阅读说明：本技术 一种注射用多西他赛胶束无菌检查方法 (Method for checking sterility of docetaxel micelle for injection ) 是由 张国林 邢以文 薛满 于 2020-04-13 设计创作，主要内容包括：本发明提供了一种注射用多西他赛胶束无菌检查方法,采用薄膜过滤法,培养物包括试验组和对照组,试验组的制备步骤包括：取注射用多西他赛胶束,用0.9%氯化钠溶液溶解后稀释作为供试液,按薄膜过滤法全量过滤至3个滤筒,同法制备6个滤筒,每个滤筒用pH7.0无菌氯化钠-蛋白胨缓冲液冲洗,在最后一次冲洗液中加入试验菌液,在每个滤筒中加入培养基培养,逐日观察。本发明针对注射用多西他赛胶束抑菌特性,确定无菌检查过程注射液原液稀释、薄膜过滤选用器械、过滤后薄膜冲洗过程,阳性对照菌、培养基、缓冲液等,有利于多西他赛胶束无菌检查。(The invention provides a docetaxel micelle sterility test method for injection, which adopts a membrane filtration method, a culture comprises a test group and a control group, and the preparation steps of the test group comprise: taking docetaxel micelle for injection, dissolving the docetaxel micelle with 0.9% sodium chloride solution, diluting the docetaxel micelle to be used as test solution, filtering the docetaxel micelle to 3 filter cylinders according to a membrane filtration method, preparing 6 filter cylinders according to the same method, washing each filter cylinder by sterile sodium chloride-peptone buffer solution with the pH value of 7.0, adding test bacterium liquid into the last washing liquid, adding culture medium into each filter cylinder for culture, and observing the test solution day by day. Aiming at the antibacterial property of docetaxel micelle for injection, the invention determines the dilution of injection stock solution, the selection of instruments for membrane filtration, the membrane washing process after filtration, positive control bacteria, culture medium, buffer solution and the like in the aseptic examination process, and is beneficial to the aseptic examination of docetaxel micelle.)

1. A docetaxel micelle sterility test method for injection adopts a membrane filtration method, and a culture comprises a test group and a control group, and is characterized in that:

taking 15 bottles of docetaxel micelles for injection, wherein each bottle is 500mg, dissolving the docetaxel micelles by using 0.9% sodium chloride solution, diluting the docetaxel micelles to 500m L to be used as test solution, filtering the docetaxel micelles to 3 filter cylinders according to the full amount of a membrane filtration method, preparing 6 filter cylinders by the same method, flushing each filter cylinder by using sterile sodium chloride-peptone buffer solution with the pH value of 7.0 for 3 times, each time for 100m L, adding test bacterium solution into flushing solution of the last time, adding culture medium 100m L into each filter cylinder, culturing, observing the filter cylinders day by day to be used as a test group;

the preparation steps of the control group comprise: the same subsequent operation was carried out after replacing docetaxel micelle test solution for injection with an equivalent amount of 0.9% sterile sodium chloride solution, and the resulting solution was used as a control.

2. The method for examining the sterility of docetaxel micelle for injection according to claim 1, wherein: the filter adopted by the membrane filtration method is a triple NS01-AS3 type totally-enclosed sterile test filter culture device, and the aperture is 0.22 mu m.

3. The method for examining the sterility of docetaxel micelle for injection according to claim 1, wherein: the culture medium is a thioglycollate fluid culture medium or a trypticase soytone liquid culture medium.

4. The method for examining the sterility of docetaxel micelle for injection according to claim 1, wherein: the test bacterial liquid is staphylococcus aureus bacterial liquid, escherichia coli bacterial liquid, bacillus subtilis bacterial liquid, clostridium sporogenes bacterial liquid, candida albicans bacterial liquid and aspergillus niger spore suspension.

5. The method for examining the sterility of docetaxel micelle for injection according to claim 3, wherein: the pH value of the thioglycolate fluid medium at 25 ℃ is 7.1 +/-0.1; the pH value of the trypticase soy peptone liquid medium at 25 ℃ is 7.3 +/-0.1.

6. The method for aseptically examining docetaxel micelle for injection as claimed in claim 4, wherein the preparation method of the Staphylococcus aureus, Escherichia coli and Bacillus subtilis solutions comprises inoculating 1m L of glycerol frozen stock solution of Staphylococcus aureus, Escherichia coli and Bacillus subtilis to tryptone Soy peptone liquid medium, culturing at 32 deg.C for 24h, and diluting with sterilized 0.9% sodium chloride solution in 10-fold gradient to obtain test bacterial solutions with bacterial count <100cfu per 1m L.

