A method for preparing lactose for injection

Abstract

The present invention provides a method for preparing lactose for injection, which uses tannic acid as a complexing agent, and obtains a finished product through steps such as dissolution, complexation, filter pressing, ultrafiltration, nanofiltration, redissolution, crystallization, centrifugation, and drying. The method of the present invention is combined with membrane separation technology to remove foreign proteins, α-albumin, β-globulin and other impurities in food-grade lactose, and prepare a lactose product that meets the demand for injection and can be used for injection. The production method disclosed in the present invention is simple, efficient, high in yield, environmentally friendly, and suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of medical supplies, in particular to a method for preparing lactose for injection. Background Art

[0002] Lactose is a reducing disaccharide, consisting of one molecule of β-D-galactose and one molecule of α-D-glucose, connected by a glycosidic bond at the β-1,4-position. It is divided into anhydrous lactose and monohydrate lactose.

[0003]

[0004] Lactose is one of the main nutrients for children's growth and development. It is very important for the intellectual development of teenagers, especially for newborn babies. It is often used to make baby food, candy, artificial milk, etc. Under natural conditions, it exists in the milk of mammals. The lactose content in cow's milk is about 4.6-4.7%, and the lactose content in human milk is about 6-8%. In industrial production, it is often extracted from whey. Crude lactose contains a small amount of glucose, galactose, fructose, protein, amino acids, etc.

[0005] In the food industry, lactose is used in the manufacture of baby food and condensed milk products. In the pharmaceutical industry, as a pharmaceutical excipient, it is mainly used as a flavoring agent, excipient, diluent, filler, dry powder inhalation carrier, stabilizer, etc.; in addition, it can also be used as a bacterial culture medium. It is widely used in tablets, capsules, granules, freeze-dried products, and dry powder inhalers.

[0006] Lactose has important applications in the medical field. As a freeze-dried agent scaffold, its biggest difference from other commonly used freeze-dried agents (such as glucose, mannitol, etc.) is that the latter are monosaccharides with relatively low molecular weight, and the glass transition temperature during freeze-drying is also low. The freeze-drying process is also a crystallization process. For some relatively "fragile" freeze-dried agents, this crystallization process has a strong destructive effect; in addition, lactose with a higher glass transition temperature does not produce crystals during the freeze-drying process, but exists in an amorphous form, which has no destructive effect on the main ingredients or certain special preparations. Therefore, in such preparations, lactose usually plays the role of "freeze-dried protective agent", especially in the application of new drug delivery systems such as nanoemulsions, liposomes, etc. It has an irreplaceable role.

[0007] However, since lactose mainly comes from animal whey, its source is relatively special and it is a by-product of milk. The traditional refining and purification processes such as activated carbon adsorption, filtration, crystallization and drying to prepare lactose cannot completely remove residual protein, colored impurities, microorganisms, endotoxins and other impurities, especially miscellaneous proteins and trace amounts of α-albumin and β-globulin. These trace amounts of miscellaneous proteins, α-albumin, and β-globulin can cause strong allergic reactions to the human body, such as urticaria, cough, fever, shock and a series of adverse reactions, which greatly limit the application of lactose with excellent performance in injections.

[0008] Comparative documents CN102516321, CN 104606679B, CN 109293715A, CN 110478490A, etc. use adsorption, ultrafiltration, nanofiltration membrane separation and other technologies for refining, and the statements on the protein removal effect are different. From the patent specifications, some of them are not reasonable. From the process point of view, there are several problems: 1. The protein contained in lactose directly enters the membrane system for separation and treatment, which is easy to cause membrane contamination, and ultimately leads to frequent membrane cleaning and low production efficiency; 2. The process flow is long and the operation is cumbersome; 3. The treatment effect on specific α-albumin and β-globulin is not ideal, and there are great safety risks for the use of lactose as an injection.

