Aseptic test method of amphotericin B liposome for injection

Abstract

The invention is applicable to the technical field of drug detection, and provides a sterility test method of amphotericin B liposome for injection, which comprises the following steps: mixing a casein tryptone liquid culture medium with Tween, ergosterol and ethanol to obtain a culture medium containing a neutralizer, mixing a 0.1% peptone buffer solution with Tween, ergosterol and ethanol to obtain a culture medium containing a neutralizer, and carrying out freeze-drying to obtain the amphotericin B liposome for injection. A buffer solution containing the neutralizer is obtained; redissolving the amphotericin B lipidosome for injection by using sterilized water to obtain a lipidosome redissolved solution, diluting and uniformly mixing; filtering the diluted amphotericin B lipidosome for injection by adopting a membrane filtration method, washing the amphotericin B lipidosome for multiple times by using a buffer solution containing a neutralizing agent, and then adding a culture medium containing the neutralizing agent; and adding a standard test strain subjected to sterility test into the culture medium, and culturing. According to the method, the ergosterol is used as a neutralizing agent, the Tween is used as a dispersing agent, and the ethanol is used as a dissolving agent of the ergosterol, so that the effectiveness of the sterility test method of the amphotericin B liposome for injection is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and in particular relates to a sterility inspection method for amphotericin B liposomes for injection. Background Art

[0002] Amphotericin B liposomes for injection are an antifungal drug mainly used to treat severe systemic fungal infections. Its antibacterial mechanism is that amphotericin B molecules bind to ergosterol on the fungal cell membrane, destroying the permeability of the fungal cell membrane, causing the leakage of potassium ions, nucleotides, amino acids, etc. in the fungal cells, thereby causing the fungal cells to rupture and die.

[0003] The sterility test method is a detection method used to confirm whether products such as medicines and medical devices contain live microorganisms. The Chinese Pharmacopoeia does not include relevant content on "Liposomal Amphotericin B for Injection", but it does include the sterility test method for "Amphotericin B for Injection", which is a membrane filtration method. However, experiments have shown that even if the flushing volume reaches the maximum amount specified in the pharmacopoeia, it cannot be completely reproduced between different laboratories. Even after using this method multiple times, the antibacterial property cannot be completely eliminated. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide a method for sterility testing of liposome amphotericin B for injection, aiming to solve the problems raised in the above background technology.

[0005] The embodiment of the present invention is achieved by a method for sterility testing of liposome amphotericin B for injection, comprising the following steps: Mixing tryptic soy broth with Tween, ergosterol, and ethanol to obtain a medium containing a neutralizer, and mixing 0.1% peptone buffer with Tween, ergosterol, and ethanol to obtain a buffer containing a neutralizer; Redissolve the amphotericin B liposomes for injection in sterile water to obtain a liposome reconstituted solution, and dilute and mix evenly; The diluted amphotericin B liposomes for injection are filtered by membrane filtration in an isolator for sterility testing, and washed several times with a buffer solution containing a neutralizing agent and a 5% sucrose solution, and then a culture medium containing a neutralizing agent is added; A standard test strain for sterility testing is added to the medium containing the neutralizer and culture is performed.

[0006] Preferably, the mass ratio of Tween to trypticase soy broth or 0.1% peptone buffer is 1-2:100, more preferably 1:100.

[0007] Preferably, the mass ratio of ergosterol to trypticase soy broth or 0.1% peptone buffer is 0.01-0.1:100, more preferably 0.01:100.

[0008] Preferably, the volume ratio of the ethanol to the trypticase soy broth or 0.1% peptone buffer is 2:100.

[0009] Preferably, in the step of diluting and mixing uniformly, 5% sucrose solution or sterile water is used for dilution; the volume ratio of the liposome reconstitution solution to 5% sucrose solution or sterile water is 0.136-1.5:1, more preferably 0.136:1.

[0010] Preferably, in the step of performing multiple flushing with a buffer solution containing a neutralizer, the flushing volume of the buffer solution containing a neutralizer is 200-1000 mL.

