CN1070380C - Application and prodn. method of HDL preparation - Google Patents

Abstract

The production method of high-density lipoprotein preparation belongs to the field of medicine. The high-density lipoprotein preparation is used to treat heart disease and arteriosclerosis. It is simple to use and has good effect. Obtained high-density lipoprotein fractions were removed by potassium oxalate to remove dextran sulfate, ultrafiltration to remove impurities, and then poison inactivation, sterilizing filtration, subpackaging, and freeze-drying. The invention has the advantages of being able to comprehensively utilize various components in blood plasma, and the production method has the advantages of low investment, simple operation and the like.

Description

Production method of high-density lipoprotein preparation

The invention belongs to the field of medicine, and relates to a production method of biochemical medicine.

The application of the high-density lipoprotein preparation of the invention is to prevent and treat coronary heart disease and arteriosclerosis. At present, coronary heart disease is characterized by the deposition of cholesterol and neutral lipids in the blood on the inner wall of blood vessels, forming fatty plaques, narrowing or even blocking blood vessel channels, and insufficient blood supply, causing angina pectoris and other symptoms. Currently used preventive and therapeutic drugs can be roughly divided into lipid-lowering drugs such as cholestyramine, and the other is vasodilator drugs, such as nitrates, which can only relieve symptoms but cannot transfer the hardened blood vessels.

The purpose of the present invention is to provide a production method of a drug high-density lipoprotein preparation that not only has the effect of lowering blood fat, but also removes the fatty plaque on the blood vessel wall, promotes the restoration of unimpeded blood vessels, and treats both symptoms and root causes.

The high-density lipoprotein preparation of the invention is used for preventing and treating atherosclerotic diseases such as coronary heart disease, phlebitis and blood vessel embolism caused by hyperlipidemia. The specific usage is that high-density lipoprotein preparations are made into ampoules or bottled freeze-dried powders. The protein content is divided into 30mg/bottle and 60mg/bottle. When used, add 1ml sterile distilled water to dissolve, intramuscular injection or intravenous drip. The dosage and usage are determined by the doctor according to the condition. Generally, intramuscular injection can be used, one (ampere) bottle at a time, three times a week, and four weeks is a course of treatment. During the treatment, there is no need to change eating habits. Treatment can be continued as necessary.

HDL preparations are produced by using ammonium sulfate or ethanol to separate lipoproteins in human plasma from other proteins that are used in the manufacture of other blood products. The lipoprotein fraction was depleted of LDL with varying concentrations of ammonium sulfate and dextran sulfate. The obtained HDL fraction was freed of dextran sulfate with potassium oxalate. Ultrafiltration removes residual potassium oxalate and other molecules. The application number for virus inactivation treatment is: 97119839.x. Sterilize and filter, aliquot and freeze-dry.

The production process of high-density lipoprotein preparations is:

(A) Dilute an appropriate amount of plasma with distilled water, add 0.1-1% sodium citrate, 0.2-1.5% sodium chloride, stir well, add saturated ammonium sulfate solution to 30-80% saturation, stir well, and let it stand The precipitation is complete, filter it with a filter cloth, or centrifuge with a continuous flow centrifuge, transfer the precipitation (primary centrifugation) for other purposes, collect the filtrate (or centrifugation supernatant), add ammonium sulfate to 50%-95% saturation, and precipitate After completion, use a filter cloth to filter or continue to centrifuge, transfer the supernatant for other purposes, and collect the precipitate (secondary precipitate) for later use.

(B) Appropriate amount of plasma was added with ethanol to reach 15-50% concentration according to Cohn’s method, followed by continuous flow centrifugation, and the precipitate was transferred for other use. The supernatant was added with ethanol to reach 20-60%, the precipitate was collected by continuous flow centrifugation, and the supernatant was transferred for other use. , precipitation (FIV) for later use.

(C) Take the secondary precipitation of (A) or the FIV of (B), add appropriate amount of water to dissolve, ultrafiltration or dialysis to remove residual ammonium sulfate (A) method or ethanol (B) method, collect ultrafiltration or dialysis internal fluid, Add dextran sulfate to 0.2-0.8% and divalent cations to 0.2M, stir until fully dissolved, place for more than 16 hours, collect the precipitate by centrifugation, transfer the supernatant to other uses, dissolve the precipitate with an appropriate concentration of oxalate, and centrifuge Or filter to remove the generated precipitate, supernatant ultrafiltration or dialysis to remove oxalate, sterilize by Pasteur method, sterilize filter, subpackage, and freeze-dry to make powder dosage form.

