Method for refining biologically extracted 3D-MPL and application thereof

Abstract

The invention provides a method for refining biologically extracted 3D-MPL, which comprises the following steps: firstly purifying a 3D-MPL crude product by anion chromatography, then eluting with dichloromethane-methanol-ammonium acetate-containing eluent, and finally adding low-concentration acid liquor into the eluent for treatment to obtain a 3D-MPL refined product. According to the method, an elution system of anion chromatography is optimized, the operation method is simplified, meanwhile, the content of penta-acyl MPL and hexa-acyl MPL with higher immunocompetence in a product can be effectively increased, the content of tetraacyl is controlled, the content of heptaacyl is reduced, and high yield can be obtained; according to the present invention, after the dichloromethane elution system and the low-concentration acid treatment are adopted, the obtained 3D-MPL refined product can be directly subjected to TEA salinization without the traditional high-concentration acidification step so as to obtain the stable MPL finished product, such that the cost can be reduced, the salinization rate can be effectively improved, and the free fatty acid content in the product can be reduced so as to improve the purity and the stability of the MPL product.

Description

Technical Field

[0001] This application belongs to the field of biomedical engineering, and specifically relates to a method for purifying biologically extracted 3D-MPL and its application. Background Technology

[0002] Lipopolysaccharide (LPS) is a major component of bacterial endotoxins, primarily found in the outer membrane of Gram-negative bacteria. It consists of three parts: lipid A, a core oligosaccharide region, and an O-antigen. LPS plays a crucial role in protecting bacteria from stress and stimulating immune responses, but its toxicity limits its application as an immune adjuvant. In the early 1980s, Edgar Ribbie demonstrated that a much less toxic molecule could be obtained from LPS through sequential acidic and alkaline hydrolysis. The molecule obtained by acid hydrolysis, called monophospholipid A (MPLA), is significantly less toxic than LPS. Ribbie and colleagues then observed that further mild alkaline hydrolysis of MPLA resulted in an MPL molecule with even lower pyrogenicity while still exhibiting adjuvant activity.

[0003] In existing methods, MPL is mainly extracted through microbial fermentation, but this process easily produces impurities such as free fatty acids and proteins, making purification difficult, complex, and costly. Furthermore, according to existing reports, non-salt forms of MPL have poor water solubility, severely limiting their use in aqueous vaccine formulations. Vaccine Adjuvants: Immunological and Clinical Principles (2006) describes a method for TEA-salting MPL, adding an acidification step before salting. Theoretically, this could protonate the MPL, allowing it to better neutralize with alkaline solutions to form salts. Theoretically, lyophilized MPL would also have better solubility, but this operation also increases time, labor, and economic costs. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for purifying bio-extracted 3D-MPL, comprising the following steps:

[0005] S1: The crude 3D-MPL product is purified by anion exchange chromatography, wherein the crude 3D-MPL product is the product obtained by acid hydrolysis and alkaline hydrolysis of LPS, and the crude 3D-MPL product is dissolved in a solution system with a water content of no more than 1%.

[0006] S2: Elute with a dichloromethane-methanol-ammonium acetate eluent and collect the elution product;

[0007] S3: The elution product is treated with a 1mM acid solution to obtain a purified 3D-MPL product.

[0008] Traditional elution methods require elution with a series of gradient elution solutions containing ammonium salts of varying concentrations (e.g., 0, 100 mM, 240 mM, and 480 mM) to fully collect MPL chromatographic products. This application improves the elution method, which can effectively collect chromatographic products in only 1 to 2 elution steps, ensuring high yield and purity, as well as MPL homolog distribution that meets standards. This method can effectively simplify operation and save costs.

[0009] Furthermore, the packing material used in the anion chromatography is DE52 packing material.

[0010] Further, in step S1, the crude 3D-MPL product is dissolved in a dichloromethane-methanol-water solution system.

[0011] Furthermore, the volume ratio of dichloromethane, methanol, and water in the mixed system is 50–70:30–50:1, preferably 60:40:1.