[0011] Preferably, in the step of performing multiple rinses with a buffer solution containing a neutralizer, a 5% sucrose solution is further added for rinsing, and the rinsing volume of the buffer solution containing a neutralizer and the 5% sucrose solution is 200-1000 mL.

[0012] Preferably, in the step of filtering the diluted amphotericin B liposomes for injection by membrane filtration in an isolator for sterility testing, the rotation speed of the peristaltic pump during filtration is 100-200 rpm, more preferably 160 rpm.

[0013] Preferably, the culture temperature is 20-25°C and the culture time is ≤ 5 days.

[0014] An embodiment of the present invention provides a sterility testing method for amphotericin B liposomes for injection. Ergosterol is used as a neutralizing agent, and the problem of ergosterol dissolution needs to be solved. Ergosterol needs to be dissolved in a suitable medium before being added to a culture medium and a buffer. The higher the ergosterol concentration, the better the neutralization effect of the amphotericin B liposomes. However, the problem of ergosterol precipitation in the buffer and culture medium needs to be solved. Tween is used as a dispersant, and ethanol is used as a solubilizer for ergosterol. This method can maintain the original properties of the culture medium and optimize the culture time of fungi (Candida albicans and Aspergillus niger) in the applicability test of the sterility testing method for amphotericin B liposomes for injection, which cannot grow for more than 5 days, to a culture time of fungi that can be detected within 3 days, thereby ensuring the effectiveness of the sterility testing method for amphotericin B liposomes for injection. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0016] The 2020 edition of the Chinese Pharmacopoeia, Section 1101 Microbial Limit Test for Non-sterile Preparations: Microbial Count Method, recommends the following neutralizers for different products: Serial number Interference Optional neutralizing agents or inactivation methods 1 Glutaraldehyde, mercury preparations Sodium bisulfite 2 Phenols, ethanol, aldehydes, adsorbents Dilution method 3 Aldehydes Glycine 4 Quaternary ammonium compounds, parahydroxybenzoic acid, diguanidine compounds Lecithin 5 Quaternary ammonium compounds, parabens Polysorbate 6 mercury Thioglycolate 7 Mercury, mercury compounds, aldehydes Thiosulfate 8 EDTA, quinolone antibiotics Magnesium or calcium ions 9 Sulfonamides p-Aminobenzoic acid 10 β-lactam antibiotics β-lactamase According to the pharmacology of amphotericin B, it is a polyene antifungal drug with in vitro antibacterial activity against a variety of pathogenic fungi. Based on the types of interferors corresponding to the neutralizers recommended by the pharmacopoeia, it can be inferred that the above neutralizers have no effective neutralizing effect on polyene antibiotics.

[0017] The information related to the culture medium, buffer solution and reagents used in the embodiment of the present invention is shown in Table 1: Table 1 name source Specification Trypticase Soy Broth Beijing Sanyao Technology Development Co., Ltd. 250g / bottle Peptone Beijing Sanyao Technology Development Co., Ltd. 250g / bottle Twain 80 Sinopharm Chemical Reagent Co., Ltd. AR500mL / bottle Ergosterol SIGMA-ALDRICH / MACKLIN 25g / bottle Anhydrous ethanol Sinopharm Chemical Reagent Co., Ltd. AR500mL / bottle Liposomal amphotericin B for injection Jiangxi Aishite Pharmaceutical Co., Ltd. 50mg / bottle In addition, the preparation method of the 0.1% peptone buffer used in the embodiment of the present invention is as follows: 1 g of peptone is uniformly dissolved in 1 L of purified water; The relevant information of the standard test strains (Candida albicans and Aspergillus niger) used in the examples of the present invention is shown in Table 2: Table 2 Strain name Strain number source Candida albicans CMCC(F)98001 China Food and Drug Inspection Institute ATCC 10231 American Type Culture Collection Aspergillus niger CMCC(F)98003 China Food and Drug Inspection Institute ATCC 16404 American Type Culture Collection The specific implementation of the present invention is described in detail below with reference to specific embodiments. Example 1