7. The method for aseptically examining docetaxel micelle for injection as claimed in claim 4, wherein the Clostridium sporogenes bacterial liquid is prepared by inoculating 1m L frozen glycerol stock solution of Clostridium sporogenes into thioglycollate fluid medium, culturing at 32 ℃ for 24h, diluting with 10-fold gradient of sterilized 0.9% sodium chloride solution, and finally preparing test bacterial liquid containing bacteria number <100cfu per 1m L.

8. The method for aseptically examining docetaxel micelle for injection as claimed in claim 4, wherein the Candida albicans solution is prepared by inoculating 1m L glycerol frozen stock solution of Candida albicans into a glucose saxatilis liquid culture medium, culturing at 23 ℃ for 24h, performing 10-fold gradient dilution with sterilized 0.9% sodium chloride solution, and finally preparing test bacterial solution containing bacteria count <100cfu per 1m L.

9. The method for inspecting the sterility of docetaxel micelle for injection according to claim 4, wherein the Aspergillus niger bacterial liquid is prepared by directly diluting Aspergillus niger spore glycerol frozen stock solution after returning to room temperature, and performing 10-fold gradient dilution with sterilized 0.9% sodium chloride solution to obtain a test bacterial liquid containing <100cfu of spores per 1m L.

10. The method for examining the sterility of docetaxel micelle for injection according to claim 8, wherein: the pH value of the Sabouraud's dextrose liquid culture medium at 25 ℃ is 5.6 +/-0.1.

Technical Field

The invention belongs to the field of medicine inspection, relates to an inspection method of chemicals, and particularly relates to a method for inspecting the sterility of docetaxel micelles for injection.

Background

Docetaxel belongs to a cycle-specific drug in the M phase, is mainly clinically used for treating advanced breast cancer, non-cell lung cancer and ovarian cancer, has pharmacological activity of promoting the polymerization of tubules into microtubules and inhibiting the degradation of the tubules, and plays a role in destroying the network structure of the microtubules mainly by reducing the number of the tubules. The micelle is an ordered assembly formed by molecules of the micelle with polar groups as outer layers and nonpolar groups as inner cores after the concentration of a surfactant solution exceeds a critical concentration, and has the effects of embedding and delivering medicaments. The micelle has the main advantages of good biocompatibility and degradability, easily modified structure and capability of improving the characteristic of poor water solubility of the antitumor drug. The docetaxel micelle for injection is required to be sterile in hygienic index, and although the docetaxel micelle is produced by adopting a sterile production process, microorganisms in a partial dormant state still exist.

The sterility test method is a method for testing the sterility of aseptic drugs, is very important for product process release and supervision after marketing, and part of antitumor drugs, such as daunorubicin, dactinomycin, adriamycin and the like, have antibacterial activity of different degrees. Meanwhile, some substances with antitumor activity also show a certain degree of antibacterial activity, and at present, no paper or patent literature discloses a method for examining the sterility of docetaxel micelles for injection. The docetaxel micelle for injection belongs to a novel drug with strong antitumor activity, and the key point is the determination of the type of a dissolving diluent, the type of a flushing fluid, the flushing frequency, the flushing amount and the selection of positive bacteria when a sterility test method is established, wherein false negative is easily caused when the flushing amount is too large, and false negative is also caused when the flushing amount is not enough and the bacteriostatic activity of the drug cannot be eliminated.

Disclosure of Invention

The technical problem to be solved is as follows: the invention aims to provide a method for inspecting the sterility of docetaxel micelles for injection, aiming at the bacteriostatic property of docetaxel, factors such as sterility inspection process, culture medium, buffer solution and the like are adjusted, and the docetaxel micelle injection can be accurately and reliably inspected for sterility.

The technical scheme is as follows:

a method for inspecting the sterility of docetaxel micelle for injection by membrane filtration method includes such steps as culturing the culture containing test group and control group,

taking 15 bottles of docetaxel micelles for injection, wherein each bottle is 500mg, dissolving the docetaxel micelles by using 0.9% sodium chloride solution, diluting the docetaxel micelles to 500m L to be used as test solution, filtering the docetaxel micelles to 3 filter cylinders according to the full amount of a membrane filtration method, preparing 6 filter cylinders by the same method, flushing each filter cylinder by using sterile sodium chloride-peptone buffer solution with the pH value of 7.0 for 3 times, each time for 100m L, adding test bacterium solution into flushing solution of the last time, adding culture medium 100m L into each filter cylinder, culturing, observing the filter cylinders day by day to be used as a test group;

the preparation steps of the control group comprise: the same subsequent operation was carried out after replacing docetaxel micelle test solution for injection with an equivalent amount of 0.9% sterile sodium chloride solution, and the resulting solution was used as a control.

