[0065] 4C, DLS data of MPL triethylamine salt solution;

[0066] 4D, DLS data of MPL tert-butylamine salt solution;

[0067] 4E. DLS data of MPL ammonium salt solution. DETAILED DESCRIPTION

[0068] The MPL used in the following examples was synthesized by our company.

[0069] The chemical synthesis process of MPL can be found in CN202010305764, CN202010306822, CN202010305752, CN202010306826, CN202010305744, CN202010305754. The quality and purity analysis results of the synthesized MPL are shown in Figure 1 .

[0070] Unless otherwise specified, other experimental materials and reagents and consumables were analytically pure or chromatographically pure raw materials purchased from the market, or obtained by conventional preparation methods in the art.

[0071] Example 1, MPL triethylamine salt (MPL-TEA) preparation process

[0072] Take 1 g of crude MPL and dissolve it completely in 10 mL of chloroform, filter it with a 0.45 μm microporous filter membrane, add 5 mL of methanol and rotary evaporate to remove the solvent so that it is evenly distributed at the bottom of the round-bottom flask.

[0073] 10 mL of 1.0% TEA aqueous solution was added to the flask and ultrasonicated at 25-30° C. for 0.5 h to uniformly suspend MPL in the solution.

[0074] The above solution was transferred into a container containing 40 mL of pure water and dispersed evenly by ultrasonication for 10 min.

[0075] Add 50 mL of 0.2% TEA aqueous solution into the above system and sonicate for 10 min.

[0076] The final liquid was 100 ml, in which MPL was 10 mg / ml and TEA concentration was 0.2%.

[0077] The above solution was homogenized twice by a high-pressure homogenizer, the D50 particle size was <100 nm, the solution was filtered through a 0.45 μm aqueous phase microporous filter membrane, transferred to a freeze dryer, and freeze-dried according to the freeze-drying curve. After the freeze-drying was completed, nitrogen was flushed into the freeze dryer, the bottle stopper was sealed, the bottle was taken out, and stored at 2-8°C.

[0078] The freeze-drying parameters are as follows:

[0079] Pre-freeze:

[0080] Set temperature(℃) Duration (min) Phase 1 -30.0 180

[0081] One-time drying

[0082] Set temperature(℃) Setting time (min) Duration (min) Vacuum degree(mbar) Phase 1 -15.0 30 480 0.1200 Phase II -5.0 30 240 0.1200 Phase 3 0.0 15 240 0.1200

[0083] Analytical drying

[0084] Set temperature(℃) Setting time (min) Duration (min) Vacuum degree(mbar) Phase 1 20.0 15 480 0.1200

[0085] The TGA analysis and DSC test results of MPL triethylamine salt lyophilized powder are shown in Figure 2 A, 2B; see gas chromatography Figure 2 C;

[0086] The gas chromatography analysis process and results are as follows

[0087] Content calculation formula: in:

[0088] Ax = test sample peak area, As = reference sample peak area (six times peak area, RSD% = 2.8%), Ms = reference sample mass, Mx = test sample mass, Vx = test sample fixed volume (5 mL), Vs = reference sample fixed volume (20 mL), fs = reference sample dilution factor (50), fx = test sample dilution multiple (1), p = 100%; the results are shown in the following table:

[0089] 1 2 3 4 5 6 Average value Peak area of ​​reference substance 121032 128135 119825 121072 119568 118624 121376 Test sample peak area Ax 86687 97948 Reference material quality Ms 25.43mg 29.77mg Test sample quality Mx 1.94mg 2.19mg ω% 4.68％ 4.69％ 4.7％

[0090] The triethylamine content is calculated to be 4.7%. According to the chemical equivalent ratio of 1:1, the triethylamine content of MPL triethylamine salt is 5.48%.

[0091] Example 2, MPL tert-butylamine salt (MPL-SDA) preparation process

[0092] Take 1 g of crude MPL and dissolve it completely in 10 mL of chloroform, filter it with a 0.45 μm microporous filter membrane, add 5 mL of methanol and rotary evaporate to remove the solvent so that it is evenly distributed at the bottom of the round-bottom flask.

[0093] Add 10 mL of 1.0% tert-butylamine aqueous solution into the flask and ultrasonicate at 25-30° C. for 0.5 h to uniformly suspend MPL in the solution.

[0094] The above solution was transferred into a container containing 40 mL of pure water and dispersed evenly by ultrasonication for 10 min.

[0095] Add 50 ml of 0.2% tert-butylammonium aqueous solution into the above system and ultrasonicate for 10 min.

[0096] Finally, 100 ml of liquid was obtained, in which MPL was 10 mg / ml and the concentration of tert-butylammonium was 0.2%.

[0097] The above solution was homogenized twice by a high-pressure homogenizer, the D50 particle size was <100 nm, the solution was filtered through a 0.45 μm aqueous phase microporous filter membrane, transferred to a freeze dryer, and freeze-dried according to the freeze-drying curve. After the freeze-drying was completed, nitrogen was flushed into the freeze dryer, the bottle stopper was sealed, the bottle was taken out, and stored at 2-8°C.

[0098] The freeze-drying parameters were the same as in Example 1.