[0018] A method for sterility testing of liposome amphotericin B for injection comprises the following steps: (1) Mixing a tryptic soytone liquid medium with Tween, ergosterol and ethanol to obtain a medium containing a neutralizer, and mixing a 0.1% peptone buffer with Tween, ergosterol and ethanol to obtain a buffer containing a neutralizer; wherein the mass ratio of Tween to the tryptic soytone liquid medium and the 0.1% peptone buffer is 1:100, the volume ratio of ethanol to the tryptic soytone liquid medium and the 0.1% peptone buffer is 2:100, and the mass ratio of ergosterol to the tryptic soytone liquid medium and the 0.1% peptone buffer is 0.01:100; (2) Redissolve the amphotericin B liposomes for injection in 12 mL of sterile water to obtain a liposome reconstituted solution, dilute it to 100 mL with 5% sucrose solution, and mix well; (3) Filter the diluted amphotericin B liposomes for injection in an isolator for sterility testing using a membrane filtration method at a peristaltic pump speed of 160 rpm, and rinse with 200 mL of buffer containing a neutralizer and 300 mL of a 5% sucrose solution at 100 mL / times, and then add 100 mL of culture medium containing a neutralizer; (4) Add less than 100 cfu of standard test strains for sterility testing (Candida albicans and Aspergillus niger) to the culture medium of step (3) and culture at 20-25°C for no more than 5 days.

[0019] Example 2 is compared with Example 1, except that the mass ratio of ergosterol to trypticase soytone liquid culture medium and 0.1% peptone buffer is adjusted to 0.05:100, respectively.

[0020] Example 3 is compared with Example 1, except that the mass ratio of ergosterol to trypticase soytone liquid culture medium and 0.1% peptone buffer is adjusted to 0.1:100, respectively.

[0021] Example 4: Compared with Example 1, the only difference is that the volume ratio of the liposome reconstitution solution to the 5% sucrose solution is adjusted to 1.5:1.

[0022] Example 5: Compared with Example 1, the only difference is that the rinsing process in step (3) is adjusted to rinsing with 300 mL of buffer solution containing a neutralizer and 200 mL of 5% sucrose solution, 100 mL / time.

[0023] Example 6: Compared with Example 1, the only difference is that the flushing process in step (3) is adjusted to flushing with 1000 mL of buffer solution containing a neutralizing agent, 100 mL / time.

[0024] Comparative Example 1 is compared with Example 1, except that ergosterol is not added in step (1), and Tween 80 is used as a neutralizing agent, and the concentration of Tween 80 is 1%.

[0025] Comparative Example 2: Compared with Example 1, the only difference is that the mass ratio of ergosterol to trypticase soytone liquid culture medium and 0.1% peptone buffer is adjusted to 0.15:100, respectively.

[0026] Comparative Example 3: Compared with Example 1, the only difference is that the volume ratio of the liposome reconstitution solution and the 5% sucrose solution is adjusted to 2:1.

[0027] Comparative Example 4: Compared with Example 1, the only difference is that the flushing process in step (3) is adjusted to flushing with 1000 mL of 0.1% peptone buffer, 100 mL / time.

[0028] Comparative Example 5: Compared with Example 1, the only difference is that the peristaltic pump speed is adjusted to 300 rpm; During the experiment, it was found that the bottom part of the membrane of the bacterial culture vessel (the consumable used when the membrane filtration method was adopted in step (3)) was damaged, resulting in the failure of the experiment.