[0009] Therefore, there is an urgent need in the art to develop a method for preparing low-impurity, safe and effective lactose for injection. Summary of the invention

[0010] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing lactose for injection.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] A method for preparing lactose for injection comprises the following steps:

[0013] 1) Dissolving: Take an appropriate amount of food-grade lactose, add an appropriate amount of purified water, stir and heat to 30-80°C to make a 5%-15% lactose aqueous solution;

[0014] 2) Complexation: Add an appropriate amount of tannic acid aqueous solution to the lactose solution, and stir the reaction at 30-70°C for 30-60 minutes to form a macromolecular complex with the protein substances in the lactose solution and the tannic acid;

[0015] 3) Filtration: The complexed liquid is filtered by a filter press to remove the protein-tannic acid complex precipitate and clarify the filtrate;

[0016] 4) Ultrafiltration: The lactose solution after filter pressing enters the ultrafiltration membrane separation device for filtration, and the retentate is collected;

[0017] 5) Nanofiltration: The permeate after ultrafiltration treatment enters the nanofiltration membrane separation device for filtration, and the retentate is collected for subsequent treatment;

[0018] 6) Redissolving: heating the collected nanofiltration retentate and adding an appropriate amount of ethanol to dissolve it;

[0019] 7) Crystallization: stirring and cooling to crystallize and precipitate crystals;

[0020] 8) Centrifugation: centrifuge the crystal slurry after crystallization to collect crystals;

[0021] 9) Drying: Dry the crystals collected by centrifugation to obtain the finished product.

[0022] In the method of the present invention, the lactose is lactose monohydrate, anhydrous lactose or a combination thereof.

[0023] In the method of the present invention, the complexing agent tannic acid used in step 2) complexation includes but is not limited to industrial grade tannic acid, food grade tannic acid, medical grade tannic acid, condensed tannic acid, and hydrolyzed tannic acid, and the molecular weight of the tannic acid is 500 to 3000 Da.

[0024] Preferably, the concentration of the tannic acid aqueous solution used in step 2) of the complexing method of the present invention is 1% to 20% (mass percentage); wherein the amount of tannic acid dry matter is 0.01% to 1.0% (tannic acid / lactose, mass percentage) of the weight of the lactose raw material.

[0025] In the method of the present invention, food-grade lactose contains protein impurities, and the protein impurities are miscellaneous proteins, α-casein, and β-globulin.

[0026] Preferably, the impurity proteins in the product include proteins, polypeptides, oligopeptides, and amino acid substances other than α-albumin and β-globulin, and the total content of these proteins is less than or equal to 100 ppm, the content of α-albumin is less than or equal to 1 ppm, and the content of β-globulin is less than or equal to 1 ppm.

[0027] In a preferred embodiment, the ultrafiltration and nanofiltration membranes include but are not limited to organic membranes, ceramic membranes and composite membranes thereof, wherein the molecular weight cutoff of the ultrafiltration membrane is 500-1000Da, and the molecular weight cutoff of the nanofiltration membrane is 200Da.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The method of the present invention uses tannic acid as a complexing agent and combines it with membrane separation technology, so that the residual impurity content of lactose produced is significantly reduced, α-albumin, β-globulin and other impurities are significantly controlled, and adverse reactions caused by heterologous proteins in clinical use can be reduced, and a series of adverse reaction problems existing when ordinary inhalation-grade lactose is used for injection can be solved. The product prepared by the method of the present invention can be directly used in injections, providing an excellent injection-grade pharmaceutical excipient for the development of new injection dosage forms in the pharmaceutical industry.

