CN100497383C - Human blood high-density lipoprotein, its production method and its application - Google Patents

Abstract

The present invention relates to a human blood high-density lipoprotein and its product, as well as a manufacturing method and application thereof. The human blood high-density lipoprotein has an apo AI content of 77-88%. The present invention uses the discarded FIV-1 precipitate after producing human plasma albumin and immunoglobulin by a low-temperature ethanol method as a raw material, extracts HDL from FIV-1 through a separation and purification step, provides a new low-temperature ethanol process for large-scale preparation of human plasma HDL, and prepares a human blood HDL preparation for intravenous infusion, which fully utilizes precious human blood resources. The human blood high-density lipoprotein and its product of the present invention have good applications in the preparation of drugs for treating multiple organ dysfunction syndrome (MODS), cardiovascular system diseases such as atherosclerosis (AS), and neutralizing bacterial toxins and other acute symptoms.

Description

Human blood high-density lipoprotein, its production method and its application

technical field

The invention relates to a human blood high-density lipoprotein product, its manufacturing method and its application in pharmacy, belonging to the field of biochemistry.

Background technique

AS is a common multiple disease characterized by thickening and hardening of the arterial wall, loss of elasticity and formation of atherosclerotic lesions due to lipid deposition, fiber and connective tissue proliferation in the arterial wall. Arterial lumen occlusion caused by AS can cause insufficient blood supply to vital organs such as the heart, brain, and kidneys, thereby seriously threatening human health and life. At present, the commonly used drugs for the treatment of AS in clinic generally include vasodilator drugs, blood lipid lowering drugs, antiplatelet drugs and thrombolytic drugs, etc. Although these drugs can fight against AS by relieving vasomotor disturbance and regulating blood lipids, none of them can prevent and eliminate AS plaques safely and effectively. And because AS is a chronic non-inflammatory, degenerative and proliferative disease, long-term use of certain drugs will cause side effects such as cholelithiasis and liver function damage.

A large number of epidemiological studies have shown that elevated blood lipids, obesity, hypertension, diabetes and smoking are the main risk factors for AS. However, there are no ideal methods and drugs for the prevention and treatment of AS at present. In addition to diet control, the current prevention and treatment of AS generally uses drugs that lower blood lipids, dilate blood vessels, or target risk factors. Therefore, it is very necessary to discover and prepare new drugs that can prevent, especially promote the reversal or regression of AS.

A large number of epidemiological investigations since the 1970s have found that plasma high-density lipoprotein (HDL) levels are negatively correlated with the incidence of atherosclerosis (AS). The Framingham survey shows that the incidence of coronary heart disease (CHD) in families or regions with high plasma high-density lipoprotein-cholesterol (HDL-C, which can represent an indicator of plasma HDL content) is significantly lower. Rifkind found in the surveyed population that the risk of CHD decreased by 2-3% for every 1mg/dL increase in plasma HDL-C content. Helsinki Heart Study (Helsinki Heart Study) also confirmed that if the plasma HDL-C level is raised with drugs, the incidence of CHD in men can be reduced by 34%. Animal experiments have also proved that animals with low plasma HDL levels, such as housewigs, monkeys, and pigs, are prone to induce AS. On the contrary, animals with high plasma HDL levels, such as minks, Peking ducks, and tree shrews, are less likely or even not to develop AS. In addition, cell culture has proved that HDL can promote the removal of cholesterol in cells (including arterial smooth muscle cells), and animal experiments have also proved that HDL can inhibit the formation of AS plaques and promote the regression of plaques. These all show that HDL has the function of fighting against AS. And because HDL is a natural drug derived from human plasma, it has incomparable advantages over other synthetic drugs in the long-term process of preventing and treating AS.

HDL is mainly composed of apolipoprotein and lipid, which is the most dense but extremely heterogeneous type of lipoprotein in plasma. HDL is a lipoprotein composed of protein and lipid. It contains 50% to 55% protein, mainly composed of apolipoprotein (apo) AI and AII, and also contains a small amount of apoAIV, CI, CII, CIII, D, E and J, HDL contains about 45%-50% lipid, mainly composed of phospholipids (PL, 40-60%), cholesterol and cholesterol esters (total cholesterol TC, 30%-40%) and a small amount of triglycerides (TG, 10%) left and right) constitute. In HDL, apoAI has the most content and is the main functional protein of HDL, so the content of apo AI can reflect the content of HDL.

At present, most blood products at home and abroad adopt Cohn’s low-temperature ethanol method to produce human serum albumin and gamma globulin. This method can divide plasma protein into five components such as I, II, III, IV and V during the separation process. Among them, the HDL-rich component IV-1 (FIV-1) is currently used as waste treatment and has not been utilized, which is actually a huge waste.

Contents of the invention

The object of the present invention is to provide a kind of human blood high-density lipoprotein and its product.

Another object of the present invention is to provide a method for producing human blood high-density lipoprotein and its products.

Another object of the present invention is to provide the above-mentioned human blood high-density lipoprotein and its products in the preparation and treatment of multiple organ dysfunction syndrome (MODS), cardiovascular system diseases, such as the application of acute symptoms such as AS and neutralizing bacterial toxins .

In the human blood high-density lipoprotein described in the present invention, the content of apo AI is 77-88%.

The molecular weight of apoAI in the above-mentioned human blood high-density lipoprotein is 27.7KD.

The human blood high-density lipoprotein product of the present invention may contain 0.1-99% of human blood high-density lipoprotein.

The above-mentioned human blood high-density lipoprotein product can be added with 9-11% sucrose and/or 0.5% albumin and/or 150mg ± 1mg/L vitamin C to make a human blood HDL preparation for intravenous infusion, which has a good stability.

The aforementioned HDLs are not limited to the HDLs described in the present invention, and HDLs in the prior art are also applicable to the aforementioned combination and interfitting.