[0012] Studies have found that adding a small amount of water to a mixed system can improve the dissolution effect compared to a completely organic solvent system, which helps the hexaacyl homolog bind to the chromatographic packing material and thus improves the subsequent yield. However, if the water content is too high, it will conversely affect the binding of the target product during the chromatography process, leading to increased flow through and thus affecting the yield.

[0013] Furthermore, the specific method for dissolving the crude 3D-MPL product using the aforementioned solution system is as follows:

[0014] Prepare a dichloromethane-methanol mixture to dissolve the dried crude 3D-MPL product. Add purified water before loading the sample, mix well, and set aside.

[0015] The applicant found that 3D-MPL has poor stability in aqueous storage systems and is prone to degradation and yield loss when stored for a long time. Therefore, in cases where chromatography cannot be performed immediately, it can be dissolved in an organic system first and water can be added temporarily before loading the sample to extend the storage time and improve stability.

[0016] Furthermore, in step S1, the loading pressure used during the anion chromatography is 0.2–0.5 MPa.

[0017] Studies have found that DE52 packing material has a good enrichment effect on both hexaacyl and pentacyl homologs, achieving high yields. Furthermore, it requires low pressure and low salt concentration during elution, saving costs and making operation more convenient.

[0018] Further, in step S2, the concentration of ammonium acetate in the eluent is 150-240 mM, for example 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM or 240 mM, or any value between any two adjacent values ​​mentioned above.

[0019] Further, in step S2, the volume ratio of dichloromethane, methanol and water in the eluent is 1.5-2.5:2.5-3.5:1, preferably 2:3:1.

[0020] Furthermore, the concentration of ammonium acetate in the eluent is 200 mM.

[0021] In practice, the applicant found that ammonium acetate solutions with concentrations above 100 mM can effectively elute MPL products. However, at lower concentrations (e.g., around 100 mM), the yield is lower, while at higher concentrations (e.g., above 250 mM), although the yield increases, the content of heptaacyl MPL is also higher. Therefore, considering both the overall yield and the content of the active ingredient, a compromise eluent concentration is selected, such as 150–240 mM, with 200 mM being a preferred embodiment.

[0022] Furthermore, in step S3, the specific method for treating the elution product with an acid solution is as follows:

[0023] Add an acid solution to the elution product;

[0024] Add acetic acid to make the concentration of acetic acid in the system 1 mM;

[0025] After thorough drying at room temperature, the refined 3D-MPL product is obtained.

[0026] In practice, to improve the stability and extend the shelf life of 3D-MPL refined products, a salting treatment (usually TEA salting) is usually added. Existing methods require an acidification treatment (the acid concentration needs to be at least 0.1M) before salting. However, this application, after the above operations (especially elution with a dichloromethane-containing elution system and treatment with a 1mM acid solution), has created suitable reaction conditions in the product system, eliminating the need for additional higher concentration acidification treatment and allowing direct salting, effectively saving costs.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention provides a method for purifying bio-extracted 3D-MPL. First, crude 3D-MPL is purified using anion exchange chromatography. Then, it is eluted with a dichloromethane-methanol-ammonium acetate eluent. Finally, a low-concentration acid solution is added to the eluted product for further treatment, yielding purified 3D-MPL. This method optimizes the elution system after anion exchange chromatography, simplifying the operation while effectively increasing the content of more immunologically active penta- and hexa-acyl MPL, controlling the tetra-acyl content, reducing the hepta-acyl content, and achieving higher yields. By using a dichloromethane elution system combined with a low-concentration acid solution, the obtained purified 3D-MPL can skip the traditional high-concentration acidification step and directly undergo TEA salification to obtain a stable MPL product. This reduces costs and effectively increases the salification rate, lowers the free fatty acid content in the product, thereby improving the purity and stability of the MPL product. Attached Figure Description

[0029] Figure 1 This is the liquid chromatogram of Example 1 in Experimental Example 1;

[0030] Figure 2 The liquid chromatogram of Example 2 in Experimental Example 1;

[0031] Figure 3 The liquid chromatogram of Example 7 in Experimental Example 3;

[0032] Figure 4 The liquid chromatogram of Example 8 in Experimental Example 3;