[0030] The method of the invention adopts tannic acid as a complexing agent, has a wide source of raw materials, is simple in post-treatment, has a simple and feasible overall refining process, has a high yield, is low in cost, is environmentally friendly, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a schematic diagram of the preparation process of lactose for injection. DETAILED DESCRIPTION

[0032] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In order to solve the problem that residual impurities, polypeptides, oligopeptides, amino acids, α-albumin, β-globulin and other allergens exist in the current medicinal lactose, which cannot meet the requirements of injections, and the freeze-drying protection effect is still insufficient, the present invention provides a method for preparing lactose for injection, which uses tannic acid as a complexing agent, and obtains the finished product through the steps of dissolution, complexation, filter pressing, ultrafiltration, nanofiltration, redissolution, crystallization, centrifugation, drying and the like. The method of the present invention is combined with membrane separation technology to remove impurities, α-albumin, β-globulin and other impurities in lactose, and prepare a lactose product that meets the requirements for injection and can be used for injection. The production method disclosed in the present invention is simple, efficient, high in yield, environmentally friendly, and suitable for industrial production.

[0034] Tannic acid, also known as tannic acid and tannin, is a complex class of high molecular weight polyphenolic compounds. It is a water-soluble polyphenolic compound that can precipitate alkaloids, gelatin and other proteins, and has a strong ability to bind to proteins. Its reaction mechanism is as follows: polyphenols first approach the surface of proteins through hydrophobic bonds, enter their hydrophobic pockets, and undergo two-point hydrogen bonding. The simultaneous action of hydrophobic bonds and hydrogen bonds causes them to form a hydrophobic layer between protein molecules in a multi-point bonding manner, causing them to aggregate and eventually precipitate. The binding of tannic acid to proteins is stable and efficient, and is easy to post-process.

[0035] The method provided by the present invention comprises the following steps: adding an appropriate amount of tannic acid solution to a lactose solution, and removing trace amounts of impurity proteins, polypeptides, oligopeptides, amino acids, α-albumin, β-globulin, tannic acid and the complex of the two by combining complexation, filter pressing and ultrafiltration / nanofiltration to obtain a high-purity lactose solution, then heating and dissolving the solution, and then using an alcohol aqueous solution for crystallization, controlling the ethanol concentration between 30% and 75%, and finally centrifuging and drying the solution to prepare lactose for injection.

[0036] In an exemplary embodiment, the present invention provides a method for preparing lactose for injection, comprising the following steps:

[0037] 1. Dissolution: Take an appropriate amount of food-grade lactose, add an appropriate amount of purified water, stir and heat to 30-80°C to make a lactose solution;

[0038] 2. Complexation: Add an appropriate amount of tannic acid aqueous solution (concentration of 1% to 20%) to the lactose solution, stir and react at 30 to 70°C for 30 to 60 minutes to form a macromolecular complex with the tannic acid and the proteins, polypeptides, oligopeptides, amino acids and other substances in the lactose solution;

[0039] 3. Filter pressing: The complexed liquid is filtered by plate and frame or other flat filter presses to remove protein-tannic acid complex precipitates. The main purpose is to remove the complex and clarify the filtrate, to provide security for the subsequent ultra-nanofiltration system, to reduce the cleaning cycle, to improve the operating efficiency and to extend the service life;

[0040] 4. Ultrafiltration: The lactose solution after filter pressing is passed into an ultrafiltration membrane separation device with a molecular weight cutoff of 500-1000Da for filtration, mainly intercepting trace proteins and tannic acid that are not completely complexed with tannic acid, to ensure that the filtrate no longer contains impurities such as protein and tannic acid;

[0041] 5. Nanofiltration: The permeate treated by ultrafiltration enters the nanofiltration separation device with a molecular weight cutoff of 200Da for filtration, and the retentate is collected for subsequent treatment. The main purpose is to concentrate and remove trace small molecular amino acids and other impurities;

[0042] 6. Redissolution: Heat the collected nanofiltration retentate and add an appropriate amount of ethanol to dissolve it;

[0043] 7. Crystallization: Stir and cool to crystallize and precipitate crystals;

[0044] 8. Centrifugation: Centrifuge the crystal slurry after crystallization to collect the crystals;

[0045] 9. Drying: Dry the crystals collected by centrifugation to obtain the finished product.

[0046] The present invention is further described below through specific embodiments.

[0047] Analysis method of protein content in lactose: The determination method is the Coomassie Brilliant Blue method commonly used for protein content determination.