The method of the present invention comprises the following steps:

A. Take the raw plasma, add 1/5~1/4 volume of 50% ethanol, adjust the pH value to 6.8-7.4, stir at -4~-2°C for 1-3 hours, let stand for 6-8 hours, and centrifuge , to obtain the supernatant; add buffer solution dropwise to the supernatant until the pH of the mixture is 6.5-7.0, add 1/5 to 1/4 of the volume of the mixture 95% ethanol, and stir at -6 to -4°C for 1-3 hours, Stand still for 6-8 hours, centrifuge to obtain the supernatant; add 45-50% volume of water for injection to the supernatant, add buffer solution dropwise to make the pH 4.5-5.5, stir at -6～-3°C for 1- 3 hours, let stand for 1-3 hours, centrifuge, discard the supernatant, and get a precipitate;

B. Dissolve the precipitate with 2-3 times the weight of 0.02-0.1M NaCl solution, adjust the pH to 5.2±0.5, stir at 0-2°C for 2-6 hours, and then centrifuge to obtain the precipitate; use 50-150 times the weight of the precipitate to dissolve Dissolve 0.1-0.2M NaCl solution, adjust the pH of the solution to 7.2±0.5, stir at 2-8°C for 6-20 hours, and filter to obtain the filtrate;

C. Add buffer to the above filtrate to make the pH 5.2±0.5, stir for 1-3 hours, stand at 2-4°C for 6-20 hours, and collect the precipitate by centrifugation; use 50-150 times the amount of 0.1-0.2 Dissolve the MNaCl solution, adjust the pH to 7.2±0.5, stir to dissolve the precipitate completely, filter to obtain the filtrate;

D. The filtrate is ultrafiltered with an ultrafiltration membrane, desalted and concentrated to obtain a solution rich in HDL, and after post-processing, HDL and its products are obtained.

In the above method, 1mol/L NaHCO _solution is used to adjust pH.

The buffer in the above method is HAC/NaAC buffer system with pH=4. After step C is finished, it can be repeated as needed, so as to achieve better purity.

The ultrafiltration membrane in step D is an 8-70KD ultrafiltration membrane. It is preferable to use a 30-50KD ultrafiltration membrane, which has the function of further purification.

The post-treatment described in step D can be: adding 9-11% sucrose for injection or 0.5% albumin to the ultrafiltration concentrate, adding 150 mg ± 1 mg of vitamin C for injection per liter, adjusting the pH to 7.2 ± 0.5 to obtain HDL solution The above-mentioned HDL solution was inactivated in a computer-controlled pasteurizer at 60±1°C for 10 hours; the HDL solution after the inactivation of the virus was sterilized and subpackaged, and stored at 2-8°C.

By adopting the method of the invention to prepare HDL, the recovery rate of apoAI can reach more than 69%, which has remarkable economic benefits.

The method of the present invention uses low-temperature ethanol method to prepare human serum albumin and the discarded FIV-1 precipitate of gamma globulin as raw material, and fully removes HDL by washing under the conditions of pH 5.1±0.5, 0-2°C and 0.03M NaCl other plasma proteins. After the crude HDL is dissolved, it is reprecipitated and purified under the conditions of pH 5.1±0.5 (isoelectric point), 2-4°C, and 0.15M NaCl. The final purified HDL solution is made by ultrafiltration, dialysis, concentration, preparation, 60±1°C, 10 hours of virus inactivation and sterilization to make pH6.4～7.4, apolipoprotein AI content≥0.5%, purity≥95%, Human blood HDL preparation containing 10% ± 1% sucrose.

In the process of HDL preparation, adjust the appropriate alcohol concentration at 6-30%, protein concentration at 1-6%, pH value at 4.5-7.5, ionic strength at 0.02-2.0kΩ·cm, and temperature at -6-6 in each step of separation. 10°C.

HDL is a lipoprotein composed of protein and lipid, which contains 50% to 55% protein, mainly composed of apolipoprotein (apo) AI (65% to 70%) and AII (20% to 25%), and also contains Small amounts of apoAIV, CI, CII, CIII, D, E and J. HDL contains about 45%-50% lipid, mainly composed of phospholipids (PL, 40-60%), cholesterol and cholesterol esters (total cholesterol TC, 30%-40%) and a small amount of triglycerides (TG, about 10%) constitute. Apo AI has the most content in HDL and is the main functional protein of HDL. Therefore, the content of apoAI can reflect the content of HDL. The inventor adopts the apolipoprotein AI detection kit (one-way immunodiffusion method) developed by the Apolipoprotein Research Office of West China Medical University to detect the apoAI content and represents the HDL content with the apoAI content in the product, and the apo AI content ≥ 0.5% is qualified .

Studies have found that there are receptors that can specifically bind HDL on the cell membranes of liver cells, arterial smooth muscle cells, and endothelial cells. The inventor used the hepatic cell membrane HDL receptor pre-coated on the microtiter plate to specifically bind to HDL, and then reacted with horseradish peroxidase-antibody conjugate HRP-anti-apoAI IgG for immunological binding, and finally passed The enzyme-catalyzed color reaction in the receptor-ligand (HDL)-anti-ligand antibody-enzyme complex adsorbed on the solid phase of the microtiter plate detects the biological activity of HDL in the product. When measuring biological activity, 1 μg apo AI was used as one HDL activity unit (U). Since the apoAI content of HDL products is ≥0.5%, which is equivalent to containing 5×105U HDL (theoretical value), the inventors use 80% of this value as the standard for judging the product’s qualification, that is, the biological activity of HDL products should be ≥4×10 ⁵ U .

Animal acute toxicity test of the HDL product of the present invention: the test results show that HDL is administered by intravenous injection and intraperitoneal injection, with the highest concentration (1%) in the extraction as the maximum administration concentration, and the maximum concentration by intravenous injection and intraperitoneal injection. Volume 0.8ml/20g (body weight) administration, continuous administration 3 times within 12h, continuous observation for 7 days after administration, no abnormalities were found in the animals, and none of the animals died after 7 days, so HDL was administered intravenously and intraperitoneally. The maximum tolerated dose is 0.024g/20g (body weight).

Stability test of the HDL product of the present invention: According to the "Compilation of Guiding Principles for Preclinical Research of New Drugs (Western Medicine) (Pharmacology, Pharmacology and Toxicology)" compiled by the Pharmaceutical Administration Bureau of the Ministry of Health of the People's Republic of China in July 1993, human blood HDL products were affected. Factor test, room temperature (25°C) 5-month sample inspection and 2-8°C 14-month sample inspection. The results show that HDL test samples can withstand the impact of changes in conditions in the circulation field and during use, and the storage conditions of the products should be strictly controlled at 2-8°C and stored in the dark, so that within one year, HDL liquid preparations can be stored in the During circulation and use, the quality standards required by the "China Biological Products Regulations" should be kept stable.

