CN114958956A - Sterility inspection method for biological product containing mercuride - Google Patents

Abstract

The invention provides a sterility inspection method of a mercuride-containing biological product, which comprises the following steps: (1) preparing a flushing liquid: fully dissolving sodium thiosulfate in 0.1% peptone water solution to prepare 0.1% peptone water solution containing sodium thiosulfate; (2) membrane filtration and washing: wetting the filter membrane by using the flushing liquid obtained in the step (1), performing membrane filtration on the biological product containing the mercuride, soaking the filter membrane for a period of time by using a proper amount of flushing liquid, filtering, and then flushing by using the flushing liquid in multiple times; (3) and (4) checking: the examination was carried out according to the general 1101 sterility test method of the four pharmacopoeias of China in 2020 edition. The invention establishes a novel sterility inspection method for biological products containing mercurides. Experiments prove that the sterility test according to the invention meets the regulations, so the method is feasible, has reliable results, and can obtain the sterility test result of the biological product containing the mercuride more quickly, effectively and accurately.

Description

Sterility inspection method for biological product containing mercuride

Technical Field

The invention belongs to the field of drug detection, and particularly relates to a sterility test method for biological products, in particular to a sterility test method for biological products containing mercuride.

Background

The sterility test is a key link of biological product quality verification, whether microbial contamination is a basic condition for judging whether the sterility test is qualified or not is an important barrier for guaranteeing the safety of biological products and the benefits of pharmaceutical enterprises, and therefore, establishing a perfect and effective sterility test scheme is of great importance for evaluating the sterility condition of a biological product production link.

Thimerosal is a preservative widely used in biological products such as vaccines and the like, and mercury ions are combined with sulfydryl of enzyme protein in thalli, so that enzyme is inactivated, and the thimerosal has strong inhibition capacity on various gram-positive bacteria, gram-negative bacteria and fungi, so as to prevent potential harm caused by microbial pollution. However, the sterility test of biological products is based on the elimination of bacteriostatic activity of the products themselves, and combines the requirement of sterility test method applicability specified in the Chinese pharmacopoeia to establish a test method suitable for the products. Thimerosal has strong bacteriostasis and is easy to remain on a filter membrane of a bacteria collecting incubator used for sterility test, and the residual bacteriostasis is easy to cause that microorganisms in a potential low-living or dormant state cannot be detected, thereby causing false negative results. The Chinese pharmacopoeia recommends that the washing fluid for sterility test is 0.1% peptone solution, sterile sodium chloride-peptone buffer solution with pH of 7.0 or 0.9% sterile sodium chloride solution, and the bacteriostatic elimination efficiency of any one of the washing fluids on the mercuride-containing biological products is very low at present. If the antibacterial activity of the product is eliminated by increasing the dosage of the flushing fluid, the experiment time is greatly increased, the filter membrane can be damaged, and the risk of microorganism damage on the filter membrane can be increased, namely false negative results are easily caused. In order to avoid the risk, the dosage of the washing liquid of a single filter membrane is generally not more than 500ml, and at most not more than 1000ml in sterility check specified by Chinese pharmacopoeia. If the washing liquid is used in a specified range to remove the bacteriostatic activity of the biological product containing mercuride, the samples can be aseptically tested in multiple groups only by reducing the amount of the samples. Thus, while avoiding the risk of filter damage, the likelihood of inter-group variability in sterility test results is increased. In addition, the grouping examination takes long time, the risk of false positive is increased, and the false positive result can bring huge loss to enterprises.

It can be seen that the conventional sterility test method has low efficiency and risks false positive or false negative results in the sterility test of the biological products containing mercurides. Based on the above-mentioned current situation, there is a need to identify an efficient and reliable method for sterility testing of a mercuride-containing biological product.

Disclosure of Invention

In the examination of the sterility of the biological product containing mercuride, the inventors found that the sterility of the biological product containing mercuride can be effectively examined by using a 0.1% peptone water solution containing sodium thiosulfate as the sterility examination rinse solution.

Therefore, the invention provides a novel method for checking the sterility of a biological product containing mercurides. The invention is realized by the following technical means:

a method of sterility testing for a mercuride-containing biological product, comprising:

a0.1% peptone water solution containing sodium thiosulfate was used as a rinse solution.

Further, the concentration of the sodium thiosulfate in the washing liquid is 0.05-0.8 mol/L.