Furthermore, the filter adopted by the membrane filtration method is a triple NS01-AS3 type totally-enclosed sterile test filtration incubator, and the pore diameter is 0.22 mu m.

Further, the culture medium is a thioglycolate fluid culture medium or a trypticase soytone liquid culture medium.

Further, the test bacterial liquid is staphylococcus aureus bacterial liquid, escherichia coli bacterial liquid, bacillus subtilis bacterial liquid, clostridium sporogenes bacterial liquid, candida albicans bacterial liquid and aspergillus niger spore suspension.

Further, the pH value of the thioglycolate fluid medium at 25 ℃ is 7.1 +/-0.1; the pH value of the trypticase soy peptone liquid medium at 25 ℃ is 7.3 +/-0.1.

Further, the preparation method of the staphylococcus aureus liquid, the escherichia coli liquid and the bacillus subtilis liquid comprises the steps of respectively taking 1m L of glycerol frozen stock solutions of staphylococcus aureus, escherichia coli and bacillus subtilis, inoculating the glycerol frozen stock solutions into tryptone soy peptone liquid culture medium, culturing the tryptone soy peptone liquid culture medium for 24 hours at 32 ℃, and carrying out 10-fold gradient dilution by using a sterilized 0.9% sodium chloride solution to respectively prepare test bacterial liquids with the bacterial number less than 100cfu per 1m L.

Further, the preparation method of the clostridium sporogenes bacterial liquid comprises the steps of inoculating 1m L of glycerol frozen stock solution of clostridium sporogenes into a thioglycollate fluid culture medium, culturing at 32 ℃ for 24h, performing 10-time gradient dilution by using sterilized 0.9% sodium chloride solution, and finally preparing test bacterial liquid containing bacteria with the number of less than 100cfu per 1m L.

Further, the preparation method of the candida albicans bacterial liquid comprises the steps of inoculating 1m L glycerol frozen stock solution of the candida albicans into a glucose liquid culture medium, culturing at 23 ℃ for 24h, performing 10-fold gradient dilution by using a sterilized 0.9% sodium chloride solution, and finally preparing test bacterial liquid containing bacteria number less than 100cfu per 1m L.

Further, the preparation method of the Aspergillus niger bacterial liquid comprises the steps of directly diluting the Aspergillus niger spore glycerol frozen stock solution after the temperature is restored to the room temperature, performing 10-fold gradient dilution by using a sterilized 0.9% sodium chloride solution, and finally preparing the test bacterial liquid containing the spore number of less than 100cfu per 1m L.

Further, the pH value of the Sabouraud's dextrose liquid culture medium at 25 ℃ is 5.6 +/-0.1.

According to the general rule <1105> of the four parts of the edition of Chinese pharmacopoeia 2015, 2 methods for aseptically inspecting docetaxel micelles for injection are established in the invention, and the result shows that each filter cylinder is washed 3 times by using sterile sodium chloride peptone buffer solution with the pH value of 7.0, the antibacterial activity of docetaxel micelles for injection is basically eliminated when the filter cylinder is washed 100m L each time, and staphylococcus aureus can be used as positive bacteria for asepsis inspection.

Has the advantages that:

1. the docetaxel micelles for injection all use staphylococcus aureus as positive control bacteria, each filter cartridge is washed 3 times by sterile sodium chloride-peptone buffer solution with the pH value of 7.0, and the sterile inspection can be carried out at 100m L each time.

2. Aiming at the antibacterial property of docetaxel micelles, the invention determines the dilution of injection stock solution, the selection of instruments for membrane filtration, the membrane washing process after filtration, positive control bacteria, culture medium, buffer solution and the like in the aseptic examination process, and is beneficial to the aseptic examination of docetaxel micelles.

3. The method can simply, effectively and accurately check the docetaxel micelles for injection, and provides a basis for product quality control and qualification rate check.

Detailed Description

1. Laboratory apparatus

The model L A2-4A1 biosafety cabinet was purchased from Esco, Singapore, the KB240 incubator was purchased from Binder, Germany, the Steritest TM Equinox bacteria-collecting instrument was purchased from Merck-Ricoh, Germany, the NS01-AS3 model totally enclosed sterile test filtration incubator was purchased from Beijing cattle GenBank, Inc., and the L ASAI III dust particle counter was purchased from PMS, USA.