The main pharmacodynamics test related to the therapeutic effect of the HDL product of the present invention: In order to verify the anti-AS effect of the human plasma HDL product of the present invention, the inventor caused the rabbit As model with high-fat feeding, and while high-fat feeding (experimental As Prevention test) and after 10 weeks of high-fat feeding to cause AS model (experimental AS treatment test), divided into low (L), medium (M) and high (H) three dose groups (10, 50, 100 mg based on apoAI content Total) Intravenous injection of human plasma HDL preparations (once a week, 10 weeks in total), from the lipid and lipid peroxide levels in the blood of the tested animals, the lipid content of the arterial wall and liver, the lipid content of gallbladder bile, and the lipid content of the arterial wall AS plaque area and other aspects to examine and verify the anti-AS effect of HDL preparations.

Since there is no anti-AS drug with the same or similar mechanism of action as HDL, the inventors selected the lipid-lowering drug fenofibrate as the positive control drug. In the selection of the negative control, because studies have confirmed immune damage, especially the repeated infusion of foreign protein in high-fat fed model animals will accelerate the occurrence and development of AS due to the occurrence of immune reactions, while animal experiments on the pharmacodynamics of human plasma HDL Inevitably, the injection of human HDL heterogeneous protein will cause allergic reaction and immune reaction of animals, which will aggravate the occurrence and development of AS in model animals fed with high fat. The influence of AS effect, the inventor is except setting the high-fat control group (C) group of injecting normal saline, also set up the human plasma albumin negative control group (A) group that is equivalent to dosage group dosage in HDL, the result shows, use human plasma HDL Preparations to prevent and treat human AS disease will not cause xenoprotein reaction.

The main results of the experiment are as follows:

(1) The results of blood lipid analysis show that: human plasma HDL preparations do not have the effect of increasing blood lipid (cholesterol TC, triglyceride (TG) and phospholipid (PL) levels in AS model animals fed with high fat (experimental AS prevention and treatment test). The effect of reducing. Therefore, the anti-AS effect of HDL is not realized by reducing the content of blood lipid (TC, TG).

(2) The results of blood lipid peroxide determination (experimental AS prevention test) found that after injection of HDL preparations, blood lipid peroxide levels (3.9, 2.5 , 3.2mmol/ml) were significantly lower than the levels of the control group (5.4 and 5.0mmol/ml) (P<0.05, P<0.01 and P<0.001), indicating that human plasma HDL preparations have the ability to resist serum lipids in rabbits fed with high fat. Oxidation of substances occurs.

(3) Measurement of thoracic aorta lipid and AS plaque area in model animals found that the lipid content of arterial wall and AS plaque area of rabbits fed with high fat increased significantly, and the negative control (A) group was the most serious. The arterial wall lipid and AS plaque area of animals injected with HDL were reduced or significantly reduced, especially in HDL medium and high dose (M, H) groups. For example: preventive test M, H group animal arterial wall TC, TG content and AS plaque area are respectively 33.8, 35.0, 28.1, 23.7mg/g dry tissue and 38.4% (M group), all compared with A group animal 54.7, 38.5 mg/g dry tissue and 57.6% were significantly reduced (P<0.05); the results of arterial wall lipid measurement in the treatment trial were similar to those in the prevention trial, and the AS plaque areas in the L, M, and H groups were 60.2%, 58.4%, and 53.7%, respectively. % was significantly less than the AS plaque area of group A (79.6%) (P<0.01 and P<0.001).

(4) The results of liver lipid assay were basically similar to those of arterial wall lipid assay. The content of TC and TG in the liver of animals in the prevention test was 135.8 and 44.0 mg/g dry tissue in group H, which was significantly lower than that in group A (192.1 and 72.8 (mg/g dry tissue)) (P<0.05); the results of the treatment test were more prominent , L, M, H group animal liver TC, TG and PL content were significantly lower than A group (P<0.05 and P<0.01).

(5) The measurement results of bile lipid content are opposite to those of arterial wall and liver lipid content. Regardless of AS prevention or treatment test, the bile lipid content of each group of animals injected with HDL showed an increasing trend compared with the control group, and the prevention test The most obvious result. In the AS prevention test, the TC content (337.5mg%, that is, 337.5mg per 100ml, the same below) in group H was significantly higher than that in group C (217.2mg%), and the PL content (978.7 and 1008.8mg%) in group M and H was significantly higher than that in group A group (688.3mg%, P<0.05).

The above results show that the injection of human plasma HDL can reduce the lipid deposition in the arterial wall and liver, increase bile lipid excretion, and reduce the AS plaque area in the arterial wall of As model animals fed with high-fat diet.

The HDL of the present invention directly observes the anti-AS effect of HDL by giving the high-fat model home a free injection of HDL preparation, and the experimental results obtained show that no matter when the high-fat model is formed or after the high-fat model causes the rabbit AS model, injection of rabbit blood HDL It can significantly reduce the lipid deposited in the arterial wall and liver of modeled animals, and the area of AS plaque on the arterial wall, which fully proves that HDL has dual functions of inhibiting the occurrence of AS and promoting the reversal or regression of AS (that is, prevention and treatment). Therefore, the HDL of the present invention has a good application prospect in the preparation of new drugs for preventing and treating AS.

Bacterial toxins are mainly divided into exotoxins and endotoxins; exotoxins of some important pathogenic bacteria that endanger human health, such as diphtheria and tetanus, have been successfully developed and effective with the successive development of their antitoxins and toxoids. Application, no longer makes people turn pale; the fight against bacterial toxins has now focused on endotoxins.

Anti-endotoxin therapy for endotoxemia has been perplexing the medical community. Despite the use of improved organ support and intensive care techniques, as well as new drugs such as anti-endotoxin antibodies, anti-inflammatory cytokine (inflammatory mediator) antibodies, or inflammatory cytokine receptor antagonists, severe Gram-negative bacterial infections caused The systemic inflammatory response syndrome (SIRS) caused by endotoxemia and the resulting multiple organ dysfunction syndrome (MODS) have a high mortality rate. Therefore, endotoxin remains a major medical problem.

The phagocytosis, degradation and removal of endotoxin in the body depends on the active participation of the immune system; in addition, the perfect detoxification mechanism in the body also plays an important role in the detoxification of endotoxin. The body's immune and detoxification systems overlap or relate to each other in many cases, but also differ from each other. After endotoxin enters the human body, lipoproteins in the blood, especially high-density lipoproteins and other protein components involved in lipid exchange and transport, bear the brunt of neutralizing and buffering endotoxins, and blood neutrophils release certain cationic proteins (such as bactericidal permeability-increasing protein) detoxifies and depolymerizes endotoxin, alkaline phosphatase widely distributed in tissues can also dephosphorylate endotoxin, and reticuloendothelial system phagocytizes and degrades endotoxin role, thereby forming the body's detoxification process for endotoxins. Therefore, lipoproteins, especially high-density lipoproteins, have the function of protecting the detoxification function of the body, and have good application prospects in the preparation of new drugs for the prevention and treatment of neutralizing bacterial toxins and MODS.

