CN103800907A

CN103800907A - Freeze-thawing resistance emulsion platform

Info

Publication number: CN103800907A
Application number: CN201210455782.9A
Authority: CN
Inventors: 邓意辉; 程晓波; 王春玲; 隋月; 付强
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2012-11-14
Filing date: 2012-11-14
Publication date: 2014-05-21

Abstract

本发明是一种耐冻融乳剂平台及其在药物制剂中的应用。本发明所提供的耐冻融乳剂平台适用于不含药乳剂，同样也适用于含药乳剂。由油溶液、乳化剂、防冻剂、抗氧剂和水相组成，油溶液、乳化剂、防冻剂、抗氧剂的用量重量比分别为2%~20%、0.5%~10%、0.5%~20%、0.5%~1.5%，余量为水。药物应占总量的0.001%~5%%用本发明所述耐冻融乳剂平台制备的乳剂能够耐受冻融，避免了乳剂产品在运输、贮存与使用过程中可能碰到的温度条件变化所导致的药品稳定性等问题，确保用药安全的同时也降低了对运输和储藏的条件要求，大大降低了用药成本。The invention is a freeze-thaw resistant emulsion platform and its application in pharmaceutical preparations. The freeze-thaw resistant emulsion platform provided by the present invention is suitable for drug-free emulsions as well as drug-containing emulsions. Composed of oil solution, emulsifier, antifreeze, antioxidant and water phase, the weight ratio of oil solution, emulsifier, antifreeze and antioxidant is 2%~20%, 0.5%~10%, 0.5% respectively ~20%, 0.5%~1.5%, the balance is water. Drugs should account for 0.001%~5% of the total amount. The emulsion prepared by using the freeze-thaw resistant emulsion platform of the present invention can withstand freeze-thaw, avoiding the temperature condition changes that the emulsion product may encounter during transportation, storage and use. The resulting problems such as the stability of the drug ensure the safety of the drug and also reduce the requirements for transportation and storage conditions, greatly reducing the cost of the drug.

Description

A kind of freeze-thawing resistant Emulsion platform

Technical field

The invention belongs to field of pharmaceutical preparations, relate to a kind of freeze-thawing resistant Emulsion platform and the application in pharmaceutical preparation thereof.

Background technology

Emulsion (emulsion) means two kinds of mutual exclusive liquid, and wherein a kind of liquid is dispersed in the heterogeneous dispersion being formed in another kind of liquid with droplet state.Emulsion, as a kind of medicine carrying dosage form, because it has slow release, controlled release, raising targeting, reduces toxicity, reduce zest and improve bioavailability medicine, has become pharmaceutical preparation study hotspot.

Existing multiple Emulsion launch in the world, as fat milk, diazepam (Diaze2muls), propofol (Diprivan), perfluorocarbon (Fluosol), etomidate (Etomidat Lipuro), PGE1 (Liple), compound liposoluble vitamins (Vitalipid), dexamethasone palmitate (Lime2thason), the intravenous injection fatty emulsions such as Alprostadil, they have also obtained splendid sales achievement in benefiting millions upon millions of patients, as the annual sales amount of Alprostadil emulsions can reach 2,000,000,000 RMB.

But the storage and transportation conditions of existing Emulsion is all harsher, need cold chain (being generally 4 ℃-8 ℃), and can not tolerate freeze thawing, because Emulsion can be destroyed in its structure after freezing, occur profit lamination, Emulsion was lost efficacy, so the temperature of Emulsion preservation environment can not be lower than 0 ℃.But the most area winter temperature of China is all below 0 ℃, and some areas are even lower than-30 ℃, and this has improved transportation and the condition of storage of Emulsion product, and then has increased drug cost.Although the storage temperature that freeze-dried emulsion has been avoided can not be lower than problem (Li Yulin, Dong Zhi, Peng Li etc., research [J] .2009 of coenzyme Q10 freeze-dried emulsion, 30 (5): 305-308 of 0 ℃; Alprostadil freeze-dried emulsion and preparation method thereof; 200510028341.0); but not only add very a large amount of freeze drying protectant (can reach weight of oil 2 times) in its prescription; and complex process cost is high; more crucial is; if freeze drying technology will be increased to the medication difficulty of user greatly for breast, does not meet the principle of practicality.So do not have so far the method for good solution Emulsion freeze thawing resistance, limited so largely the extensive use of Emulsion.