[0033] Figure 5 The liquid chromatogram of Example 9 in Experimental Example 3;

[0034] Figure 6 The liquid chromatogram of Example 10 in Experimental Example 3;

[0035] Figure 7 These are TLC images from Examples 7-9 of Experimental Example 3;

[0036] Figure 8 This is the liquid chromatogram of Example 11 in Experimental Example 4;

[0037] Figure 9 The liquid chromatograms of Example 12 in Experimental Example 4;

[0038] Figure 10 The liquid chromatogram of Example 13 in Experimental Example 4;

[0039] Figure 11 This is the liquid chromatogram of Example 14 in Experimental Example 4;

[0040] Figure 12 These are TLC images from Examples 11-12 of Experimental Example 4;

[0041] Figure 13 These are TLC images from Examples 13-14 of Experimental Example 4.

[0042] Figure 14 The content of TEA in 3D-MPL refined products prepared by different methods in Experimental Example 5 after salting. Detailed Implementation

[0043] The present invention will be further illustrated below by means of non-limiting embodiments. Those skilled in the art will recognize that many modifications can be made to the present invention without departing from its spirit, and such modifications also fall within the scope of the present invention. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention, as the implementations are necessarily diverse. The terminology used in this specification is only for illustrating specific embodiments and is not intended to be limiting; the scope of the invention is defined in the appended claims.

[0044] Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Preferred methods and materials of the invention are described below; however, any methods and materials similar to or equivalent to those described in this specification may be used to practice or test the invention. Unless otherwise specified, the experimental methods described below are conventional methods or methods described in product manuals, and the experimental materials used are readily available from commercial companies.

[0045] Terminology Definition

[0046] In this application, lipopolysaccharide (LPS) is a major component of bacterial endotoxins, mainly found in the external leaflet of the outer membrane of Gram-negative bacteria. It is approximately 10,000 Da in size and consists of three covalently linked regions: 1) an O-specific polysaccharide chain (O-antigen) in the outer region; 2) a core oligosaccharide central region; and 3) an innermost region where lipid A-acts as a hydrophobic anchor, which includes glucosamine disaccharide units that carry long-chain fatty acids. LPS can prevent serum complement and phagocytes from destroying bacteria and is associated with bacterial colonization adhesion. It can specifically activate the immune response of Toll-like Receptor 4 (TLR4) and its signaling cascade on the surface of host cell membranes, thereby triggering a series of intracellular physiological and biochemical reactions. It upregulates the expression level of co-stimulatory molecules on antigen-presenting cells and the secretion of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-8, thereby enhancing the Th1 immune response against antigens. This property makes it a potential candidate for development as a vaccine adjuvant, but its application has been limited by its toxicity.

[0047] In this application, the term "MPL" refers to Monophosphoryl lipid A, a chemical derivative obtained by acid hydrolysis of LPS, which removes one or more acyl chains, polysaccharide side chains, and two of the three phosphate groups. Its toxicity level is only about 0.1% of that of lipopolysaccharide, but most or even all of its immunomodulatory activity is retained. Therefore, it is widely used as an adjuvant in vaccines, allergy medications, and immunotherapy to enhance the immune response.

[0048] In this application, the term "3D-MPL" refers to 3-O-deacylated monophosphoryl lipid A (3D-MPL), a product obtained by further mild alkaline hydrolysis of MPL, which is 3-deacylated and has 6 acyl side chains and 1 phosphate group. Compared with LPS, it significantly lacks polysaccharide side chains and is less toxic than MPL, making it a clinical adjuvant-grade MPL. Compositions containing 3D-MPL were eventually commercialized by Corixa. It has been used as a key component in GSK's AS adjuvant system.

[0049] The crude 3D-MPL used in this application can be prepared according to the method described in the applicant's patent CN115792075B, or according to the method described in other applicants' relevant patents (including acid hydrolysis and alkaline hydrolysis steps).

[0050] In this application, "acid solution" can be any stable organic or inorganic acid, such as formic acid, acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, etc. Unless otherwise specified, the "acid solution" used in the following examples is hydrochloric acid.