The invention utilizes the discarded FIV-1 precipitate after producing human plasma albumin and gamma globulin by the low-temperature ethanol method as a raw material, and extracts HDL products from FIV-1 through separation and purification steps on the basis of a large number of experiments, providing a large-scale A new low-temperature ethanol process for preparing human plasma HDL, and successfully prepared a safe, stable, non-toxic, pyrogen-free, sterile and virus-free, pH 6.4-7.4, apolipoprotein AI content ≥ 0.5%, HDL purity ≥ 95%, containing 10% ± 1% sucrose of human blood HDL preparations for intravenous infusion, lipid and apolipoprotein composition and content analysis shows that the lipid and apolipoprotein composition and content of HDL products are consistent with the natural HDL value It is basically the same, and does not contain low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) that can promote atherosclerosis. It is a full use of precious human blood resources.

Description of drawings

Figure 1 shows the preparation process of HDL-rich FIV-1 precipitate (cohn's 6 method, 1946)

E: ethanol concentration

T: temperature

γ/2: ionic strength

P: protein concentration

Figure 2 is the HDL refining process

Figure 3 is the result of HDL immunoelectrophoresis, in which:

1. Normal human plasma

2. Goat anti-human apoAI antiserum

3. HDL products

Figure 4 is the result of HDL lipoprotein agarose gel electrophoresis, wherein:

1. Normal human plasma

2. HDL products

Fig. 5 is the result of SDS-PAGE of HDL preparation, wherein:

1, 2, 4, 5, 6, 7 are HDL products

3 is the molecular weight standard

Figure 6 is the HDL biological activity ELISA procedure

Fig. 7 is human blood HDL biological activity standard curve, wherein

■—■: HDL

●—●: LDL

▲—▲: VLDL

Detailed ways

Example 1

Take 1000L of raw plasma, add 190.5L of 50% ethanol, adjust the pH value to 7.1, protein concentration to 5.6%, and ionic strength to 0.15, stir at -2°C for 2 hours, let stand for 8 hours, use a 142-type continuous centrifuge, Centrifuge at 14000r/min to obtain 1190L of supernatant and 12Kg of precipitate;

Add 5000 mL of HAC/NaAC mixed solution with a pH of 4.0 dropwise to the supernatant to make the pH of the mixed solution 6.90, add 263 L of 95% ethanol to make the protein concentration 3.4%, and the ionic strength 0.09, stir at -4°C for 2 hours, and statically Set aside for 8 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min to obtain a supernatant of 1450L and a precipitate of 40Kg;

Add 682 L of water for injection to the supernatant, dropwise add 14.8 L of HAC/NaAC mixed solution with a pH of 4.0, so that the pH is 4.9, the protein concentration is 1.7%, and the ionic strength is 0.15. Place for 2 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard the supernatant, and obtain a precipitate of 10.2Kg;

Add 20.4L of 30mmol/L sodium chloride solution to the precipitate, add dropwise 1mol/L _NaHCO3 solution to adjust the pH to 5.5, stir at 0°C for 4 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard In the supernatant, other plasma proteins and soluble impurities were removed to obtain a precipitate of 4.65Kg;

Add 465L of 160mmol/L sodium chloride solution to the precipitate to dissolve, so that the pH of the solution is 5.21, add dropwise 400ml of 1mol/L NaHCO ₃ solution, adjust the pH of the solution to 7.02, stir at 7°C for 6 hours, filter to obtain the filtrate 600L;

Add 120ml of HAC/NaAC mixed solution with a pH of 4.0 to the filtrate dropwise to make the pH of the reaction solution 5.15, stir at 2°C for 1 hour, let stand for 6 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min, Discard the supernatant to obtain a precipitate of 3.95Kg;

Add 395L of 160mmol/L sodium chloride solution to the precipitate. At this time, the pH of the solution is 5.1. Add 350ml of 1mol/L NaHCO solution dropwise to make _the pH of the solution 7.00. Stir at 2°C for 6 hours and filter to obtain 510L of filtrate ;

Use 8-10KD ultrafiltration membrane for ultrafiltration and concentration to obtain 380L of concentrated solution. Add 38Kg sucrose to the concentrated solution, the protein concentration is 18g/L, the ethanol content is 0.02%, and the conductivity is 0.4Ms; The virus was inactivated at 60±1°C for 10 hours in the sterilizer; the content of apoAI in HDL after the inactivation of the virus was 88%, and the recovery rate of apoAI was 71%.

Sterilize the finished product in 50ml×7550 bottles, and store at 2°C.

Example 2

Take 1000L of raw plasma, add 190.5L of 50% ethanol, adjust the pH value to 7.3, protein concentration to 5.2%, and ionic strength to 0.18. Stir at -2°C for 2 hours, let stand for 8 hours, and use a 142-type continuous centrifuge. Centrifuge at 14000r/min to get supernatant 1100L and precipitate 12Kg;

Add 5000 mL of HAC/NaAC mixed solution with a pH of 4.0 dropwise to the supernatant to make the pH of the mixed solution 6.90, add 243 L of 95% ethanol to make the protein concentration 4.0%, and the ionic strength 0.15, stir at -6°C for 2 hours, and statically Set aside for 8 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min to obtain a supernatant of 1449L and a precipitate of 39Kg;

Add 682 L of water for injection to the supernatant, dropwise add 14.8 L of HAC/NaAC mixed solution with a pH of 4.0, so that the pH is 5.0, the protein concentration is 2.0%, and the ionic strength is 0.09. Place for 2 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard the supernatant, and obtain a precipitate of 9.8Kg;

Add 19.6L of 30mmol/L sodium chloride solution to the precipitate, add dropwise 1mol/L _NaHCO3 solution to adjust the pH to 5.6, stir at 2°C for 4 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard clear, 4.42Kg of precipitate was obtained;

Add 442L of 160mmol/L sodium chloride solution to the precipitate to dissolve, so that the pH of the solution _is 5.20, add dropwise 410ml of 1mol/L NaHCO solution, adjust the pH of the solution to 7.1, stir at 8°C for 6 hours, and filter to obtain Filtrate 610L;

Add 130ml of HAC/NaAC mixed solution with a pH of 4.0 to the filtrate dropwise to make the pH of the reaction solution 5.20, stir at 3°C for 1 hour, let stand for 6 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min, Discard the supernatant to obtain a precipitate of 3.80Kg;

Add 380L of 160mmol/L sodium chloride solution to the precipitate. At this time, the pH of the solution is 5.17. Add 360ml of 1mol/L _NaHCO solution dropwise to make the pH of the solution 7.10. Stir at 6°C for 7 hours and filter to obtain 500L of filtrate ;

Use 8-10KD ultrafiltration membrane for ultrafiltration and concentration to obtain 390L of concentrated solution. Add 39Kg sucrose to the concentrated solution, the protein concentration is 19g/L, the ethanol content is 0.02%, and the conductivity is 0.45Ms; The virus was inactivated at 60±1°C for 10 hours in the sterilizer; the content of apo AI in HDL after inactivating the virus was 77%, and the recovery rate of apo AI was 78%.

