A sterility test method for amphotericin B liposomes for injection

Abstract

This invention relates to the field of pharmaceutical testing technology and provides a method for sterility testing of amphotericin B liposomes for injection, comprising the following steps: mixing tryptic soy peptone liquid culture medium with Tween, ergosterol, and ethanol to obtain a culture medium containing a neutralizing agent; mixing 0.1% peptone buffer with Tween, ergosterol, and ethanol to obtain a buffer solution containing a neutralizing agent; reconstituteing the amphotericin B liposomes for injection with sterile water to obtain a liposome reconstituted solution, and diluting and mixing it evenly; filtering the diluted amphotericin B liposomes for injection using a membrane filtration method, rinsing repeatedly with the buffer solution containing the neutralizing agent, and then adding the culture medium containing the neutralizing agent; adding a standard test strain for sterility testing to the culture medium and culturing. This invention uses ergosterol as a neutralizing agent, Tween as a dispersant, and ethanol as a solvent for ergosterol, ensuring the effectiveness of the sterility testing method for amphotericin B liposomes for injection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology, and in particular relates to a method for sterility testing of injectable amphotericin B liposomes. Background Technology

[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, disrupting the permeability of the fungal cell membrane, leading to the leakage of potassium ions, nucleotides, amino acids, etc. from the fungal cell, thereby causing the fungal cell to rupture and die.

[0003] The sterility test method is used to confirm whether products such as drugs and medical devices contain live microorganisms. The Chinese Pharmacopoeia does not include content related to "Amphotericin B Liposome for Injection", but it does include a sterility test method for "Amphotericin B for Injection", which is a membrane filtration method. However, experiments have shown that even if the number of rinses reaches the maximum amount specified in the pharmacopoeia, it cannot be completely reproduced between different laboratories. Even after multiple uses of this method, the antibacterial properties still cannot be completely eliminated. Summary of the Invention

[0004] The purpose of this invention is to provide a method for sterility testing of amphotericin B liposomes for injection, aiming to solve the problems mentioned in the background art.

[0005] The present invention is implemented as follows: a method for sterility testing of amphotericin B liposomes for injection includes the following steps:

[0006] Tryptic soybean liquid medium was mixed with Tween, ergosterol and ethanol to obtain a medium containing a neutralizing agent. 0.1% peptone buffer was mixed with Tween, ergosterol and ethanol to obtain a buffer containing a neutralizing agent.

[0007] The amphotericin B liposomes for injection were reconstituted with sterile water to obtain a liposome reconstituted solution, which was then diluted and mixed thoroughly.

[0008] The diluted amphotericin B liposomes for injection were filtered in a sterile testing isolator using a membrane filtration method, and then rinsed multiple times with a buffer solution containing a neutralizing agent and a 5% sucrose solution before adding culture medium containing a neutralizing agent.

[0009] Add the standard test strain for sterility testing to the culture medium containing the neutralizing agent and culture it.

[0010] Preferably, the mass ratio of Tween to tryptic soy peptone liquid culture medium or 0.1% peptone buffer is 1-2:100, more preferably 1:100.

[0011] Preferably, the mass ratio of ergosterol to tryptic soybean liquid culture medium or 0.1% peptone buffer is 0.01-0.1:100, more preferably 0.01:100.

[0012] Preferably, the volume ratio of ethanol to tryptic soy peptone liquid culture medium or 0.1% peptone buffer is 2:100.

[0013] Preferably, in the step of dilution and uniform mixing, a 5% sucrose solution or sterile water is used for dilution; the volume ratio of the liposome reconstitution solution to the 5% sucrose solution or sterile water is 0.136-1.5:1, more preferably 0.136:1.

[0014] Preferably, in the step of rinsing multiple times with a buffer solution containing a neutralizing agent, the rinsing volume of the buffer solution containing the neutralizing agent is 200-1000 mL.