[0051] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0052] In this application, the term "around" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0053] The main instruments and equipment used in the following embodiments are as follows:

[0054] Table 1 Main Instruments and Equipment

[0055]

[0056] The main reagents used in the examples are as follows:

[0057] Table 2 Main Reagents

[0058]

[0059] The main solution formulations used in the examples are as follows:

[0060] Table 3 Preparation of the main solutions

[0061]

[0062] *Preparation and mixing order: ammonium acetate → dichloromethane → methanol → purified water, mix thoroughly.

[0063] Example 1. Preparation of 3D-MPL Refined Products

[0064] Weigh 1.00 g of dried crude MPL and dissolve it thoroughly in a dissolving solution (dichloromethane:methanol = 4:1) to a concentration of approximately 35 mg / mL. Pack a 26 mm * 10 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with a equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 100 mM, 240 mM, and 480 mM ammonium acetate (where the volume ratio of dichloromethane, methanol, and purified water is 2:3:1), eluting for 3–5 CV for each gradient. Collect the eluent for each gradient separately. The operating pressure is 0.08–0.09 MPa.

[0065] The collected eluents were concentrated by rotary evaporation at a reaction temperature of 50–55°C and a vacuum of 800 mbar gradually reduced and maintained for 10–15 min until no more condensate was produced. The evaporated samples were centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was air-dried and stored for later use.

[0066] Example 2.3 Preparation of D-MPL Refined Products

[0067] Weigh 1.00 g of dried crude MPL and dissolve it thoroughly in a dissolving solution (dichloromethane:methanol = 4:1) to a concentration of approximately 35 mg / mL. Pack a 26 mm * 30 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with a equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 100 mM, 240 mM, and 480 mM ammonium acetate (where the volume ratio of dichloromethane, methanol, and purified water is 2:3:1), eluting for 3–5 CV for each gradient. Collect the eluent for each gradient separately. The operating pressure is 0.08–0.09 MPa.

[0068] The collected eluents were concentrated by rotary evaporation at a reaction temperature of 50–55°C and a vacuum of 800 mbar gradually reduced and maintained for 10–15 min until no more condensate was produced. The evaporated samples were centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was air-dried and stored for later use.

[0069] Example 3.3 Preparation of D-MPL Refined Product

[0070] Weigh 1.00 g of dried crude MPL and dissolve it thoroughly in a dissolving solution (dichloromethane:methanol = 4:1) to a concentration of approximately 35 mg / mL. Pack a 25 mm * 14 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with a equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 100 mM, 240 mM, and 480 mM ammonium acetate (where the volume ratio of dichloromethane, methanol, and purified water is 2:3:1), eluting for 3–5 CV for each gradient. Collect the eluent for each gradient separately. The operating pressure is 0.08–0.09 MPa.

[0071] The collected eluents were concentrated by rotary evaporation at a reaction temperature of 50–55°C and a vacuum of 800 mbar gradually reduced and maintained for 10–15 min until no more condensate was produced. The evaporated samples were centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was air-dried and stored for later use.

[0072] Example 4.3 Preparation of D-MPL Refined Product

[0073] Weigh 1.00 g of dried crude MPL and dissolve it thoroughly in a dissolving solution (dichloromethane:methanol = 4:1) to a concentration of approximately 35 mg / mL. Pack a 25 mm * 7 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with a equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 100 mM, 240 mM, and 480 mM ammonium acetate (where the volume ratio of dichloromethane, methanol, and purified water is 2:3:1), eluting for 3–5 CV for each gradient. Collect the eluent for each gradient separately. The operating pressure is 0.08–0.09 MPa.

[0074] The collected eluents were concentrated by rotary evaporation at a reaction temperature of 50–55°C and a vacuum of 800 mbar gradually reduced and maintained for 10–15 min until no more condensate was produced. The evaporated samples were centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was air-dried and stored for later use.