Summary of the invention

The serious problems that exist for above-mentioned existing Emulsion---do not tolerate freeze thawing, the object of the invention is to propose a kind of freeze-thawing resistant Emulsion platform and the application in pharmaceutical preparation thereof.

So far, there is no freeze-thawing resistant Emulsion product both at home and abroad.

For realizing object of the present invention, inventor provides following technical scheme:

A kind of freeze-thawing resistant Emulsion platform and the application in pharmaceutical preparation thereof.

Freeze-thawing resistant Emulsion platform provided by the present invention is applicable to not pastille Emulsion, is equally also applicable to pastille Emulsion.A kind of freeze-thawing resistant Emulsion platform, it is characterized in that: be made up of oil solution, emulsifying agent, antifreezing agent, antioxidant and water, the consumption weight ratio of its Chinese medicine, oil solution, emulsifying agent, antifreezing agent, antioxidant is respectively 2% ~ 20%, 0.5% ~ 10%, 0.5% ~ 20%, 0.5% ~ 1.5%, surplus is water.

When freeze-thawing resistant Emulsion platform provided by the present invention is during for pastille freeze-thawing resistant Emulsion platform not, it is made up of oil solution, emulsifying agent, antifreezing agent, antioxidant and water.

In the time that freeze-thawing resistant Emulsion platform provided by the present invention is pastille freeze-thawing resistant Emulsion platform, it is made up of medicine, oil solution, emulsifying agent, antifreezing agent, antioxidant and water.

When freeze-thawing resistant Emulsion platform provided by the present invention is pastille freeze-thawing resistant Emulsion platform, described medicine should comprise all medicines that are prepared into Emulsion.Medicine should account for 0.001% ~ 5% % of total amount

Oil solution in freeze-thawing resistant Emulsion platform of the present invention comprises a kind of or several mixture in MCT Oil (MCT), long chain triglyceride (LCT), medicinal soybean oil, medicinal oil with hydrogenated soybean, refined maize oil, medicinal olive oil, medicinal tea oil, medicinal pine-seed oil, medicinal Oleum Helianthi, medicinal castor oil, medicinal flower oil generation, medicinal almond oil, medicinal Oleum Gossypii semen, medicinal cotmar etc., and its consumption is 2% ~ 20%.

Emulsifying agent in freeze-thawing resistant Emulsion platform of the present invention comprises soybean phospholipid, lecithin, hydrogenated soya phosphatide, hydrolecithin, polysorbas20 (TWEEN-20), tween 21 (TWEEN-21), tell 40 (TWEEN-40), polysorbate60 (TWEEN-60), Tween61 (TWEEN-61), Tween 80 (TWEEN-80), sorbimacrogol oleate100 (TWEEN-81), polysorbate85 (TWEEN-85), Myrij (Myrij), Brij (Brij), medical cane sugar fatty acid ester (SE30, SE40), PLURONICS F87 (F68), poloxamer188 (F127), poloxamer 237(F87), myristyl benzene sulfonic acid sodium salt (ABS), triethanolamine oleate (FM), enuatrol, potassium oleate, 15-hydroxy stearic acid Polyethylene Glycol (HS15), Oleum Ricini gathers hydrocarbon oxygen ester (Cremophor EL), Oleum Ricini gathers hydrocarbon oxygen ester (purification level) (Cremophor ELP), a kind of or several mixture in Cremophor RH 40 (RH40) etc., its consumption is 0.5% ~ 10%.

Antifreezing agent in freeze-thawing resistant Emulsion platform of the present invention comprises a kind of or several mixture in glucose, xylose, fructose, sucrose, maltose, lactose, galactose, trehalose, mannitol, xylitol, maltol, propylene glycol, glycerol, PEG200, PEG300, PEG400, PEG600, PEG1000, PEG2000, PEG4000, PEG6000 etc., and its consumption is 0.5% ~ 20% of prescription total amount.

Antioxidant in freeze-thawing resistant Emulsion platform of the present invention comprises a kind of or several mixture in sulphite, bisulfites, dithiocar-bamate, ascorbic acid, citric acid, malic acid, sorbitol, ascorbic acid cetylate, hydrogen basic note legumin, vitamin E, ethanolamine, fabaceous lecithin, cephalin, propyl gallate, butylated hydroxyarisol (BHA), ditertbutylparacresol (BHT), nordihydroguaiaretic acid (NDGA), EDTA etc., and its consumption is 0.5% ~ 1.5%.