[0048] Analysis method for α-albumin content in lactose: α-lactalbumin (α-LA) enzyme-linked immunosorbent assay (ELISA).

[0049] Principle of analysis: The α-lactalbumin (α-LA) level in the sample is determined by enzyme-linked immunosorbent assay. The microplate is coated with purified α-lactalbumin (α-LA) antibody to prepare a solid-phase antibody. α-lactalbumin (α-LA) and HRP-labeled α-lactalbumin (α-LA) antigen are added to the microwells coated with the monoclonal antibody to make them compete for binding. After thorough washing, the substrate TMB is added for color development. The depth of the sample color is negatively correlated with the content of α-lactalbumin (α-LA) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme marker, and the content of α-lactalbumin (α-LA) in the sample is calculated using the standard curve.

[0050] Analysis method for β-lactoglobulin content in lactose: β-lactoglobulin (β-Lg) enzyme-linked immunosorbent assay (ELISA).

[0051] Principle of analysis: The β-lactoglobulin (β-Lg) level in the sample is determined by enzyme-linked immunosorbent assay. The microplate is coated with purified β-lactoglobulin (β-Lg) antibody to prepare a solid-phase antibody. β-lactoglobulin (β-Lg) and HRP-labeled β-lactoglobulin (β-Lg) antigen are added to the microwells coated with the monoclonal antibody to make them compete for binding. After thorough washing, the substrate TMB is added for color development. The depth of the sample color is negatively correlated with the content of β-lactoglobulin (β-Lg) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme marker, and the content of β-lactoglobulin (β-Lg) in the sample is calculated using the standard curve.

[0052] The analytical method for endotoxin content in lactose is carried out in accordance with the bacterial endotoxin test method in Appendix XI E of Part II of the Chinese Pharmacopoeia.

[0053] Tannic acid content detection method: high performance liquid chromatography (chromatographic column: Hypersil ODS (125×4.0 mm, 5 μm); mobile phase: 80% CH 3 OH, 20%H 2 O; detection wavelength: 275nm; flow rate: 0.5ml / min).

[0054] Example 1

[0055] 1. Weigh 1.0 kg of food-grade lactose, add 7.0 L of purified water, heat to 70 ° C and keep for 30 min to completely dissolve it to form a lactose solution;

[0056] 2. Add 10 ml of 1% tannic acid solution to the lactose solution and stir for complexation at 50°C for 30 minutes;

[0057] 3. Transfer the complexed lactose solution to a flat filter press, use a 0.22 μm filter membrane as the filter medium, and filter at a pressure of 3.0 bar;

[0058] 4. Transfer the filtered lactose solution to an ultrafiltration device with a molecular weight cutoff of 1000 Da for filtration at an operating pressure of 8.0 bar, and collect the filtrate;

[0059] 5. The filtrate after ultrafiltration treatment enters a nanofiltration separation device with a molecular weight cutoff of 200Da for filtration at an operating pressure of 15bar, and the retentate is collected;

[0060] 6. Add 1L of ethanol to the film-treated liquid and heat it until the lactose is completely dissolved into a clear and transparent solution;

[0061] 7. After the dissolution is completed, the liquid is crystallized with a stirring speed of 10r / min, and the temperature is slowly lowered to 10°C and maintained for 2h;

[0062] 8. Use a Buchner funnel to vacuum filter the crystal slurry after crystallization to collect the crystals;

[0063] 9. The collected crystals were transferred to an oven and dried under vacuum at 70°C for 2 h to obtain 843 g of the finished product.