[0075] Example 5.3 Preparation of D-MPL Refined Product

[0076] Weigh 1.52 g of dried crude MPL product and dissolve it thoroughly in 100 ml of equilibration buffer (dichloromethane:methanol:water = 2:3:1) until no precipitate forms. Divide the sample into two equal portions for later use. Pack a 26 mm * 30 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 150 mM and 240 mM ammonium acetate (dichloromethane, methanol, and purified water in a volume ratio of 2:3:1), eluting 3–5 CV for each gradient. Collect the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0077] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0078] Example 6.3 Preparation of D-MPL Refined Product

[0079] Weigh 1.52 g of dried crude MPL and dissolve it thoroughly in 100 ml of equilibration buffer (dichloromethane:methanol:water = 2:3:1) until no precipitate forms. Pack a 26 mm * 30 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min and elute in one step with eluent containing 200 mM ammonium acetate (dichloromethane, methanol, and purified water in a volume ratio of 2:3:1). Elute for 3–5 CVs, collecting the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0080] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0081] Example 7.3 Preparation of D-MPL Refined Product

[0082] Weigh 1.52 g of dried crude MPL product and dissolve it thoroughly in 100 ml of equilibration buffer (dichloromethane:methanol:water = 2:3:1) until no precipitate forms. Divide the sample into two equal portions for later use. Pack a 26 mm * 10 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 150 mM and 240 mM ammonium acetate (dichloromethane, methanol, and purified water in a volume ratio of 2:3:1), eluting 3–5 CV for each gradient. Collect the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0083] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0084] Example 8.3 Preparation of D-MPL Refined Product

[0085] Weigh 258 mg of dried crude MPL and dissolve it thoroughly in 50 ml of dissolving buffer (dichloromethane:methanol:water = 60:40:10) until no precipitate remains. Pack a 26 mm * 10 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 150 mM and 240 mM ammonium acetate (dichloromethane, methanol, and purified water in a volume ratio of 2:3:1), eluting 3–5 CV for each gradient. Collect the eluent for each gradient separately. Operating pressure ≤ 0.3 MPa.

[0086] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0087] Example 9.3 Preparation of D-MPL Refined Product

[0088] Weigh 258 mg of dried crude MPL and dissolve it thoroughly in 50 ml of dissolving buffer (dichloromethane:methanol:water = 60:40:10) until no precipitate remains. Then add 0.5 ml of 1 M TEA solution and mix well. Pack a 26 mm * 10 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute sequentially with eluents containing 150 mM and 240 mM ammonium acetate (where the volume ratio of dichloromethane, methanol, and purified water is 2:3:1), eluting 3–5 CV for each gradient. Collect the eluent for each gradient separately. Operating pressure ≤ 0.3 MPa.

[0089] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0090] Example 10.3 Preparation of D-MPL Refined Product

[0091] Weigh 1.8 g of dried crude MPL and dissolve it thoroughly in 360 ml of dissolving buffer (dichloromethane:methanol:water = 60:40:1) until no precipitate remains. Pack a 50 mm * 30 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min and elute in one step with eluent containing 200 mM ammonium acetate (dichloromethane, methanol, and purified water in a volume ratio of 2:3:1). Elute for 3–5 CVs, collecting the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0092] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0093] Example 11.3 Preparation of D-MPL Refined Product

[0094] Weigh 1.8 g of dried crude MPL and dissolve it in 147 ml of dichloromethane and 59 ml of methanol. Pack a 50 mm * 10 cm chromatography column with DE52 packing material treated with ammonium acetate and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Before loading the sample, take 112 ml of sample, add 1.12 ml of purified water and mix thoroughly. Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute in one step with eluent containing 200 mM ammonium acetate (where the volume ratio of dichloromethane, methanol, and purified water is 2:3:1). Collect the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0095] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0096] Example 12.3 Preparation of D-MPL Refined Product

[0097] Weigh 1.8 g of dried crude MPL and dissolve it in 147 ml of dichloromethane and 59 ml of methanol. Pack a DE52 chromatography column (26 mm * 30 cm) with ammonium acetate-treated packing material and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Before loading the sample, take 84 ml of sample, add 0.84 ml of purified water and mix thoroughly. Load the sample onto the column at a flow rate of 0.5–1.0 cm / min, and elute in one step with eluent containing 200 mM ammonium acetate (where the volume ratio of dichloromethane, methanol, and purified water is 2:3:1). Elute for 3–5 CVs, and collect the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0098] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0099] Example 13.3 Preparation of D-MPL Refined Product