Sterilize the finished product in 50ml×7850 bottles, and store at 4°C.

Example 3

Take 1000L of raw plasma, add 190.5L of 50% ethanol, adjust the pH value to 6.8, protein concentration to 4.0%, and ionic strength to 0.10, stir at -2°C for 2 hours, let stand for 6 hours, use a 142-type continuous centrifuge, Centrifuge at 14000r/min to obtain 1190L of supernatant and 13Kg of precipitate;

Add dropwise 5000 mL of HAC/NaAC mixed solution with a pH of 4.0 to the supernatant to make the pH of the mixed solution 6.90, add 263 L of 95% ethanol to make the protein concentration 2.0%, ionic strength 0.05, stir at -6°C for 2 hours, statically Set aside for 6 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min to obtain a supernatant of 1450L and a precipitate of 38Kg;

Add 658 L of water for injection to the supernatant, dropwise add 14.3 L of HAC/NaAC mixed solution with a pH of 4.0, so that the pH is 4.5, the protein concentration is 1.0%, and the ionic strength is 0.05. Stir at -3°C for 2 hours, and statically Place for 2 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard the supernatant, and obtain a precipitate of 10.1Kg;

Add 20.2L of 30mmol/L sodium chloride solution to the precipitate, add dropwise 1mol/L NaHCO ₃ solution to adjust the pH to 5.15, stir at 0°C for 4 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard clear, 4.51Kg of precipitate was obtained;

Add 451L of 160mmol/L sodium chloride solution to the precipitate to dissolve, so that the pH of the solution _is 5.18, add dropwise 390ml of 1mol/L NaHCO solution, adjust the pH of the solution to 7.02, stir at 5°C for 6 hours, and filter to obtain the filtrate 560L;

Add 110ml of HAC/NaAC mixed solution with pH 4.0 dropwise to the filtrate to make the pH of the reaction solution 5.15, stir at 2°C for 1 hour, let stand for 6 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, Discard the supernatant to obtain a precipitate of 3.75Kg;

Add 375L of 160mmol/L sodium chloride solution to the precipitate. At this time, the pH of the solution is 5.15. _Add 330ml of 1mol/L NaHCO solution dropwise to make the pH of the solution 7.00. Stir at 2°C for 6 hours and filter to obtain 500L of filtrate ;

Use 8-10KD ultrafiltration membrane for ultrafiltration and concentration to obtain 370L of concentrated solution. Add 37Kg sucrose to the concentrated solution, the protein concentration is 18g/L, the ethanol content is 0.03%, and the conductivity is 0.5Ms; Inactivate the virus at 60±1°C for 10 hours in the sterilizer; the content of apo AI in HDL after inactivating the virus is 81%, and the recovery rate of apo AI is 69%.

Sterilize the finished product in 50ml×7350 bottles, and store at 6°C.

Example 4

Take 1000L of raw plasma, add 190.5L of 50% ethanol, adjust the pH value to 7.4, protein concentration to 6.0%, and ionic strength to 0.2, stir at -2°C for 2 hours, let stand for 8 hours, and use a 142-type continuous centrifuge. Centrifuge at 14000r/min to get supernatant 1250L and precipitate 13.8Kg;

Add 5050 mL of HAC/NaAC mixed solution with a pH of 4.0 dropwise to the supernatant to make the pH of the mixed solution 6.95, add 272 L of 95% ethanol to make the protein concentration 4.0%, and the ionic strength 0.15, stir at -6°C for 2 hours, statically Set aside for 8 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min to obtain a supernatant of 1580L and a precipitate of 45Kg;

Add 743 L of water for injection to the supernatant, and dropwise add 16.1 L of HAC/NaAC mixed solution with a pH of 4.0 to make the pH to 5.5, the protein concentration to 2.0%, and the ionic strength to 0.15. Stir at -6°C for 2 hours, statically Place for 2 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard the supernatant, and obtain a precipitate of 10.3Kg;

Add 20.6L of 30mmol/L sodium chloride solution to the precipitate, add dropwise 1mol/L _NaHCO3 solution to adjust the pH to 5.0, stir at 0°C for 4 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard In the supernatant, 4.61Kg of precipitate was obtained;

Add 461L of 160mmol/L sodium chloride solution to the precipitate to dissolve, so that the pH of the solution is 5.15, add dropwise 420ml of 1mol/L NaHCO ₃ solution, adjust the pH of the solution to 7.7, stir at 10°C for 6 hours, filter to obtain the filtrate 620L;

Add 130ml of HAC/NaAC mixed solution with a pH of 4.0 to the filtrate dropwise, make the pH of the reaction solution 5.25, stir at 2°C for 1 hour, let stand for 6 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, Discard the supernatant to obtain a precipitate of 3.92Kg;

Add 392L of 160mmol/L sodium chloride solution to the precipitate. At this time, the pH of the solution is 5.16. Add 360ml of 1mol/L _NaHCO solution dropwise to make the pH of the solution 7.09. Stir at 8°C for 8 hours and filter to obtain 560L of filtrate ;

Use 8-10KD ultrafiltration membrane for ultrafiltration and concentration to obtain 400L of concentrated solution, add 40Kg sucrose to the concentrated solution, the protein concentration is 19.5g/L, the ethanol content is 0.02%, and the conductivity is 0.3Ms; The virus was inactivated in a sterilizer at 60±1°C for 10 hours; the content of apoAI in HDL after the inactivation of the virus was 85%, and the recovery rate of apoAI was 75%.

Sterilize the finished product in 50ml×7900 bottles, and store at 8°C.