[0099] Example 3, MPL ammonium salt (MPL-NH3) and preparation process

[0100] Take 1 g of crude MPL and dissolve it completely in 10 mL of chloroform, filter it with a 0.45 μm microporous filter membrane, add 5 mL of methanol and rotary evaporate to remove the solvent so that it is evenly distributed at the bottom of the round-bottom flask.

[0101] Add 10 mL of 1.0% ammonia solution into the flask and ultrasonicate at 25-30°C for 0.5 h to evenly suspend MPL in the solution.

[0102] The above solution was transferred into a container containing 40 mL of pure water and dispersed evenly by ultrasonication for 10 min.

[0103] Add 50 ml of 0.2% ammonia solution into the above system and ultrasonicate for 10 min.

[0104] The final liquid was 100 ml, in which MPL was 10 mg / ml and TEA concentration was 0.2%.

[0105] The above solution was homogenized twice by a high-pressure homogenizer, the D50 particle size was <100 nm, the solution was filtered through a 0.45 μm aqueous phase microporous filter membrane, transferred to a freeze dryer, and freeze-dried according to the freeze-drying curve. After the freeze-drying was completed, nitrogen was flushed into the freeze dryer, the bottle stopper was sealed, the bottle was taken out, and stored at 2-8°C.

[0106] The freeze-drying parameters were the same as in Example 1.

[0107] Example 4: Comparative verification of solubility of MPL base and its salt after reconstitution

[0108] Take 5 mg of MPL and add 1 mL of deionized water to disperse, and dissolve by ultrasonic for 10 min. Similarly, take 5 mg of each type of MPL salt (prepared in Examples 1-3) and add 5 mL of deionized water to disperse, and dissolve by ultrasonic for 10 min.

[0109] Prepare reconstituted solutions of different concentrations and test them by appearance and DLS.

[0110] The testing equipment is: PSS particle size analyzer, equipment model is AccuSizer A9000 FX.

[0111] The DLS test results are shown in the following table and Figure 3 As shown, except for MPL triethylamine salt, other forms of MPL are insoluble in water, emulsions, and have a micrometer scale, so the test cannot be completed.

[0112] D50 D90 PI MPL-Base (water system) - - - MPL triethylamine salt (water system) 925.8nm 2233.2nm 0.627 MPL tert-butylamine salt (water system) - - - MPL amine salt (water system) - - -

[0113] Example 5: Comparative verification of the solubility of MPL base and its salt after solubilization and re-dissolution with a small amount of DMSO

[0114] Take 5 mg of MPL (or various types of MPL salts prepared in Examples 1-3) and dissolve it in 0.5 mL of DMSO, slowly drop it into 5 mL of water for dispersion, and dissolve it by ultrasonic for 10 minutes. The appearance and DLS tests were performed using a PSS particle size analyzer, and the equipment model was AccuSizer A9000 FX.

[0115] The DLS test results are shown in the following table and Figure 4 As shown, it can be seen that MPL triethylamine salt still has the best solubility and the best solvent dispersion effect.

[0116] D50 D90 PI MPL-Base 1195.6nm 4189.8nm 2.143 MPL triethylamine salt 127.3nm 218.4nm 0.216 MPL tert-butylamine salt 364.7nm 692.8nm 0.271 MPL amine salt 309.8nm 671.9nm 0.491

[0117] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims

1. A method for preparing a monophosphoryl ester A triethylamine salt, characterized in that: The method comprises the following steps: (1) The crude monophosphoryl ester A was completely dissolved in 10% by mass chloroform, filtered through a 0.45 μm microporous membrane, and the solvent was removed by vacuum rotary evaporation to make it evenly distributed at the bottom of a round-bottom flask; (2) Add 1.0% TEA aqueous solution and suspend the monophosphoryl ester A uniformly in the solution by ultrasonication at room temperature; (3) Transfer the above solution into pure water and disperse it evenly by ultrasonication; (4) Add 0.2% TEA aqueous solution to the above system and sonicate for 10 min; (5) The reaction solution of step (4) was homogenized by a high-pressure homogenizer and then filtered through a 0.45㎛ aqueous microporous filter membrane; (6) Lyophilizing the monophosphoryl ester A triethylamine salt solution obtained in step (5), wherein the specific lyophilization steps are as follows: Prefreezing: -30.0℃, 180 min; One-time drying: 1) The first stage: -15.0℃, cooling for 30 minutes, lasting for 480 minutes; 2) The second stage: -5.0℃, heating for 30min, lasting for 240min; 3) The third stage: 0℃, heating for 15min, lasting for 240min; Desorption drying: 20.0℃, heating for 15min, lasting for 480min; After freeze-drying, flush with nitrogen, seal and store at 2-8°C.

2. The method according to claim 1, characterized in that In step (2), the amount of TEA aqueous solution added is 10 times the amount of crude monophosphoryl ester A by mass ratio; In step (3), the amount of pure water added is 40 times the mass ratio of the crude monophosphoryl ester A; In step (4), the amount of 0.2% TEA aqueous solution added is 50 times the mass ratio of the crude monophosphoryl ester A.

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