[0015] Preferably, in the step of rinsing multiple times with a buffer solution containing a neutralizing agent, a 5% sucrose solution is also added for rinsing, and the rinsing volume of the buffer solution containing the neutralizing agent and the 5% sucrose solution is 200-1000 mL.

[0016] Preferably, in the step of filtering the diluted amphotericin B liposomes for injection in a sterile testing isolator using a membrane filtration method, the rotation speed of the peristaltic pump during filtration is 100-200 rpm, more preferably 160 rpm.

[0017] Preferably, the culture temperature is 20-25℃ and the time is ≤5 days.

[0018] This invention provides a sterility testing method for amphotericin B liposomes for injection, using ergosterol as a neutralizing agent. The solution involves addressing the solubility of ergosterol, requiring the use of a suitable medium to dissolve it before adding it to the culture medium and buffer solution. Higher ergosterol concentrations result in better neutralization of amphotericin B liposomes. However, the issue of ergosterol precipitation in the buffer and culture medium must also be addressed. Tween is used as a dispersant, and ethanol as a solubilizer for ergosterol. This method maintains the original properties of the culture medium and optimizes the detection of fungi (Candida albicans and Aspergillus niger) in the sterility detection method applicability test of amphotericin B liposomes for injection, which previously failed to grow for more than 5 days, to detectable within 3 days, thus ensuring the effectiveness of the sterility testing method for amphotericin B liposomes for injection. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] In the 2020 edition of the Chinese Pharmacopoeia, section 1101, Microbial Limit Tests for Non-Sterile Preparations: Microbial Counting Method, the following recommendations are made for neutralizing agents for different products:

[0021] 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, p-hydroxybenzoic acid, bipolar compounds Lecithin 5 Quaternary ammonium compounds, benzoic acid, p-hydroxybenzoic acid Polysorbate 6 mercury mercaptoacetate 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

[0022] 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 interfering agents corresponding to the neutralizing agents recommended in the pharmacopoeia, it can be inferred that the above neutralizing agents have no effective neutralizing effect on polyene antibiotics.

[0023] Information regarding the culture medium, buffer solution, and reagents used in the embodiments of this invention is shown in Table 1:

[0024] Table 1

[0025] name source Specification Tryptic soy peptone liquid culture medium 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 Amphotericin B Liposome for Injection Jiangxi Aishite Pharmaceutical Co., Ltd. 50mg / bottle

[0026] In addition, the 0.1% peptone buffer solution used in this embodiment of the invention is prepared by dissolving 1g of peptone in 1L of purified water evenly.

[0027] The relevant information of the standard test strains (Candida albicans and Aspergillus niger) used in the embodiments of this invention is shown in Table 2:

[0028] Table 2

[0029] strain name strain number source Candida albicans CMCC(F)98001 China National Institutes for Food and Drug Control ATCC 10231 American Culture Collection Aspergillus niger CMCC(F)98003 China National Institutes for Food and Drug Control ATCC 16404 American Culture Collection

[0030] The specific implementation of the present invention will be described in detail below with reference to specific embodiments. Example 1

[0031] A method for sterility testing of injectable amphotericin B liposomes includes the following steps:

[0032] (1) Mix tryptic soy broth with Tween, ergosterol and ethanol to obtain a medium containing a neutralizing agent. Mix 0.1% peptone buffer with Tween, ergosterol and ethanol to obtain a buffer containing a neutralizing agent. The mass ratio of Tween to tryptic soy broth and 0.1% peptone buffer is 1:100, the volume ratio of ethanol to tryptic soy broth and 0.1% peptone buffer is 2:100, and the mass ratio of ergosterol to tryptic soy broth and 0.1% peptone buffer is 0.01:100.

[0033] (2) The liposomes for injection were reconstituted with 12 mL of sterile water to obtain a liposome reconstituted solution, and then diluted with 5% sucrose solution to 100 mL and mixed evenly.