Example 5

Take 1000L of raw plasma, add 190.5L of 50% ethanol, adjust the pH value to 7.2, protein concentration to 5.5%, and ionic strength to 0.19, stir at -2°C for 2 hours, let stand for 7 hours, and use a 142-type continuous centrifuge. Centrifuge at 14000r/min to obtain 1300L of supernatant and 14Kg of precipitate;

Add dropwise 5090 mL of HAC/NaAC mixed solution with a pH of 4.0 to the supernatant to make the pH of the mixed solution 6.50, add 287 L of 95% ethanol to make the protein concentration 3.8%, and the ionic strength 0.12, stir at -5°C for 2 hours, statically Set aside for 8 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min to obtain a supernatant of 1600L and a precipitate of 49Kg;

Add 752 L of water for injection to the supernatant, and dropwise add 16.3 L of HAC/NaAC mixed solution with a pH of 4.0 to make the pH to 5.1, the protein concentration to 1.9%, and the ionic strength to 0.08. Stir at -4°C for 2 hours and statically Place for 2 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard the supernatant, and obtain a precipitate of 10.2Kg;

Add 20.4L of 30mmol/L sodium chloride solution to the precipitate, add dropwise 1mol/L _NaHCO3 solution to adjust the pH to 6.0, stir at 2°C for 4 hours, use a 142-type continuous centrifuge, centrifuge at 14000r/min, discard In the supernatant, 4.58Kg of precipitate was obtained;

Add 458L of 160mmol/L sodium chloride solution to the precipitate to dissolve, so that the pH of the solution is 5.19, add dropwise 410ml of 1mol/L NaHCO ₃ solution, adjust the pH of the solution to 7.12, stir at 8°C for 6 hours, filter to obtain the filtrate 600L;

Add 115ml of HAC/NaAC mixed solution with a pH of 4.0 to the filtrate dropwise to make the pH of the reaction solution 5.21, stir at 2°C for 1 hour, let stand for 6 hours, use a 142-type continuous centrifuge, and centrifuge at 14000r/min, Discard the supernatant to obtain a precipitate of 3.85Kg;

Add 385L of 160mmol/L sodium chloride solution to the precipitate. At this time, the pH of the solution is 5.20. Add 360ml of 1mol/L _NaHCO solution dropwise to make the pH of the solution 7.70. Stir at 4°C for 8 hours and filter to obtain 570L of filtrate ;

Use 8-10KD ultrafiltration membrane for ultrafiltration and concentration to obtain 415L of concentrated solution. Add 41.5Kg sucrose to the concentrated solution, the protein concentration is 19.3g/L, the ethanol content is 0.03%, and the conductivity is 0.5Ms; The virus was inactivated in a sterilizer at 60±1° C. for 10 hours; the content of apoAI in HDL after the inactivation of the virus was 78%, and the recovery rate of apoAI was 73%.

Sterilize and subpackage the finished product in 50ml×8150 bottles, and store at 5°C.

Experimental example 1

This experimental example is the verification data of the product HDL of the present invention.

1. Method

1.1 Identification and purity analysis of HDL: The prepared HDL was identified by immunoelectrophoresis and lipoprotein agarose gel electrophoresis, and its purity was scanned and analyzed according to the results of lipoprotein agarose gel electrophoresis.

1.2 Determination of protein content: Determination by the modified Lowry method of Markwell et al.

1.3 Determination of lipid and apolipoprotein content: triglyceride (TG) and total cholesterol (TC) in HDL were determined by Beijing Zhongsheng Beikong Biotechnology Co., Ltd. enzymatic kit, and phospholipid (PL) was determined by improved ascorbic acid reduction method Determination. Apolipoprotein (apo) AI, AII, B100, CII, CIII, and E were determined by one-way immunodiffusion kits from West China Apolipoprotein Laboratory, Sichuan University.

1.4 Determination of molecular weight of apoAI: determined by SDS-PAGE method.

1.5 Determination of HDL biological activity: The human blood HDL biological activity assay method established by Xu Yanhua was used for determination. According to each 1μgapoAI is 1 HDL activity unit (U).

1.6 Sterility test: According to the "Pharmacopoeia of the People's Republic of China" 2005 edition three appendix "sterility test method".

1.7 Bacterial endotoxin inspection: According to the three appendices "Bacterial endotoxin inspection method" of "Pharmacopoeia of the People's Republic of China" in 2005 edition.

1.8 Pyrogen inspection: According to the "Pyrogen Inspection Method" in the three appendices of the 2005 edition of the Pharmacopoeia of the People's Republic of China.

1.9 Examination of abnormal toxicity: according to the "Pharmacopoeia of the People's Republic of China" 2005 edition three appendices "abnormal toxicity inspection method"

2. Results

2.1 Identification and purity analysis of HDL

(1) Immunoelectrophoresis, see Figure 3; it can be seen from Figure 3 that HDL products and normal human plasma have precipitation lines at the same position.

(2) lipoprotein agarose gel electrophoresis, see Fig. 4; Visible by Fig. 4, HDL product presents a zone in lipoprotein agarose gel electrophoresis, Biorad gel imaging scanning system scanning analysis, its purity is 100% .

2.2 Lipid and apolipoprotein composition (%) in HDL, see the table below; As can be seen from the table below, HDL product apoAI content of the present invention is higher.

2.3 Determination of molecular weight of apoAI, see Figure 5, its molecular weight is 27.7KD;

2.4 Determination of biological activity of HDL preparations, see the table below:

2 Examples pH value apoAI content (mg/dL) Biological activity (U/ml) 1 6.96 1289 13750 2 6.45 1274 12500

3 6.41 1304 13750 4 6.60 1297 13125 5 6.40 1312 13000

Experimental example 2

This experimental example is the research on the assay method of anti-AS biological activity of human blood HDL, the content is as follows:

1. Materials and reagents

Japanese big-eared white rabbits; normal human plasma is collected from blood donors; enter the plasma HDL preparation, prepared according to the method described in the present invention; HRP (RZ=3.0), Sigma company product; purify rabbit liver cell membrane, goat anti-human apoAI IgG, HRP -Anti-apoAI IgG, human LDL and VLDL, self-made; TG, TC enzymatic assay kit, Beijing Zhongsheng Beikong Biotechnology Co., Ltd.; apolipoprotein AI, B100, CII, CIII and E immune one-way diffusion kit , West China Apolipoprotein Laboratory, Sichuan University; the rest of the reagents were of domestic analytical grade.

96-well polystyrene microplate plate, product of Corning Company; Model 550 microplate reader, product of Bio-RAD Company; L8-55 ultracentrifuge, Beckman Company; Sebris scanner, product of Sebris Company.