2. Test strains and treatment method

Respectively inoculating 1m L glycerol frozen stock solutions of staphylococcus aureus, escherichia coli and bacillus subtilis into a trypticase soytone liquid culture medium, culturing at 32 ℃ for 24 hours for later use, inoculating 1m L glycerol frozen stock solution of clostridium sporogenes into a thioglycolate fluid culture medium, culturing at 32 ℃ for 24 hours for later use, inoculating 1m L candida albicans glycerol frozen stock solution into a glucose liquid culture medium, culturing at 23 ℃ for 24 hours for later use, directly diluting the aspergillus niger spore glycerol frozen stock solution after recovering the room temperature, treating in the above way, diluting the cultured bacterial suspension (or thawed spore suspension) with a sterilized 0.9% sodium chloride solution in a gradient manner by 10 times, and finally preparing test bacterial solutions containing the bacterial number (or spore number) of less than 100cfu per 1m L.

TABLE 1 test strains for sterility test method verification

Bacterial species name	Strain numbering	Using algebra
			Staphylococcus aureus	CMCC（B）26003	3 rd generation culture
Bacillus subtilis	CMCC（B）63501	3 rd generation culture
			Escherichia coli	CMCC（B）44102	3 rd generation culture
Clostridium sporogenes	CMCC（B）64941	3 rd generation culture
			Candida albicans	CMCC（F）98001	3 rd generation culture
Aspergillus niger	CMCC（F）98003	2 nd generation culture

3. Test article operating process

Test group, taking 15 bottles and 500 mg/bottle of the product, dissolving the product by using 0.9% sodium chloride solution, diluting the solution to 500m L to be used as test solution, filtering the solution to 3 filter cylinders according to a membrane filtration method, preparing 6 filter cylinders according to the same method, washing each filter cylinder by using sterile sodium chloride-peptone buffer solution with the pH value of 7.0 for 3 times, each time 100m L, adding 6 test bacteria solutions with the concentration of less than 100cfu into the washing solution of the last time, adding 100m L culture medium into each filter cylinder, culturing and observing the test solution day by day.

Control group: the same subsequent operation was carried out after replacing docetaxel micelle test solution for injection with an equivalent amount of 0.9% sterile sodium chloride solution, and the resulting solution was used as a control.

4. Environmental monitoring

The study monitors items such as temperature and humidity, ventilation times, external pressure difference and dust particle number (the method is referred to GB/T16292-2010) and settlement bacteria (the method is referred to GB/T16294-2010) of an environment verified by a sterility test methodology.

5. Suitability verification

The docetaxel micelles for injection A come from Wuxi BD drug research Co Ltd (specification 500 mg/bottle, batch No. DXTS 20131123), docetaxel micelles for injection B come from Suzhou HTBA biotechnology Co Ltd (specification 500 mg/bottle, batch No. 20141110), membrane filtration is carried out by adopting a triple NS01-AS3 type totally-enclosed sterile test filtration incubator, each filter cartridge is washed for 3 times by pH7.0 sterile sodium chloride peptone buffer solution, the bacteriostatic activity of the drug is basically eliminated after each 100m L treatment, the sterility test can be carried out, and the result is feasible.

5.1 environmental monitoring results

The number of dust particles in the biological safety cabinet is 0/m, the number of the dust particles is more than or equal to 5 microns, and the number of the dust particles in the biological safety cabinet is more than or equal to 0.5 microns³The detection result of the sedimentary bacteria is 0 cfu/vessel, the external pressure difference of the sterile room is 10Pa, the environmental temperature is 23 ℃, the relative humidity is 50 percent, the ventilation times are 41 times/h, and the requirement of methodology verification is met.

5.2 docetaxel micelle sterility test method for injection verification test result

According to the research, each filter cylinder is washed 3 times, after 100m L treatment each time, the bacterial liquid is added into the last washing liquid, the thioglycolate fluid culture medium is added for culture, and 6 verification strains in the docetaxel micelle A for injection and the docetaxel micelle B for injection grow well within 48 hours, namely the docetaxel micelle for injection to be verified has no antibacterial activity or the antibacterial activity can be ignored under the treatment condition, and the table 2 shows.

TABLE 2 docetaxel micelle sterility test results for injection

The invention discloses a method for detecting the activity of docetaxel micelle for injection, wherein the docetaxel micelle for injection is washed by 3 times of sterile sodium chloride buffer solution with the pH value of 100m L each time, and the antibacterial activity of the docetaxel micelle for injection is basically eliminated, so that the docetaxel micelle for injection has stronger antibacterial activity, and the bacterial strain for injection can be used as a selective method for detecting the activity of the docetaxel for injection, which has no effect on staphylococcus aureus, has no effect on the staphylococcus aureus, bacillus subtilis, proteus, and the like, and has no effect on the staphylococcus aureus.