Water in freeze-thawing resistant Emulsion platform of the present invention comprises the one in purified water, distilled water, water for injection, sterilizing purified water, sterilized water for injection etc.As preferred version, the water in described prescription is decided to be purified water and sterilizing purified water.

The benefit that the present invention brings:

Can tolerate freeze thawing with Emulsion prepared by freeze-thawing resistant Emulsion platform of the present invention, avoid the temperature conditions that Emulsion product may be encountered in transportation, storage and use procedure to change the problems such as the stability of drug products causing, when guaranteeing drug safety, also reduce the conditional request to transportation and storage, greatly reduced drug cost.

The specific embodiment

For a more clear understanding of the present invention, the present invention is described in further detail for the embodiment completing according to technical scheme of the present invention providing below in conjunction with inventor.

Be below the embodiment that the inventor provides, the present invention is not limited to these embodiment, and any pro forma accommodation that the present invention is made and/or change all will fall into protection domain of the present invention.

embodiment 1:the ubiquinone of preparing with common emulsifying agent emulsifying separately ₁₀emulsion and freezing-thawing test thereof

	Prescription 1	Prescription 2	Prescription 3	Prescription 4
					Ubiquinone ₁₀	75 mg	75 mg	75 mg	75 mg
Tween-80	600 mg
					RH40		600 mg
HS15			600 mg
					Cremophor EL				600 mg
MCT	3000 mg	3000 mg	3000 mg	3000 mg
					Distilled water	24 ml	24 ml	24 ml	24 ml

Technique:

Take the ubiquinone of recipe quantity ₁₀, Tween-80/RH40/HS15/Cremophor EL, MCT form oil phase; Measure the distilled water of recipe quantity as water, be biphasely heated to respectively 55 ℃.After material in oil phase dissolves completely, under the condition of magnetic agitation, water is slowly added in oil phase, continue to stir 20 min, water for injection is diluted to 30 mL, crosses 0.22 μ m microporous filter membrane, subpackage, inflated with nitrogen, jump a queue, aluminium lid sealing, with 100 ℃, 30 min condition sterilizings, to obtain final product.

The freeze-thaw stability of Emulsion is investigated

According to " chemicals stability study technological guidance principle ", for being easily separated, viscosity reduces, the medicine that precipitates or assemble, need verify the stability in its transportation or use procedure by low temperature or freezing-thawing test.Freezing-thawing test is as a part for influence factor's test, and concrete grammar is as follows:

Get the ubiquinone preparing ₁₀the some bottles of Emulsion, freezing 48 h under-20 ℃ of conditions then place 48 h as a circulation under 40 ℃ of acceleration environments, circulate altogether m time (m>=1, m=3 in this experiment), often complete a circulation and survey its particle diameter.

The results are shown in following table:

Figure 2012104557829100002DEST_PATH_IMAGE001

Freezing-thawing test result shows, this ubiquinone ₁₀emulsion is unable to bear freezing-thawing test, and freeze-thaw stability is poor.

embodiment 2:with common emulsifying agent emulsifying and add ubiquinone prepared by antifreezing agent ₁₀emulsion and freezing-thawing test thereof (antifreezing agent is not commensurability glycerol)

	Prescription 5	Prescription 6	Prescription 7	Prescription 8
					Ubiquinone ₁₀	75 mg	75 mg	75 mg	75 mg
Tween-80	600 mg
					RH40		600 mg
HS15			600 mg
					Cremophor EL				600 mg
Glycerol	150 mg	300 mg	450 mg	600 mg
					MCT	3000 mg	3000 mg	3000 mg	3000 mg
Distilled water	24 ml	24 ml	24 ml	24 ml

Technique:

Take the ubiquinone of recipe quantity ₁₀, Tween-80/RH40/HS15/Cremophor EL, MCT form oil phase; The glycerol that measures recipe quantity is dissolved in distilled water as water, is biphasely heated to respectively 55 ℃.After material in oil phase dissolves completely, under the condition of magnetic agitation, water is slowly added in oil phase, continue to stir 20 min, water for injection is diluted to 30 mL, crosses 0.22 μ m microporous filter membrane, subpackage, inflated with nitrogen, jump a queue, aluminium lid sealing, with 100 ℃, 30 min condition sterilizings, to obtain final product.