[0034] (3) The diluted amphotericin B liposomes for injection were filtered in a sterile test isolator using a membrane filtration method with a peristaltic pump speed of 160 rpm. The liposomes were rinsed with 200 mL of buffer solution containing neutralizing agent and 300 mL of 5% sucrose solution, 100 mL each time. Then, 100 mL of culture medium containing neutralizing agent was added.

[0035] (4) Add less than 100 cfu of standard test strains (Candida albicans and Aspergillus niger) for sterility testing to the culture medium in step (3) and incubate at 20-25°C for no more than 5 days.

[0036] Example 2, compared with Example 1, differs only in that the mass ratio of ergosterol to tryptic soybean liquid culture medium and 0.1% peptone buffer is adjusted to 0.05:100.

[0037] Example 3, compared with Example 1, differs only in that the mass ratio of ergosterol to tryptic soybean liquid culture medium and 0.1% peptone buffer is adjusted to 0.1:100.

[0038] Example 4: Compared with Example 1, the only difference is that the volume ratio of the liposome reconstituted solution to the 5% sucrose solution is adjusted to 1.5:1.

[0039] Example 5: Compared with Example 1, the only difference is that the rinsing process in step (3) is adjusted to rinse with 300 mL of buffer solution containing neutralizing agent and 200 mL of 5% sucrose solution, 100 mL / time.

[0040] Example 6: Compared with Example 1, the only difference is that the rinsing process in step (3) is adjusted to rinse with 1000 mL of buffer solution containing neutralizing agent, 100 mL / time.

[0041] Comparative Example 1: Compared with Example 1, the only difference is that ergosterol was not added in step (1), and Tween 80 was used as a neutralizing agent with a concentration of 1%.

[0042] Comparative Example 2, compared with Example 1, differed only in that the mass ratio of ergosterol to tryptic soybean liquid culture medium and 0.1% peptone buffer was adjusted to 0.15:100.

[0043] Comparative Example 3, compared with Example 1, differs only in that the volume ratio of liposome reconstitution solution to 5% sucrose solution is adjusted to 2:1.

[0044] Comparative Example 4: Compared with Example 1, the only difference is that the rinsing process in step (3) is adjusted to rinse with 1000 mL of 0.1% peptone buffer, 100 mL / time.

[0045] Comparative Example 5: Compared with Example 1, the only difference is that the peristaltic pump speed is adjusted to 300 rpm;

[0046] During the experiment, it was found that the membrane at the bottom of the bacterial culture device (the consumable used when the membrane filtration method is adopted in step (3)) was damaged, which led to the failure of the experiment.

[0047] After culturing the culture media obtained from Examples 1-6 and Comparative Examples 1-4, the growth of the specific test strains is shown in Table 3 below:

[0048] Table 3

[0049] project Detection time of test strains Example 1 Candida albicans: fungus grows on the third day; Aspergillus niger: fungus grows on the third day. Example 2 Candida albicans: Day 1 growth, culture medium partially cloudy; Aspergillus niger: Day 1 growth, culture medium partially cloudy. Example 3 Candida albicans: Day 1 growth, culture medium partially cloudy; Aspergillus niger: Day 1 growth, culture medium partially cloudy. Example 4 Candida albicans: fungus develops on the fifth day; Aspergillus niger: fungus develops on the fourth day. Example 5 Candida albicans: fungus grows on the third day; Aspergillus niger: fungus grows on the third day. Example 6 Candida albicans: fungus develops on the fourth day; Aspergillus niger: fungus develops on the third day. Comparative Example 1 Candida albicans: No fungal growth on day 5; Aspergillus niger: No fungal growth on day 5 Comparative Example 2 Candida albicans: Turbid culture medium makes it impossible to observe bacterial growth. Aspergillus niger: Turbid culture medium makes it impossible to observe bacterial growth. Comparative Example 3 Candida albicans: No fungal growth on day 5; Aspergillus niger: No fungal growth on day 5 Comparative Example 4 Candida albicans: No fungal growth on day 5; Aspergillus niger: No fungal growth on day 5