Coating buffer is pH 9.6, 50mmol/L Na ₂ CO ₃ -NaHCO ₃ buffer; washing and dilution buffer is pH 7.4, 20mmol/L PBS containing 0.05% Tween-20; enzyme reaction matrix solution is pH 5 .6, 0.1 mol/L citric acid-Na ₂ HPO ₄ buffer solution containing 0.04% o-phenylenediamine and 0.05% H ₂ O ₂ (prepared just before use).

2. Method

2.1 Preparation of rabbit liver cell membrane

Rabbit liver cell membranes were purified by sucrose density gradient ultracentrifugation. The 37%-41% sucrose density interface membrane fraction was collected, diluted with buffer solution (50mmol/L Tris-HCl, 100mmol/L NaCl, 0.5mmol/L CaCl ₂ , pH7.5), and centrifuged at 27000×g for 20 minutes. Collect the precipitate, add a small amount of buffer, and process it in an ice bath with an MSE-150 ultrasonic instrument for 2×10 seconds with an amplitude of 14. The prepared liver cell membranes were stored at -70°C. The membrane yield is 1.5-2.0 mg/g wet liver tissue, and the 5'-nucleotidase activity is increased by 6-10 times compared with liver homogenate.

2.2 Preparation of LDL and VLDL

Separate human plasma lipoproteins by one-time density gradient centrifugation to collect LDL components with d=1.030～1.050g/mL and VLDL components with d=0.95～1.006g/mL, for 10mmol/L Tris-HCl, 1mmol/L EDTA, 50mmol/L NaCl, pH 7.4 buffer solution for dialysis and desalting, and stored at 4°C for later use.

Membrane proteins and lipoproteins were determined by the method of Markwell et al.

2.3 Processing of HDL (embodiment 2)

The human blood HDL preparation obtained in Example 2 of the present invention was used as a raw material, and was prepared by ultracentrifugation after ultrafiltration and concentration. During preparation, the HDL preparation was adjusted to a density of 1.21 g/mL with solid NaBr, and centrifuged in an L8-55 ultracentrifuge at 50,000 rpm and 10°C for 24 hours. Collect the fraction with d≤1.21g/mL in the upper layer, dialyze, sterilize and aliquot, and store at 4°C for future use.

HDL (Example 2) TG and TC content were measured by enzymatic kit; phospholipid (PL) was measured by ascorbic acid reduction method; apoAI, AII, B100, CII, CIII and E content were measured by immunounidirectional diffusion kit.

2.4 HDL biological activity detection test

2.4.1 Determination steps

Carry out according to the procedure of HDL receptor ELISA, see Figure 6;

When measuring, the coating concentration of membrane protein and the dilution of HRP-anti-apoAI IgG were determined by checkerboard titration to be 25 μg membrane protein/ml and 1:250 respectively.

2.4.2 HDL biological activity standard curve

Taking the prepared HDL (Example 2) as a standard, according to 1 μ gapoAI as 1 HDL activity unit (U), the HDL reference product was diluted to 80, 40, 20, 10 and 5 (U/mL) with diluent, and then Measured according to the above steps, draw the standard curve of light absorption value and HDL biological activity, and obtain the corresponding double-logarithmic regression equation.

2.4.3 Sample determination

After the sample to be tested is diluted, its light absorbance value is measured in the same microtiter plate used to make the standard curve. Finally, the activity units of HDL preparations were calculated by the regression equation of the standard curve and the sample dilution factor.

3. Results

3.1 Identification of HDL (Example 2)

3.1.1 HDL (embodiment 2) lipid and apolipoprotein composition, see the following table:

As can be seen from the above table, the apoAI content of HDL (Example 2) is higher than the HDL literature value.

3.1.2 Lipoprotein prestaining electrophoresis: HDL (Example 2) presents a zone, which is scanned and analyzed by a Sebris scanner, and its purity is 99.8%.

3.1.3 Immunoelectrophoresis: see Figure 3. HDL (Example 2) and normal human serum have precipitation lines at the same position.

3.2 HDL biological activity standard curve: HDL standard curve results show that the logarithm of the light absorption value at 492nm and the number of HDL activity units has a linear relationship within the range of 1 to 16 activity units, as shown in Figure 7.

3.3 Specificity test: It can be seen from Figure 7 that the purified human LDL and VLDL have no competitive effect on the enzyme-antibody cross-linked product with the enzyme plate coated with the liver cell membrane, indicating that human LDL and VLDL and the enzyme-antibody cross-linked product No cross-reactivity.

3.4 Repeatability test:

3.4.1 Intra-plate repeatability: the same HDL preparation was diluted 250 and 500 times respectively, and the measured intra-plate variation coefficients were 7.6% and 9.5% (n=10).

3.4.2 Inter-plate repeatability: Three ELISA plates were used to measure the same HDL preparation (250-fold dilution), and the test was completed within 3 days. The inter-plate variation coefficient was 12.3%.

Experimental example 3

This experimental example is the anti-AS biological activity stability test of HDL preparation.

The HDL preparations prepared in Example 1 were respectively placed at 2-8°C, 25°C and 40°C, samples were taken regularly, and their pH value, apoAI content and anti-AS biological activity were measured, and the effects of storage temperature and storage time on the anti-AS biological activity of HDL preparations were observed. effect on activity. The results are shown in the table below:

It can be seen from the above table that the anti-AS biological activity of HDL preparations did not change significantly when placed at 2-8°C for 18 months and at 25°C for 5 months, but when placed at 40°C for one month, its anti-AS biological activity That is, it is significantly reduced; it shows that under low temperature conditions (2-8°C), the anti-AS biological activity of HDL preparations can remain stable for a long time, while under high temperature (40°C) conditions, the anti-AS biological activity of HDL preparations will significantly decrease; therefore, A suitable ambient temperature for HDL formulations is 2-8°C.

Experimental example 4

This experimental example shows that the HDL preparation of the present invention has an anti-endotoxemia effect.

200 normal rabbits were taken and randomly divided into experimental group and control group, with 100 rabbits in each group.

Two groups of rabbits were injected with endotoxin (2ng/kg) to cause endotoxemia.

3.5 hours before endotoxin injection, the HDL preparation prepared in Example 3 (10 mg/kg, based on apoAI content) was intravenously infused into the experimental group, and the instillation was completed in 30 minutes.