[0064] Example 2

[0065] 1. Weigh 10.0 kg of food-grade lactose, add 190 L of purified water, heat to 70 ° C and keep for 40 min to completely dissolve it to form a lactose solution;

[0066] 2. Add 50 ml of 10% tannic acid solution to the lactose solution and stir for complexation at 70°C for 30 minutes;

[0067] 3. Transfer the complexed lactose solution to a flat filter press, use a 0.45 μm filter membrane as the filter medium, and filter at a pressure of 3.0 bar;

[0068] 4. Transfer the filtered lactose solution to an ultrafiltration device with a molecular weight cutoff of 500 Da for filtration at an operating pressure of 7.0 bar, and collect the filtrate;

[0069] 5. The filtrate after ultrafiltration treatment enters a nanofiltration separation device with a molecular weight cutoff of 200 Da for filtration at an operating pressure of 12.0 bar, and the retentate is collected;

[0070] 6. Add 20L of ethanol to the film-treated liquid and heat it until the lactose is completely dissolved into a clear and transparent solution;

[0071] 7. After the dissolution is completed, the liquid is crystallized with a stirring speed of 15r / min, and the temperature is slowly lowered to 15°C and maintained for 3h;

[0072] 8. Centrifuge the crystal slurry after crystallization to collect the crystals;

[0073] 9. The collected crystals were transferred to an oven and dried under vacuum at 50°C for 4 h to obtain 8.29 kg of finished product.

[0074] Example 3

[0075] 1. Weigh 25.0 kg of food-grade lactose, add 300 L of purified water, heat to 65 °C and keep for 60 min to completely dissolve it to form a lactose solution;

[0076] 2. Add 625 ml of 20% tannic acid solution to the lactose solution and stir for complexation at 60°C for 50 minutes;

[0077] 3. Transfer the complexed lactose solution to a flat filter press, use a 0.22 μm filter membrane as the filter medium, and filter at a pressure of 3.0 bar;

[0078] 4. Transfer the filtered lactose solution to an ultrafiltration device with a molecular weight cutoff of 1000 Da for filtration at an operating pressure of 8.0 bar, and collect the filtrate;

[0079] 5. The filtrate after ultrafiltration treatment enters a nanofiltration separation device with a molecular weight cutoff of 200Da for filtration at an operating pressure of 13bar, and the retentate is collected;

[0080] 6. Add 37.5L of ethanol to the film-treated liquid and heat it until the lactose is completely dissolved into a clear and transparent solution;

[0081] 7. After the dissolution is completed, the liquid is crystallized with a stirring speed of 10r / min, and the temperature is slowly lowered to 5°C and maintained for 2h;

[0082] 8. Centrifuge the crystal slurry after crystallization to collect the crystals;

[0083] 9. The collected crystals were transferred to an oven and vacuum dried at 60°C for 3 h to obtain 20.74 kg of finished product.

[0084] Example 4

[0085] 1. Weigh 10.0 kg of food-grade lactose, add 120 L of purified water, heat to 70 ° C and keep for 60 min to completely dissolve it to form a lactose solution;

[0086] 2. Add 1L of 10% tannic acid solution to the lactose solution and stir at 60°C for 40 minutes;

[0087] 3. Transfer the complexed lactose solution to a flat filter press, use a 0.22 μm filter membrane as the filter medium, and filter at a pressure of 3.0 bar;

[0088] 4. Transfer the filtered lactose solution to an ultrafiltration device with a molecular weight cutoff of 500 Da for filtration at an operating pressure of 8.0 bar, and collect the filtrate;

[0089] 5. The filtrate after ultrafiltration treatment enters a nanofiltration separation device with a molecular weight cutoff of 200Da for filtration at an operating pressure of 12bar, and the retentate is collected;

[0090] 6. Add 20L of ethanol to the film-treated liquid and heat it until the lactose is completely dissolved into a clear and transparent solution;

[0091] 7. After the dissolution is completed, the liquid is crystallized with a stirring speed of 10r / min, and the temperature is slowly lowered to 10°C and maintained for 2h;

[0092] 8. Centrifuge the crystal slurry after crystallization to collect the crystals;

[0093] 9. The collected crystals were transferred to an oven and vacuum dried at 50°C for 4 h to obtain 8.27 kg of finished product.

[0094] The lactose products produced in each embodiment were subjected to quality inspection, and the inspection results are shown in Table 1.