[0100] Weigh 0.6 g of dried crude MPL and dissolve it in 120 ml of dissolving buffer (dichloromethane:methanol:water = 60:40:1). Pack a 50 mm * 10 cm chromatography column with ammonium acetate-treated DE52 packing material and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min and elute in one step with eluent containing 200 mM ammonium acetate (dichloromethane, methanol, and purified water in a volume ratio of 2:3:1). Elute for 3–5 CVs, collecting the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0101] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0102] Example 14.3 Preparation of D-MPL Refined Product

[0103] Weigh 0.6 g of dried crude MPL and dissolve it in 120 ml of dissolving buffer (dichloromethane:methanol:water = 60:40:1). Pack a 50 mm * 23 cm chromatography column with ammonium acetate-treated DE52 packing material and pre-equilibrate with equilibration buffer (dichloromethane:methanol:water = 2:3:1). Load the sample onto the column at a flow rate of 0.5–1.0 cm / min and elute in one step with eluent containing 200 mM ammonium acetate (dichloromethane, methanol, and purified water in a volume ratio of 2:3:1). Elute for 3–5 CVs, collecting the eluent from each gradient separately. Operating pressure ≤ 0.3 MPa.

[0104] Add 1M acetic acid to the eluent to make the final concentration of acetic acid in the mixture 1mM. Stir magnetically for 15min, then dry by rotary evaporation. Place in a desiccator and continue to air dry before storage.

[0105] Experimental Example 1. Effect of column bed specifications on chromatography efficiency

[0106] The same batch of crude MPL was purified by chromatography according to the methods provided in Examples 1 and 2. The effects of different column bed specifications (especially column height) on the distribution and yield of homologous products were investigated. The results are as follows: Figures 1-2 As shown in Table 4, the homolog distribution results show that in Example 1 (column height 10 cm), the homolog distribution of the 100 mM ammonium acetate eluent basically meets the EU standard. However, the content of pentacyl and hexaacyl homologs in the 240 mM ammonium acetate eluent is significantly reduced, reaching the lower limit of the EU standard, while the tetraacyl homolog is significantly increased, exceeding the upper limit of the standard. At the same time, heptaacyl homologs are also detected, and the total product yield is only 22.4%. In Example 2 (column height 30 cm), the situation is exactly the opposite. The content of tetraacyl homologs in the 100 mM ammonium acetate eluent is too high, and the content of pentacyl and hexaacyl homologs is too low. However, the homolog distribution of the 240 mM ammonium acetate eluent fully meets the EU standard, and the total product yield is significantly improved, reaching 36.1%. No MPL pentacyl or hexaacyl homologs were detected in either group of 480 mM ammonium acetate eluents, indicating that the 240 mM elution step was sufficient to achieve elution.

[0107] In summary, preliminary findings suggest that changes in column height affect the elution position of the target product. Increasing column height delays the elution position in MPL, possibly because the increased column height lengthens the binding-dissociation path. Simultaneously, increasing column height significantly improves product yield, likely due to the longer path allowing for more complete binding and elution. Considering all these factors, a column height of 15–30 cm is recommended.

[0108] Table 4. Detection results of high-purification samples from different columns

[0109]

[0110] Experimental Example 2. Effect of Elution System on Chromatographic Efficiency

[0111] The same batch of crude MPL product was purified by chromatography according to the methods provided in Examples 5 and 6, focusing on the effects of different elution systems and strategies on the distribution and yield of homologous products. The results are shown in Table 5. The homologous product distribution patterns at different concentrations in Example 5 were basically consistent with those in Example 1. The tetraacyl homologous content was low and the hexaacyl homologous content was high in the 150 mM ammonium acetate eluent. In the 240 mM ammonium acetate eluent, the tetraacyl, pentaacyl, and hexaacyl homologous contents all met EU standards, but the heptaacyl homologous content remained consistently high. The homologous product distribution pattern in Example 6 was the same as that in the 240 mM ammonium acetate eluent of Example 5, also showing an excess of heptaacyl homologous content despite the main components meeting the standards. The recoveries of both examples exceeded those of Example 1, but were still significantly lower than those of Example 2. It can be seen that both elution systems can obtain MPL products that basically meet the standards and achieve high yields; however, in view of the remaining problems, it was found through comparison that the main difference between Examples 5 and 6 and Example 2 lies in the different sample dissolution systems. Examples 5 and 6 contain water, but Example 2 does not. Therefore, the next step is to start from the dissolution system and study the effect of water content on the chromatography effect.

