WO2023142723A1

WO2023142723A1 - Method for fast sterility detection in sterile drug substances

Info

Publication number: WO2023142723A1
Application number: PCT/CN2022/138250
Authority: WO
Inventors: 周玉岩; 付钊; 李聪; 冀亚坤; 安丽康; 李挥; 高燕霞; 刘雪莉; 舒晨景; 任庆瑜
Original assignee: 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心)
Priority date: 2022-01-25
Filing date: 2022-12-10
Publication date: 2023-08-03
Also published as: CN114480555A; CN114480555B

Abstract

The present invention relates to the technical field of sterility detection in sterile drug substances, and in particular to a method for fast sterility detection of sterile drug substances. The method comprises: after 48 hours of culture, aseptically adding 1-10 mL of a culture broth from each of an anaerobic bacterium collector and a fungus or aerobic bacterium collector into an anaerobic bacterium culture flask containing hemolysin or a standard aerobic culture flask in an automatic microorganism culture/detection system, and cultivating in the automatic microorganism culture/detection system for 5 days; and continuing the anaerobic bacterium collector and the fungus or aerobic bacterium collector cultivation for 14 days at 30-35 °C or 20-25 °C. On the basis of no changes in the existing method for sterility detection in sterile drug substances and minimized changes in system applicability, the period for sterile detection is reduced to 7 days.

Description

A method for rapid detection of sterility of sterile raw materials

technical field

The invention belongs to the technical field of sterility detection of sterile raw materials, in particular to a method for rapid detection of sterility of sterile raw materials.

Background technique

Raw materials refer to various powders, crystals, extracts, cells, etc. prepared by chemical synthesis, plant extraction or biotechnology, which are used to produce drugs and cannot be directly used by patients. Sterile APIs refer to APIs that do not contain any active microorganisms, such as molds, bacteria, and viruses, used in pharmaceutical manufacturing. The sterility test method is a method used to check the sterility of pharmaceuticals, biological products, medical devices, raw materials, excipients and other varieties that require sterility in the Pharmacopoeia.

The sterility test method is written in detail under the four general rules or monographs of the 2020 edition of the Chinese Pharmacopoeia. Disadvantages of this method: 1. The cultivation period is long and at least 14 days, which makes it difficult to meet the urgent time requirements of drug emergencies, and at the same time severely limits the efficiency of internal production and testing in the enterprise; It is greatly affected by the subjective factors of the operator; 3. Repeated observation is required during the entire inspection process, the operation is cumbersome, the degree of automation is low, and there is a lack of certain traceability.

Contents of the invention

The purpose of the present invention is to address the above problems and provide a method for rapid detection of the sterility of sterile raw materials. Based on the change of system suitability, the sterility test time was shortened to 7 days.

In order to achieve the above object, the present invention provides the following technical scheme: a method for rapid detection of sterility of sterile raw materials, comprising the following steps:

(1) Sterile raw materials are filtered by membrane filtration and put into two fungal collectors, namely the anaerobic bacteria group and the fungal or aerobic bacteria group. Add 100mL of the corresponding culture medium respectively, and set 30 to 35 for the anaerobic bacteria group. Cultivate at ℃, culture fungi or aerobic bacteria at 20-25℃, and cultivate for 48 hours;

(2) Aseptic operation Take 1-10mL culture solution from the anaerobic bacteria collection device and the fungi or aerobic bacteria collection device respectively, and add them to the hemolysin anaerobic culture bottle in the automatic microbial culture detection system respectively and standard aerobic culture bottle, cultivated in the automatic microbial culture detection system for 5 days;

(3) The anaerobic bacteria collection device in step (2) is kept at 30-35°C, and the fungus or aerobic bacteria collection device is continued to be cultivated at 20-25°C until 14 days;

(4) If the result of the automatic microbial culture detection system is negative, it is determined that the sterility of the sterile API is qualified, and the API can be quickly released for subsequent aseptic subpackage production; if the result of the automatic microbial culture detection system is positive, Or if the observation and test results of the fungal or aerobic bacteria collectors of the anaerobic bacteria group and the fungi are positive, it will be judged as unqualified, and the final product will be subject to quality investigation.

Preferably, the medium added to the anaerobic bacteria group in the step (1) is a thioglycolate fluid medium.

Preferably, the culture medium added by the fungus or aerobic bacteria group in the step (1) is tryptone liquid culture medium.

Preferably, in the step (2), 10 mL of culture solution is added to the fully automatic microbial culture detection system.

Preferably, the culture temperature of the anaerobic bacteria collector in the step (3) is 35°C, and the culture temperature of the fungi or aerobic bacteria collector is 25°C.

Preferably, the automatic microbial culture detection system adopts BACTECTM FX40 automatic system.