The freeze-thaw stability of Emulsion is investigated

The results are shown in following table:

Freezing-thawing test result shows, this ubiquinone ₁₀emulsion holds out against freezing-thawing test, and freeze-thaw stability is good.

Use xylitol, mannitol, glucose, trehalose, sucrose, propylene glycol, PEG200, PEG300, PEG400, the PEG600 of same amount to replace glycerol to obtain basically identical experimental result, can tolerate freezing-thawing test.

embodiment 3:cucurbitacin Emulsion and the freezing-thawing test thereof prepared with common emulsifying agent emulsifying separately

	Prescription 9	Prescription 10	Prescription 11	Prescription 12
					Cucurbitacin	15 mg	15 mg	15 mg	15 mg
Tween-80	600 mg
					RH40		600 mg
HS15			600 mg
					Cremophor EL				600 mg
MCT	3000 mg	3000 mg	3000 mg	3000 mg
					Distilled water	24 ml	24 ml	24 ml	24 ml

Technique:

The cucurbitacin, Tween-80/RH40/HS15/Cremophor EL, the MCT that take recipe quantity form oil phase; Measure the distilled water of recipe quantity as water, be biphasely heated to respectively 55 ℃.After material in oil phase dissolves completely, under the condition of magnetic agitation, water is slowly added in oil phase, continue to stir 20 min, water for injection is diluted to 30 mL, crosses 0.22 μ m microporous filter membrane, subpackage, inflated with nitrogen, jump a queue, aluminium lid sealing, with 100 ℃, 30 min condition sterilizings, to obtain final product.

The freeze-thaw stability of Emulsion is investigated

Get the some bottles of the cucurbitacin Emulsion preparing, freezing 48 h under-20 ℃ of conditions then place 48 h as a circulation under 40 ℃ of acceleration environments, circulate altogether m time (m >=1, m=3 in this experiment), often complete a circulation and survey its particle diameter.

The results are shown in following table:

Figure 2012104557829100002DEST_PATH_IMAGE003

Freezing-thawing test result shows, this cucurbitacin Emulsion is unable to bear freezing-thawing test, and freeze-thaw stability is poor.

embodiment 4:take common emulsifying agent emulsifying and add cucurbitacin Emulsion and freezing-thawing test (antifreezing agent is as not commensurability sucrose) thereof prepared by antifreezing agent

	Prescription 13	Prescription 14	Prescription 15	Prescription 16
					Cucurbitacin	15 mg	15 mg	15 mg	15 mg
Tween-80	600 mg
					RH40		600 mg
HS15			600 mg
					Cremophor EL				600 mg
Sucrose	150 mg	450 mg	750 mg	900 mg
					MCT	3000 mg	3000 mg	3000 mg	3000 mg
Distilled water	24 ml	24 ml	24 ml	24 ml

Technique:

The cucurbitacin, Tween-80/RH40/HS15/Cremophor EL, the MCT that take recipe quantity form oil phase; The sucrose that measures recipe quantity is dissolved in distilled water as water, is biphasely heated to respectively 55 ℃.After material in oil phase dissolves completely, under the condition of magnetic agitation, water is slowly added in oil phase, continue to stir 20 min, water for injection is diluted to 30 mL, crosses 0.22 μ m microporous filter membrane, subpackage, inflated with nitrogen, jump a queue, aluminium lid sealing, with 100 ℃, 30 min condition sterilizings, to obtain final product.

The freeze-thaw stability of Emulsion is investigated

The results are shown in following table:

Freezing-thawing test result shows, this cucurbitacin Emulsion holds out against freezing-thawing test, and freeze-thaw stability is good.