[0095] Table 1 Lactose quality test results

[0096]

[0097]

[0098] From the above examples and test results, it can be seen that the lactose prepared by the present invention can significantly control residual protein (including α-albumin, β-globulin, miscellaneous proteins), endotoxin, microbial limits and impurity levels, and can meet the standards for injection. In summary, the present invention adopts a technology combining tannic acid complexation with membrane separation technology, and the lactose prepared completely solves the problem of high protein residue in lactose, which leads to potential safety hazards in the use of lactose in injections. At the same time, the crystallization solvent of the present invention is ethanol aqueous solution crystallization, and the product has the characteristics of controllable quality risk, higher safety level, more stable product, and low safety risk; the process has the characteristics of simple production process, environmental friendliness, and easy operation of industrial production; it can be directly used for injection.

[0099] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0100] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing lactose for injection, characterized in that: The method comprises the following steps: 1) Dissolution: Take an appropriate amount of food-grade lactose, add an appropriate amount of purified water, stir and heat to 30-80°C to make a 5%-15% lactose aqueous solution; 2) Complexation: Add an appropriate amount of 1% to 20% tannic acid aqueous solution to the lactose solution, stir and react at 30 to 70°C for 30 to 60 minutes to form a macromolecular complex with the tannic acid, wherein the dry amount of tannic acid is 0.01% to 1.0% of the weight of the lactose raw material; 3) Filtration: The complexed liquid is filtered by a filter press to remove the protein-tannic acid complex precipitate and clarify the filtrate; 4) Ultrafiltration: The lactose solution after filter pressing is passed into an ultrafiltration membrane separation device with a molecular weight cutoff of 500-1000Da for filtration, and the retentate is collected; 5) Nanofiltration: The permeate after ultrafiltration treatment enters the nanofiltration membrane separation device with a molecular weight cutoff of 200Da for filtration, and the retentate is collected for subsequent treatment; 6) Redissolution: Heat the collected nanofiltration retentate and add an appropriate amount of ethanol to dissolve it; 7) Crystallization: Stir and cool to crystallize and precipitate crystals; 8) Centrifugation: Centrifuge the crystal slurry after crystallization to collect the crystals; 9) Drying: The crystals collected by centrifugation are dried to obtain the finished product.

2. The method for preparing lactose for injection according to claim 1, characterized in that: The lactose is lactose monohydrate, lactose anhydrous or a combination thereof.

3. The method for preparing lactose for injection according to claim 1, characterized in that: Step 2) The complexing agent tannic acid used in the complexing is selected from industrial grade tannic acid, food grade tannic acid, medical grade tannic acid, condensed tannic acid, and hydrolyzed tannic acid, and the molecular weight of the tannic acid is 500 to 3000 Da.

4. The method for preparing lactose for injection according to claim 1, characterized in that: Step 2) The concentration of the tannic acid aqueous solution used in the complexation is 1% to 20% by mass; wherein the amount of tannic acid dry matter is 0.01% to 1.0% by weight of the lactose raw material.

5. The method for preparing lactose for injection according to claim 1, characterized in that: Food-grade lactose is used as a raw material, which contains protein impurities, and the protein impurities are α-albumin, β-globulin and other miscellaneous proteins except α-albumin and β-globulin.

6. The method for preparing lactose for injection according to claim 5, characterized in that: The impurity proteins in the product are selected from proteins, polypeptides, oligopeptides, and amino acid substances other than α-albumin and β-globulin, and the total content of the impurity proteins is less than or equal to 100 ppm, the α-albumin content is less than or equal to 1 ppm, and the β-globulin content is less than or equal to 1 ppm.

7. The method for preparing lactose for injection according to claim 1, characterized in that: The ultrafiltration and nanofiltration membranes are selected from organic membranes, ceramic membranes and composite membranes, wherein the molecular weight cutoff of the ultrafiltration membrane is 500-1000Da, and the molecular weight cutoff of the nanofiltration membrane is 200Da.

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