After 10 hours, it was found that the release of inflammatory mediators such as TNF-α and IL-6 in the experimental group was small, and the infection symptoms of rabbits in the experimental group were mild, indicating that intravenous infusion of HDL has the effect of fighting endotoxemia, as shown in the following table:

group Number of cases IL-6(ng/L) TNF-α(ng/L) control group 100 280.36±57.29 501.82±99.08 test group 100 82.18±7.63 155.61±20.06

Compared with the control group: P<0.05

This experimental example fully proves that the HDL preparation of the present invention has an anti-endotoxemia effect. The inventors believe that the phospholipid layer of the HDL molecular shell can be tightly combined with the lipid A in the endotoxin molecule, and the combined endotoxin no longer binds to the CD14 receptor, thus blocking the endotoxin from stimulating monocytes/macrophages Cells release inflammatory mediators.

Experimental example 5

This experimental example shows that HDL treatment can effectively reduce inflammatory mediators in patients with multiple organ dysfunction syndrome (MODS).

1. Objective: To compare the therapeutic effects of HDL therapy and continuous renal replacement therapy (CRRT) on inflammatory mediators (interleukin-1β, interleukin-6, tumor necrosis factor-α, etc.) in patients with MODS.

2. Methods: 40 critically ill patients with MODS were selected. The diagnosis of MODS was in line with the diagnostic criteria established by the American Society of Critical Care Diseases in 1992. The patients were randomly divided into the experimental group and the control group according to the order of admission. The odd number was the experimental group, 20 cases, and the experimental group received HDL treatment; the even number was the control group, 20 cases, who received continuous renal replacement therapy (CRRT). Blood was drawn for examination in the two groups at the time of diagnosis of MODS and 24 hours after treatment.

3. Determination: (The radioimmunotherapy kit was purchased from the Institute of Radioimmunity, Science and Technology Development Center, General Hospital of the Chinese People's Liberation Army) Determination of interleukin-1β and interleukin-6 (IL-1β, IL-6): take 2ml of venous blood, inject After coagulation in the test tube, separate the serum, store it at -20°C, and use the equilibrium method for primary monitoring; Tumor necrosis factor-α (TNF-α): collect 2ml of venous blood, use 10% EDTA disodium as anticoagulant, separate After the plasma was stored, it was stored at -20°C and monitored by the liquid phase competition method.

4. Results: Interleukin-1β, interleukin-6 and tumor necrosis factor-α in the experimental group were significantly lower than those in the control group after 24 hours of HDL treatment. See the table below:

Compared with the control group: P<0.01

The systemic inflammatory response (SIR) is the basis of the pathogenesis of MODS. When the body suffers severe blows such as infection or trauma, bacteria/toxins or tissue damage will stimulate inflammatory cells such as macrophages to release inflammatory mediators (tumor necrosis factor-α , interleukin-1β and interleukin-6, etc.), tumor necrosis factor is one of the earliest released inflammatory mediators, which can further stimulate and activate macrophages, granulocytes, lymphocytes and endothelial cells, releasing a large number of inflammatory mediators Sexual media, forming a "cascade-like" chain reaction mediated by inflammatory mediators, like dominoes amplified step by step, making the inflammatory response out of control. If its development is not effectively blocked, it will lead to multiple organ dysfunction syndrome (MODS), or even multiple organ system failure (MOSF), resulting in death of the patient.

This experimental example proves that HDL treatment can significantly reduce the concentration of inflammatory mediators such as tumor necrosis factor-α, interleukin-1β and interleukin-6 in patients with multiple organ dysfunction syndrome, and effectively reduce the mortality of patients.

Claims (6)

1, a kind of manufacture method of human blood high-density lipoprotein and product thereof, it is characterized in that, described method comprises the following steps: A. Take the raw plasma, add 1/5~1/4 volume of 50% ethanol, adjust the pH value to 6.8-7.4, stir at -4~-2°C for 1-3 hours, let stand for 6-8 hours, centrifuge , to obtain the supernatant; add buffer solution dropwise to the supernatant until the pH of the mixed solution is 6.5-7.0, add 1/5 to 1/4 of the mixed solution volume of 95% ethanol, and stir at -6-4°C for 1-3 hours, Stand still for 6-8 hours, centrifuge to obtain the supernatant; add 45-50% volume of water for injection to the supernatant, add buffer solution dropwise to make the pH 4.5-5.5, stir at -6--3°C for 1- 3 hours, let stand for 1-3 hours, centrifuge, discard the supernatant, and get a precipitate; B. Dissolve the precipitate with 2-3 times the weight of 0.02-0.1M NaCl solution, adjust the pH to 5.2±0.5, stir at 0-2°C for 2-6 hours, and then centrifuge to obtain the precipitate; use 50-150 times the weight of the precipitate Dissolve 0.1-0.2M NaCl solution, adjust the pH of the solution to 7.2±0.5, stir at 2-8°C for 6-20 hours, and filter to obtain the filtrate; C. Add buffer to the above-mentioned filtrate to make the pH 5.2±0.5, stir for 1.3 hours, stand at 2-4°C for 6-20 hours, and collect the precipitate by centrifugation; use 50-150 times the amount of 0.1-0.2M NaCl for the precipitate Dissolve the solution, adjust the pH to 7.2±0.5, stir to dissolve the precipitate completely, filter to obtain the filtrate; D. The filtrate is ultrafiltered with an ultrafiltration membrane, desalted and concentrated to obtain a solution rich in HDL, and after post-processing, HDL and its products are obtained. 2. The manufacturing method according to claim 1, characterized in that 1 mol/L NaHCO ₃ solution is used to adjust the pH in the method steps and/or the buffer is HAC/NaAC buffer system with pH=4. 3. The manufacturing method according to claim 1, characterized in that the method step C is repeated after the end of the method. 4. The manufacturing method according to claim 1, characterized in that the ultrafiltration membrane in step D of the method is an 8-70KD ultrafiltration membrane. 5. The manufacturing method according to claim 4, characterized in that the ultrafiltration membrane in step D of the method is a 30-50KD ultrafiltration membrane. 6. The manufacturing method according to claim 1, characterized in that the post-treatment in step D of the method is: add 9-11% sucrose for injection or 0.5% albumin to the ultrafiltration concentrate, and add 150mg± 1 mg of vitamin C for injection, adjust the pH to 7.2±0.5 to obtain HDL solution; the above-mentioned HDL solution was inactivated in a computer-controlled pasteurizer at 60±1°C for 10 hours; the HDL solution after the inactivation of the virus was removed Bacteria were aliquoted and stored at 2-8°C.

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