Use xylitol, mannitol, glucose, trehalose, glycerol, propylene glycol, PEG200, PEG300, PEG400, the PEG600 of same amount to replace sucrose to obtain basically identical experimental result, can tolerate freezing-thawing test.

embodiment 5:vitamin K1 Emulsion and the freezing-thawing test thereof prepared with common emulsifying agent emulsifying separately

	Prescription 17	Prescription 18	Prescription 19	Prescription 20
					Vitamin K1	60 mg	60 mg	60 mg	60 mg
Tween-80	600 mg
					RH40		600 mg
HS15			600 mg
					Cremophor EL				600 mg
LCT	3000 mg	3000 mg	3000 mg	3000 mg
					Distilled water	24 ml	24 ml	24 ml	24 ml

Technique:

The vitamin K1, Tween-80/RH40/HS15/Cremophor EL, the LCT that take recipe quantity form oil phase; Measure the distilled water of recipe quantity as water, be biphasely heated to respectively 55 ℃.After material in oil phase dissolves completely, under the condition of magnetic agitation, water is slowly added in oil phase, continue to stir 20 min, water for injection is diluted to 30 mL, crosses 0.22 μ m microporous filter membrane, subpackage, inflated with nitrogen, jump a queue, aluminium lid sealing, with 100 ℃, 30 min condition sterilizings, to obtain final product.

The freeze-thaw stability of Emulsion is investigated

Get the some bottles of the vitamin K1 Emulsion preparing, freezing 48 h under-20 ℃ of conditions then place 48 h as a circulation under 40 ℃ of acceleration environments, circulate altogether m time (m >=1, m=3 in this experiment), often complete a circulation and survey its particle diameter.

The results are shown in following table:

Figure 2012104557829100002DEST_PATH_IMAGE005

Freezing-thawing test result shows, this vitamin K1 Emulsion is unable to bear freezing-thawing test, and freeze-thaw stability is poor.

embodiment 6:take common emulsifying agent emulsifying and add vitamin K1 Emulsion and freezing-thawing test (antifreezing agent is as not commensurability propylene glycol) thereof prepared by antifreezing agent

	Prescription 21	Prescription 22	Prescription 23	Prescription 24
					Vitamin K1	60 mg	60 mg	60 mg	60 mg
Tween-80	600 mg
					RH40		600 mg
HS15			600 mg
					Cremophor EL				600 mg
Propylene glycol	150 mg	450 mg	750 mg	900 mg
					LCT	3000 mg	3000 mg	3000 mg	3000 mg
Distilled water	24 ml	24 ml	24 ml	24 ml

Technique:

The vitamin K1, Tween-80/RH40/HS15/Cremophor EL, the LCT that take recipe quantity form oil phase; The propylene glycol that measures recipe quantity is dissolved in distilled water as water, is biphasely heated to respectively 55 ℃.After material in oil phase dissolves completely, under the condition of magnetic agitation, water is slowly added in oil phase, continue to stir 20 min, water for injection is diluted to 30 mL, crosses 0.22 μ m microporous filter membrane, subpackage, inflated with nitrogen, jump a queue, aluminium lid sealing, with 100 ℃, 30 min condition sterilizings, to obtain final product.

The freeze-thaw stability of Emulsion is investigated

The results are shown in following table:

Freezing-thawing test result shows, this vitamin K1 Emulsion holds out against freezing-thawing test, and freeze-thaw stability is good.

Use xylitol, mannitol, glucose, trehalose, glycerol, sucrose, PEG200, PEG300, PEG400, the PEG600 of same amount to replace propylene glycol to obtain basically identical experimental result, can tolerate freezing-thawing test.

embodiment 7:the preparation of freeze-thawing resistant fat milk and freezing-thawing test thereof

	Prescription 25	Prescription 26	Prescription 27	Prescription 28
					SPC	600 mg	300 mg	300 mg	300 mg
RH40		600 mg
					HS15			600 mg
Cremophor EL				600 mg
					Glycerol	600mg	600 mg	600 mg	600 mg
MCT	3000 mg	3000 mg	3000 mg	3000 mg
					Distilled water	24 ml	24 ml	24 ml	24 ml

Technique:

Take SPC/RH40/HS15/Cremophor EL, the MCT composition oil phase of recipe quantity; The glycerol that measures recipe quantity is dissolved in distilled water as water, is biphasely heated to respectively 55 ℃.After material in oil phase dissolves completely, under the condition of magnetic agitation, water is slowly added in oil phase, continue to stir 20 min, water for injection is diluted to 30 mL, crosses 0.22 μ m microporous filter membrane, subpackage, inflated with nitrogen, jump a queue, aluminium lid sealing, with 100 ℃, 30 min condition sterilizings, to obtain final product.