[0112] Table 5. Detection results of purified samples using different elution systems.

[0113]

[0114] Experimental Example 3. The Influence of Sample Dissolution System on Chromatographic Results

[0115] The same batch of crude MPL was purified by chromatography according to the methods provided in Examples 7-9, and the effects of different solutions on the distribution and yield of homologous products were investigated. TLC images of flow-through and eluted samples are shown below. Figure 7As shown, in Example 7 (1% water content), almost no flow-through was detected, and the bands of hexacyl and pentacyl homologs in the elution product were relatively obvious. In Examples 8 (10% water content) and 9 (10% water content + TEA), obvious flow-through was detected (with Example 9 showing the largest flow-through). At the same time, the band of hexacyl homolog in the elution product was faint, while the band of tetraacyl homolog was very obvious, indicating that the elution effect was better when there was a low dose of water and no alkali.

[0116] Specific test results are as follows Figures 3-5 As shown in Table 6, the content of tetraacyl homologs in Example 7 was lower than the standard while the content of hexaacyl homologs was higher. The homolog distributions in Examples 8 and 9 were the same, with the highest content of pentaacyl homologs and the lowest content of hexaacyl homologs, but both were within the standard range. In terms of yield, the yield of Example 7 was lower and the yield of Example 8 was the highest, but both were significantly higher than that of Example 1. It is speculated that a low water content environment is more conducive to the binding of hexaacyl homologs, which can increase the loading, but elution is relatively difficult; while for tetraacyl homologs, the opposite is true; in a high water content environment, the binding strength is lower and the elution difficulty is relatively lower.

[0117] Table 6. Detection results of purified samples using different dissolution and elution systems.

[0118]

[0119] The elution method was adjusted based on Example 7, and a scale-up experiment was conducted (i.e., Example 10). The results are shown in Table 6 and... Figure 6 As shown, the overall yield was significantly improved, reaching 44%, significantly higher than the other groups. The total content of penta- and hexa-acyl MPL was also increased compared to Examples 8 and 9. This indicates that the dissolution system and elution method in this example can effectively improve the distribution of homologs in the product, effectively increase the content of the more immunologically active penta- and hexa-acyl MPL in the product, control the content of tetraacyl groups, reduce the content of hepta-acyl groups, and obtain a high yield. Accordingly, a water content of 1% (dichloromethane:methanol:water = 60:40:1) was selected as the optimal dissolution system, and a single-elution method using 200mM ammonium acetate was selected in conjunction with it.

[0120] Experimental Example 4. Scale-up Experiment of Chromatography Process

[0121] The same batch of crude MPL was purified by chromatography according to the methods provided in Examples 11-14, and the changes in the distribution and yield of homologous products after process scale-up (scale-up of column diameter in Examples 11, 12, and 14) were observed. The results are shown in Table 7 and... Figures 8-11As shown, Example 12 (water added alone in the solution) had the highest yield, followed by Example 11, and Example 13 had the lowest yield. Furthermore, Example 13 had a lower content of tetraacyl homolog and a higher content of hexaacyl homolog. The contents of the other three homologs were within the standard range. This indicates that the homolog distribution in Examples 13 and 14 generally met the standard; the main problem was the overall product loss. TLC results are as follows... Figures 12-13 As shown, three main bands (i.e., tetraacyl, pentacyl, and hexaacyl homologs) were detected in each group in 200 mM eluent. Examples 11, 12, and 14 all showed obvious bands, while the band in Example 13 was fainter, which is consistent with the yield.