The positive effects of the present invention are: various components in blood plasma can be comprehensively utilized, the production scale can be expanded arbitrarily, the operation is simple and convenient, advanced equipment such as low-temperature continuous flow centrifuge, ultrafilter, etc. can be applied, and filtration, dialysis, etc. can also be used with less investment. Low-cost equipment can obtain products that meet pharmaceutical standards.

Embodiments of the present invention are as follows: high-density protein uses high-density lipoprotein preparations as ampoules or bottled freeze-dried powders, and the protein content is divided into two types: 30mg/bottle and 60mg/bottle. When used, add 1ml sterile distilled water to dissolve , intramuscular injection or intravenous drip, the dosage and usage are determined by the doctor according to the condition, generally, intramuscular injection can be used, one (ampere) bottle at a time, three times a week, four weeks is a course of treatment), rest for 2-4 days, and start the next course of treatment , a total of three courses of treatment, no need to change eating habits during treatment. Treatment can be continued as necessary.

Production method of the present invention:

(I) Ammonium sulfate method

Frozen human plasma, melted at room temperature, filtered with gauze, collected 40,000ml of plasma, added 40,000ml of sterile distilled water to mix, then added 0.4% sodium citrate, 0.85% sodium chloride and stirred for 30 minutes, then mixed with 80,000ml of saturated ammonium sulfate, stirred , filtered after standing.

Take the supernatant and add 28160g of ammonium sulfate, stir to make it completely dissolved, let it sit and filter, add appropriate amount of water to dissolve the precipitate, dialyze for 72 hours, add D.S99g and calcium chloride 400g to the dialysate to dissolve all, overnight Centrifuge at 4000rpm at 4°C for 20 minutes every day, collect the precipitate, add 2000ml of potassium oxalate until completely dissolved, and then centrifuge for 20 minutes under the same centrifugation conditions as above, collect the supernatant, concentrate it by ultrafiltration, inactivate the virus, and filter it aseptically. Aliquot and lyophilize.

(II) Using ethanol fraction IV (FIV) method

Add ethanol to human plasma to a concentration of 25%, centrifuge to remove sections I, II and III of the combination, add ethanol to the supernatant to reach a concentration of 40%, and precipitate FIV.

Take 100g of FIV, add 1000ml of water, stir to dissolve, centrifuge at 4000rpm, 4°C for 20 minutes, take the supernatant, add 5.5g of D.S, 2.2g of calcium chloride, centrifuge again under the above centrifugal conditions, take the precipitate and add 300ml of 10% potassium oxalate, Until it is completely dissolved, centrifuge as above, collect the supernatant and concentrate it by ultrafiltration, inactivate the virus, sterilize and filter, and freeze-dry in aliquots.

Claims (1)

1, the production method of high density lipoprotein preparation, it is characterized in that: (1) Dilute an appropriate amount of plasma with distilled water, add 0.1-1% sodium citrate, 0.2-1.5% sodium chloride, stir well, add saturated ammonium sulfate solution to 30-80% saturation, stir well, let stand The precipitation is complete, filter with filter cloth, or centrifuge with a continuous flow centrifuge, and transfer the precipitation (primary centrifugation) for other purposes, collect the filtrate (or centrifugal supernatant), add ammonium sulfate to 50%-95% saturation, After the precipitation is complete, filter it with a filter cloth or centrifuge in continuous flow, transfer the supernatant for other purposes, and collect the precipitate (secondary precipitation) for later use. (2) Appropriate amount of plasma was added with ethanol to reach 15-50% concentration according to Cohn’s method, followed by continuous flow centrifugation, and the precipitate was transferred for other use, and the supernatant was added with ethanol to reach 20-60%, the precipitate was collected by continuous flow centrifugation, and the supernatant was transferred for other use , precipitation (FIV) for later use. (3) Take the secondary precipitation of (1) or the FIV of (2), add appropriate amount of water to dissolve, remove residual ammonium sulfate by ultrafiltration or dialysis (1) method or ethanol (2) method, collect ultrafiltration or dialysis internal fluid, Add dextran sulfate to 0.2-0.8% and divalent cations to 0.2M, stir until completely dissolved, let it stand for more than 16 hours, collect the precipitate by centrifugation, transfer the supernatant for other use, dissolve the precipitate with an appropriate concentration of oxalate, and centrifuge Or filter to remove the generated precipitate, supernatant ultrafiltration or dialysis to remove oxalate, sterilize by Pasteur method, sterilize filter, subpackage, and freeze-dry to make powder dosage form.