[0050] As can be seen from Examples 1-3 and Comparative Examples 1-2, when only the neutralizing agent Tween 80 was added, none of the test strains grew on the fifth day, indicating that Tween 80 could not neutralize the antibacterial effect of injectable amphotericin B liposomes. When ergosterol was added, all the test strains grew on the fifth day, indicating that ergosterol could neutralize the antibacterial effect of injectable amphotericin B liposomes. At the same time, as the proportion of ergosterol increased, precipitation gradually occurred, making the culture medium unobservable.

[0051] As can be seen from Examples 1 and 4 and Comparative Example 3, when the volume ratio of liposome solution to 5% sucrose solution is 1.5, the test strains can still be detected within five days. However, when the volume ratio is increased to 2 in Comparative Example 3, the test strains cannot be detected within five days.

[0052] As can be seen from Examples 1, 5, 6 and Comparative Example 4, when the test strains were washed with a buffer solution containing the neutralizing agent ergosterol, they could grow well within 5 days. However, in Comparative Example 4, which did not contain a neutralizing agent, the test strains could not be detected after 5 days of culture.

[0053] 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 within the protection scope of the present invention.

Claims

1. A method for sterility testing of amphotericin B liposome for injection, characterized by, The method comprises the following steps: Mixing the pancreas tryptose soy broth liquid medium with Tween, ergosterol and ethanol to obtain a medium containing a neutralizing agent, and mixing the 0.1% peptone buffer with Tween, ergosterol and ethanol to obtain a buffer containing a neutralizing agent; Resolving the amphotericin B liposome for injection with sterile water to obtain a liposome resolution solution, and diluting and mixing uniformly; Filtering the diluted amphotericin B liposome for injection in a sterile test isolator by using a membrane filtration method, and washing multiple times with the buffer containing the neutralizing agent, and then adding the medium containing the neutralizing agent; Adding standard test strains for sterile inspection to the medium containing the neutralizing agent, and culturing; In the step of diluting and mixing uniformly, 5% sucrose solution is used for dilution; the volume ratio of the liposome resolution solution to the 5% sucrose solution is 0.136-1.5:

1.

2. The amphotericin B liposome for injection sterility test method according to claim 1, characterized by, The mass ratio of the Tween to the pancreas tryptose soy broth liquid medium or the 0.1% peptone buffer is 1-2:

100.

3. The amphotericin B liposome for injection sterility test method according to claim 1, characterized by, The mass ratio of the ergosterol to the pancreas tryptose soy broth liquid medium or the 0.1% peptone buffer is 0.01-0.1:

100.

4. The amphotericin B liposome for injection sterility test method according to claim 1, characterized by, The volume ratio of the ethanol to the pancreas tryptose soy broth liquid medium or the 0.1% peptone buffer is 2:

100.

5. The amphotericin B liposome for injection sterility test method according to claim 1, characterized by, In the step of washing multiple times with the buffer containing the neutralizing agent, the washing amount of the buffer containing the neutralizing agent is 200-1000 mL.

6. The amphotericin B liposome for injection sterility test method according to claim 1, characterized by, In the step of washing multiple times with the buffer containing the neutralizing agent, 5% sucrose solution is also added for washing, and the washing amount of the buffer containing the neutralizing agent and the 5% sucrose solution is 200-1000 mL.

7. The amphotericin B liposome for injection sterility test method according to claim 1, characterized by, In the step of filtering the diluted amphotericin B liposome for injection in the sterile test isolator by using the membrane filtration method, the rotation speed of the peristaltic pump during filtering is 100-200 rpm.

8. The amphotericin B liposome for injection sterility test method according to claim 1, characterized by, The culture temperature is 20-25°C, and the culture time is ≤5 days.