[0122] The significant differences in yield among the groups may be due to two main reasons: First, crude MPL exhibits poor stability when stored in liquid form, particularly in polar systems containing water. Examples 13 and 14 both used pre-prepared aqueous solutions for dissolution, and the experiments were not conducted immediately after dissolution, potentially leading to MPL loss due to the pause. Second, the high loading pressure of 0.4 MPa used in Examples 13 and 14 caused packing material breakage, resulting in further MPL loss. Examples 11 and 12 avoided these problems, thus achieving better results. Therefore, Examples 11 and 12 provide a feasible approach for process scale-up.

[0123] Table 7. Detection results of purified samples from different reaction systems

[0124]

[0125] Experimental Example 5. Effect of Chromatography Process on Salting Effect of 3D-MPL

[0126] The crude MPL product was purified by chromatography according to the method provided in Example 12. Four groups of experiments were set up and adjusted according to the method shown in Table 8. Different chromatography solutions and different acid concentrations were used to prepare 3D-MPL purified products. TEA salting was performed according to the method provided in CN116199727A. The homologous distribution, TEA content and other indicators of each group were detected. The experiment was repeated multiple times to compare the effects of different acid hydrolysis extraction solutions and the presence or absence of acidification steps on the TEA content in 3D-MPL (the higher the TEA content, the better the solubility and the better the quality of the MPL product).

[0127] Table 8 3D-MPL Refined Product Preparation Scheme

[0128]

[0129] Experimental results are as follows Figure 14As shown, the TEA contents of the four groups of salinized samples were 4.80%, 4.44%, 4.55%, and 3.99%, respectively. It is evident that the TEA content in the dichloromethane system was significantly higher than that in the trichloromethane system (Group 1 was higher than Group 3, and Group 2 was higher than Group 4). Although high-concentration acidification (100 mM) effectively improved the salinization level, the TEA content of Group 2 (1 mM acid concentration) did not show a significant difference from that of Group 3 (trichloromethane system + 100 mM acid concentration). This indicates that even with a 100-fold reduction in acid concentration, the dichloromethane extraction system still achieves an effect comparable to traditional trichloromethane extraction combined with high-concentration acidification.

[0130] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for purifying biologically extracted 3D-MPL, characterized in that, Includes the following steps: S1: The crude 3D-MPL product is purified by anion exchange chromatography, wherein the crude 3D-MPL product is the product obtained by acid hydrolysis and alkaline hydrolysis of LPS, and the crude 3D-MPL product is dissolved in a solution system with a water content of no more than 1%. S2: Elute with dichloromethane-methanol-ammonium acetate eluent and collect the elution product; S3: The elution product is treated with a 1mM acid solution to obtain a purified 3D-MPL product.

2. The method as described in claim 1, characterized in that, In step S1, the packing material used in the anion chromatography is DE52 packing material.

3. The method as described in claim 2, characterized in that, In step S1, the crude 3D-MPL product is dissolved in a dichloromethane-methanol-water solution system.

4. The method as described in claim 3, characterized in that, The volume ratio of dichloromethane, methanol, and water in the solution system is 50–70:30–50:1, preferably 60:40:

1.

5. The method as described in claim 4, characterized in that, The specific method for dissolving the crude 3D-MPL product using the aforementioned solution system is as follows: Prepare a dichloromethane-methanol mixture to dissolve the dried crude 3D-MPL product. Add purified water before loading the sample, mix well, and set aside.

6. The method as described in claim 1, characterized in that, In step S2, the concentration of ammonium acetate in the eluent is 150–240 mM.

7. The method as described in claim 6, characterized in that, In step S2, the volume ratio of dichloromethane, methanol and water in the eluent is 1.5-2.5:2.5-3.5:1, preferably 2:3:

1.

8. The method as described in claim 7, characterized in that, The concentration of ammonium acetate in the eluent is 200 mM.

9. The method according to any one of claims 1 to 8, characterized in that, In step S3, the specific method for treating the elution product with an acid solution is as follows: An acid solution was added to the elution product to make the acid concentration in the system 1 mM; After thorough stirring at room temperature, the product is dried to obtain the refined 3D-MPL product.

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