Preferably, in the step (4), the automatic microbial culture detection system judges the result by comparing the _CO2 fluorescence value, colorimetric value, and air pressure value.

Preferably, if the CO ₂ fluorescence value is between 0.38 and 0.88 and presents the typical characteristics of a bacterial growth curve, the automatic microbial culture detection system will determine that the result is positive.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. Strong economy and high timeliness. The use of a fully automatic microbial culture detection system shortens the original 14-day detection time to 7 days, which greatly improves the production and detection efficiency of the company's internal product self-inspection and shortens the company's delivery period. For sterile raw materials Production enterprises have strong economical practicability.

2. The system has strong applicability. On the basis of not changing the original method of sterility testing of sterile raw materials and minimizing the change of system applicability, the sterility testing time is shortened to 7 days.

3. High sensitivity and strong automation. Reduce the influence factors of personnel's subjective experience, and can still automatically identify and judge through chemical and optical signals in the case of extremely small amounts of microbial contamination.

Description of drawings

Fig. 1 is the implementation flowchart;

Fig. 2 is the relation figure of time and fluorescence value of Staphylococcus aureus S.a in culture bottle 1;

Fig. 3 is the time and fluorescence value relation figure of Staphylococcus aureus S.a in culture bottle 2;

Fig. 4 is the time and fluorescence value relation diagram of Escherichia coli E.c in culture bottle 1;

Fig. 5 is the time and fluorescence value relation diagram of Escherichia coli E.c in culture bottle 2;

Fig. 6 is the time and fluorescence value relation diagram of Clostridium sporogenes C.s in culture bottle 1;

Fig. 7 is the time and fluorescence value relation diagram of Clostridium sporogenes C.s in culture bottle 2;

Fig. 8 is the time and fluorescence value relation diagram of Bacillus subtilis B.s in culture bottle 1;

Fig. 9 is the relationship diagram between the time and the fluorescence value of Bacillus subtilis B.s in culture bottle 2;

Fig. 10 is the time and fluorescence value relation diagram of Candida albicans C.a in culture bottle 1;

Fig. 11 is the time and fluorescence value relation diagram of Candida albicans C.a in culture bottle 2;

Fig. 12 is the time and fluorescence value relation diagram of Aspergillus niger A.b in culture bottle 1;

Fig. 13 is the time and fluorescence value relation diagram of Aspergillus niger A.b in culture bottle 2;

Fig. 14 is the relationship between time and fluorescence value of QS22110122 batch of ceftriaxone sodium bulk drug after being cultivated in the anaerobic bottle containing hemolysin for 5 days;

Fig. 15 is the relationship between the time and the fluorescence value of the QS22110132 batch of ceftriaxone sodium bulk drug after being cultured in the anaerobic bottle containing hemolysin for 5 days;

Figure 16 is a graph showing the relationship between the time and the fluorescence value of QS22110116 batches of ceftriaxone sodium bulk drug after being cultured in the anaerobic bottle containing hemolysin for 5 days;

Fig. 17 is the time and fluorescence value relation figure of QS22110122 batch ceftriaxone sodium raw material medicine after being cultivated in standard aerobic medium for 5 days;

Fig. 18 is the relationship between the time and the fluorescence value of the QS22110132 batch of ceftriaxone sodium bulk drug after being cultivated in standard aerobic conditions for 5 days;

Figure 19 is a graph showing the relationship between the time and the fluorescence value of the QS22110116 batch of ceftriaxone sodium bulk drug after being cultured in standard aerobic conditions for 5 days.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

Select the ceftriaxone sodium raw material, put it into the bacteria collector after filtering by membrane filtration method, add 100mL of corresponding medium respectively, and then add about 2cfu test bacteria (Staphylococcus aureus S.a, Escherichia coli E.c, Clostridium sporogenes C.s, Bacillus subtilis B.s, Candida albicans C.a, Aspergillus niger A.b) were cultured at 30-35°C or 20-25°C for 48 hours in a fungus collection container equipped with FTM or TSB medium.

Aseptic operation Take 10mL of culture solution from the anaerobic bacteria collection device and the fungi or aerobic bacteria collection device respectively, and add them to the hemolysin anaerobic culture bottle and the standard aerobic culture culture bottle in the automatic microbial culture detection system respectively. bottle, cultured in a fully automatic microbial culture detection system.

The test results and detection time are shown in Table 1:

Table 1 The detection time of each test bacteria in the automatic microbial culture detection system

Using the automatic microbial culture detection system, all the added test bacteria can be detected within 5 days.