The freeze-thaw stability of Emulsion is investigated

Get the some bottles of the Emulsion preparing, freezing 48 h under-20 ℃ of conditions then place 48 h as a circulation under 40 ℃ of acceleration environments, circulate altogether m time (m >=1, m=3 in this experiment), often complete a circulation and survey its particle diameter.

The results are shown in following table:

Figure 2012104557829100002DEST_PATH_IMAGE007

Freezing-thawing test result shows, this Emulsion holds out against freezing-thawing test, and freeze-thaw stability is good.

Use xylitol, mannitol, glucose, trehalose, propylene glycol, sucrose, PEG200, PEG300, PEG400, the PEG600 of same amount to replace glycerol to obtain basically identical experimental result, can tolerate freezing-thawing test.

Claims

1. A freeze-thaw resistant emulsion platform is characterized in that: it is made up of oil solution, emulsifier, antifreeze, antioxidant and water phase, and the consumption weight ratio of oil solution, emulsifier, antifreeze, antioxidant is respectively 2%~20%, 0.5%~10%, 0.5%~20%, 0.5%~1.5%, the balance is water.

2. The freeze-thaw resistant emulsion platform according to claim 1, characterized in that: all drugs that can be prepared into emulsions can be added to the freeze-thaw resistant emulsion platform, and the drugs should account for 0.001%~5% of the total amount.

3. The freeze-thaw resistant emulsion platform according to claims 1 and 2, wherein the oil solution includes medium-chain fatty acid triglycerides, long-chain triglycerides, medicinal soybean oil, medicinal hydrogenated soybean oil, refined corn oil, medicinal olive oil, medicinal tea oil, medicinal pine nut oil, medicinal sunflower oil, medicinal castor oil, medicinal peanut oil, medicinal almond oil, medicinal cottonseed oil, medicinal hydrogenated cottonseed oil species or a mixture of several species.

4. The freeze-thaw resistant emulsion platform according to claim 1 and 2, wherein the emulsifier includes soybean lecithin, lecithin, hydrogenated soybean lecithin, hydrogenated lecithin, Tween 20, Tween 21, Tween 40, Tween Wen 60, Tween 61, Tween 80, Tween 81, Tween 85, Maize, Benze, Medicinal Sucrose Fatty Acid Ester, Poloxamer 188, Poloxamer 407, Poloxamer 237, Sodium Tetradecylbenzene Sulfonate, Triethanolamine Oleate, Sodium Oleate, Potassium Oleate, Polyethylene Glycol 15-Hydroxystearate, Castor Oil Polyoxylate, Castor Oil Polyoxylate, Polyoxyethylene One or a mixture of hydrogenated castor oil.

5. The freeze-thaw resistant emulsion platform according to claim 1 and 2, wherein the antifreeze agent comprises glucose, xylose, fructose, sucrose, maltose, lactose, galactose, trehalose, mannitol, xylitol, One or a mixture of maltitol, propylene glycol, glycerin, PEG200, PEG300, PEG400, PEG600, PEG1000, PEG2000, PEG4000, PEG6000.

6. freeze-thaw resistant emulsion platform as claimed in claim 1, is characterized in that: wherein antioxidant comprises: sulfite, bisulfite, dithiocarbamate, ascorbic acid, citric acid, malic acid , sorbitol, ascorbic acid palmitate, hydrocoumarin, vitamin E, ethanolamine, soybean lecithin, cephalin, propyl gallate, tert-butyl p-hydroxyanisole, di-tert-butyl p-cresol, One or a mixture of nordihydroguaiaretic acid and EDTA.

7. The freeze-thaw resistant emulsion platform according to claim 1, wherein the water phase comprises one of purified water, distilled water, water for injection, sterilized purified water, and sterilized water for injection.

8. A method for preparing the freeze-thaw resistant emulsion platform as claimed in claim 1, characterized in that: take the recipe amount of coenzyme Q ₁₀ , Tween-80/RH40/HS15/Cremophor EL, MCT to form the oil phase; Take the prescribed amount of distilled water as the water phase, and heat the two phases to 55°C respectively. After the substances in the oil phase are completely dissolved, slowly add the water phase into the oil phase under magnetic stirring, continue stirring for 20 min, and dilute with water for injection. to 30 mL, pass through a 0.22 μm microporous membrane, aliquot, fill with nitrogen, stopper, seal, and sterilize at 100°C for 30 min.