[0029] After culturing the sample culture medium obtained in Examples 1-6 and Comparative Examples 1-4, the specific growth conditions of the test strains are shown in Table 3 below: Table 3 project Detection time of test strains Example 1 Candida albicans: growth on the third day Aspergillus niger: growth on the third day Example 2 Candida albicans: On the first day of growth, the culture medium is partially turbid. Aspergillus niger: On the first day of growth, the culture medium is partially turbid. Example 3 Candida albicans: On the first day of growth, the culture medium is partially turbid. Aspergillus niger: On the first day of growth, the culture medium is partially turbid. Example 4 Candida albicans: growth on the fifth day Aspergillus niger: growth on the fourth day Example 5 Candida albicans: growth on the third day Aspergillus niger: growth on the third day Example 6 Candida albicans: growth on the fourth day Aspergillus niger: growth on the third day Comparative Example 1 Candida albicans: No bacteria grew on the fifth day Aspergillus niger: No bacteria grew on the fifth day Comparative Example 2 Candida albicans: The culture medium is turbid and the growth of bacteria cannot be observed. Aspergillus niger: The culture medium is turbid and the growth of bacteria cannot be observed. Comparative Example 3 Candida albicans: No bacteria grew on the fifth day Aspergillus niger: No bacteria grew on the fifth day Comparative Example 4 Candida albicans: No bacteria grew on the fifth day Aspergillus niger: No bacteria grew on the fifth day According to Examples 1-3 and Comparative Examples 1-2, it can be seen that when only the neutralizing agent Tween 80 was added, the test strains did not grow on the fifth day, indicating that Tween 80 could not neutralize the antibacterial effect of amphotericin B liposomes for injection; when ergosterol was added, the test strains all grew on the fifth day, indicating that ergosterol could neutralize the antibacterial effect of amphotericin B liposomes for injection; at the same time, as the proportion of ergosterol increased, precipitation gradually occurred, making the culture medium unobservable; According to Examples 1, 4, and Comparative Example 3, it can be seen that the volume ratio of the liposome solution to the 5% sucrose solution is 1.5, and the test strain can still be detected within five days. However, when the volume ratio is increased to 2 in Comparative Example 3, the test strain cannot be detected within five days. According to Examples 1, 5, 6 and Comparative Example 4, it can be seen that when the buffer containing the neutralizing agent ergosterol is used for washing, the test strain can grow well within 5 days. However, in Comparative Example 4 where no neutralizing agent is added, the test strain cannot be detected after 5 days of culture.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for sterility testing of liposome amphotericin B for injection, characterized in that: The following steps are involved: Mixing tryptic soy broth with Tween, ergosterol, and ethanol to obtain a medium containing a neutralizer, and mixing 0.1% peptone buffer with Tween, ergosterol, and ethanol to obtain a buffer containing a neutralizer; Redissolve the amphotericin B liposomes for injection in sterile water to obtain a liposome reconstituted solution, and dilute and mix evenly; The diluted amphotericin B liposomes for injection are filtered by membrane filtration in an isolator for sterility testing, and washed multiple times with a buffer solution containing a neutralizing agent, and then a culture medium containing a neutralizing agent is added; A standard test strain for sterility testing is added to the medium containing the neutralizer and culture is performed.

2. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: The mass ratio of the Tween to the trypticase soytone liquid culture medium or 0.1% peptone buffer is 1-2:

100.

3. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: The mass ratio of the ergosterol to the trypticase soytone liquid culture medium or 0.1% peptone buffer is 0.01-0.1:

100.

4. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: The volume ratio of the ethanol to the trypticase soytone liquid culture medium or 0.1% peptone buffer is 2:

100.

5. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: In the step of diluting and mixing, 5% sucrose solution or sterilized water is used for dilution; the volume ratio of the liposome reconstitution solution to the 5% sucrose solution or sterilized water is 0.136-1.5:

1.

6. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: In the step of performing multiple flushing with the buffer solution containing the neutralizer, the flushing volume of the buffer solution containing the neutralizer is 200-1000 mL.

7. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: In the step of performing multiple rinses with the buffer solution containing a neutralizer, a 5% sucrose solution is also added for rinsing. The rinse volume of the buffer solution containing a neutralizer and the 5% sucrose solution is 200-1000 mL.

8. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: In the step of filtering the diluted amphotericin B liposomes for injection in an isolator for sterility testing using a membrane filtration method, the rotation speed of the peristaltic pump during filtration is 100-200 rpm.

9. The method for sterility inspection of liposome amphotericin B for injection according to claim 1, characterized in that: The culture temperature is 20-25° C. and the culture time is ≤ 5 days.