Example 2

Select 3 batches of ceftriaxone sodium raw materials, filter them by membrane filtration method, put them into two bacteria collectors respectively as anaerobic bacteria group and fungi or aerobic bacteria group, add 100mL of corresponding culture medium respectively, anaerobic bacteria group Cultivate at 30-35°C, and culture fungi or aerobic bacteria at 20-25°C for 48 hours. Do positive and negative controls at the same time.

Aseptic operation Take 10mL of culture solution from the anaerobic bacteria collection device and the fungi or aerobic bacteria collection device respectively, and add them to the hemolysin anaerobic culture bottle and the standard aerobic culture culture bottle in the automatic microbial culture detection system respectively. Bottles were cultured for 5 days in a fully automatic microbial culture detection system.

After sampling, the bacteria-collectors of the anaerobic bacteria group and fungi or aerobic bacteria-groups were continued to be cultured under the original culture conditions for up to 14 days.

If the result of the automatic microbial culture detection system is negative, it is judged that the sterility of the sterile API is qualified, and the API can be quickly released for subsequent aseptic sub-package production, and continue to observe the bacteria collector and fungi of the anaerobic group. Aerobic bacteria group bacteria collector, observe the test results for aseptic growth, see Table 2, the test results of the 3 batches are qualified.

Table 2 Ceftriaxone Sodium bulk drug sterility test results

As can be seen from Table 2, the present invention shortens the original 14-day sterility detection duration to 7 days without changing the original method of sterility inspection of aseptic raw materials and minimizing the change of system applicability. It has greatly improved the production and testing efficiency of the company's internal product self-inspection, shortened the company's delivery period, and has strong economic practicability for sterile raw material drug manufacturers.

As can be seen from the accompanying drawings 2-13, the positive is a rising S-shaped curve; as can be seen from the accompanying drawings 14-19, the negative is a descending curve.

It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is a relationship between these entities or operations. There is no such actual relationship or order between them. Furthermore, the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus comprising a set of elements includes not only those elements, but also includes elements not expressly listed. other elements of or also include elements inherent in such a process, method, article, or device.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims

A method for rapid detection of sterility of sterile raw materials, characterized in that: comprising the following steps:

(1) Sterile raw materials are filtered by membrane filtration and put into two fungal collectors, namely the anaerobic bacteria group and the fungal or aerobic bacteria group. Add 100mL of the corresponding culture medium respectively, and set 30 to 35 for the anaerobic bacteria group. Cultivate at ℃, culture fungi or aerobic bacteria at 20-25℃, and cultivate for 48 hours;

(2) Aseptic operation Take 1-10mL culture solution from the anaerobic bacteria collection device and the fungi or aerobic bacteria collection device respectively, and add them to the hemolysin anaerobic culture bottle in the automatic microbial culture detection system and standard aerobic culture bottle, cultivated in the automatic microbial culture detection system for 5 days;

(3) The anaerobic bacteria collection device in step (2) is kept at 30-35°C, and the fungus or aerobic bacteria collection device is continued to be cultivated at 20-25°C until 14 days;

(4) If the result of the automatic microbial culture detection system is negative, it is determined that the sterility of the sterile API is qualified, and the API can be quickly released for subsequent aseptic subpackage production; if the result of the automatic microbial culture detection system is positive, Or if the observation and test results of the fungal or aerobic bacteria collectors of the anaerobic bacteria group and the fungi are positive, it will be judged as unqualified, and the final product will be subject to quality investigation.
The method for rapidly detecting the sterility of a sterile bulk drug according to claim 1, wherein the culture medium added by the anaerobic bacteria group in the step (1) is a thioglycolate fluid culture medium.
The method for rapid detection of the sterility of a kind of aseptic raw material drug according to claim 1, is characterized in that, the substratum that fungus or aerobic bacteria group adds in the described step (1) is tryptic soytone liquid culture medium .
The method for rapidly detecting the sterility of a sterile raw material drug according to claim 1, wherein in the step (2), each 10 mL of culture solution is taken and added to a fully automatic microbial culture detection system.
The method for rapidly detecting the sterility of a sterile bulk drug according to claim 1, wherein the culture temperature of the anaerobic bacteria collector in the step (3) is 35° C. The culture temperature of the bacteria collector of the oxygen bacteria group is 25°C.
The method for rapidly detecting the sterility of a sterile bulk drug according to claim 1, wherein the automatic microbial culture detection system adopts the BACTECTM FX40 automatic system.
The method for rapid detection of the sterility of a kind of aseptic raw material drug according to claim 1, is characterized in that, in described step (4), automatic microbial culture detection system is by comparing CO Fluorescent value, colorimetric value, air pressure value to judge the result.
A method for rapidly detecting the sterility of sterile raw materials according to claim 7, wherein the CO2 fluorescence value is between 0.38 and 0.88 and presents typical characteristics of bacterial growth curves, then fully automatic microbial culture The detection system judged the result as positive.