Further, the sterility test method comprises:

(1) preparing a flushing liquid: weighing sodium thiosulfate pentahydrate crystals, fully dissolving with 0.1% peptone water solution, and obtaining a flushing liquid for later use;

(2) membrane filtration and washing: wetting the filter membrane by using flushing fluid, performing membrane filtration on the biological product containing mercurides, soaking the filter membrane for a period of time by using proper amount of flushing fluid, filtering, and then flushing by using the flushing fluid for multiple times, wherein the flushing process is slow and ensures that the flushing fluid covers the whole filter membrane;

(3) and (4) checking: the test was carried out according to the general 1101 sterility test method of the four pharmacopoeia of China, 2020 edition, with Staphylococcus aureus as a positive control.

Further, the concentration of sodium thiosulfate in the washing liquid in the step (1) is 0.05-0.8mol/L, preferably 0.1mol/L, and the concentration is the ratio of the amount of the sodium thiosulfate in mol to the volume of the washing liquid in L.

Further, the dosage of the filter membrane flushing solution in the step (2) is 100-500 ml/filter membrane.

Further, the proper amount of flushing liquid in the step (2) is 10-30 mL/membrane flushing liquid; the time for soaking the filter membrane by the flushing liquid is 3-5 minutes.

Further, the proper amount of flushing liquid is 20 mL; the time for soaking the filter membrane by the flushing liquid is 5 minutes.

The invention also discloses an application of the sterility test method in the biological products containing mercurides.

Further, the application comprises: any biological product containing mercuride is selected, 0.1% peptone water solution containing sodium thiosulfate is used as a flushing liquid, and the dosage of the required flushing liquid is determined according to the guidelines of the applicability of the aseptic inspection method of the four-part rules 1101 of the Chinese pharmacopoeia of 2020 edition, so that the aseptic inspection requirements of the biological product are met.

In the context of the present invention, sodium thiosulfate, CAS number: 10102-17-7, molecular weight: 248.18.

in the context of the present invention, "mol/L" is used to refer to the ratio of the amount (mol) of a substance to the volume (L) of the solute in a solute/solution system, unless otherwise stated.

In the context of the present invention, the adopted four parts of the 2020 edition Chinese pharmacopoeia are the four parts of the 2020 edition Chinese people's republic of China pharmacopoeia, compiled by the Council of national pharmacopoeia, published by the Chinese medicine science and technology publisher, ISBN 978-7-5214-.

In the context of the present invention, the sodium thiosulfate-containing 0.1% peptone water solution employed in step (1) may also be referred to as "flush solution", both of which may be used interchangeably; the 0.1% peptone water solution containing sodium thiosulfate adopted in the step (2) can be called as 'flushing liquor', and the two can be used interchangeably.

As described above, since step (3) of the sterility test method of the present invention is performed by a membrane filtration method in the sterility test method 1101 of the general rule of the four pharmacopoeias of china pharmacopoeia of 2020 edition, it is not necessary to describe the details of the test in step (3). The entire contents of the aforementioned 2020 edition chinese pharmacopoeia general rules 1101 sterility test method are incorporated herein by reference.

Preferably, in the step (1) and the step (2) of the sterility test method provided by the invention, the 0.1% peptone water solution containing sodium thiosulfate is subpackaged and autoclaved at 121 ℃ for 15min after preparation.

According to an embodiment of the present invention, the sterility test method of the present invention comprises the steps of:

A. taking a small amount of flushing fluid, namely 0.1% peptone water solution containing sodium thiosulfate, and wetting the filter membrane;

B. taking a proper amount of test sample, namely a mercuride-containing biological product, uniformly distributing all the test sample into a filter cylinder of a triple bacteria-collecting incubator, continuously filtering, and draining;

C. soaking the filter membrane for 3-5 minutes by 10-30 mL/membrane flushing fluid to neutralize mercury ions or mercurides remained on the filter membrane; then washing and filtering by using washing liquid for multiple times, and filtering to dry;

D. adding thioglycollate fluid culture medium into 2 bacteria collecting incubators as test sample groups, culturing at 30-35 deg.C and 20-25 deg.C respectively, adding trypticase soytone fluid culture medium into the other 1 bacteria collecting incubator as test sample groups, culturing at 20-25 deg.C, and observing at specified time.

The invention has the beneficial effects that:

the invention establishes a novel sterile inspection method for biological products containing mercurides by a verification test of a sterile inspection method, particularly by screening and optimizing a neutralizer diluent, a flushing liquid, a flushing amount, a neutralizer concentration, neutralization reaction time and the like and investigating the whole process of the sterile inspection method (a membrane filtration method). Experiments prove that the method according to any technical scheme of the invention shows that the sterility test meets the regulations, so the method is feasible, has reliable results and can be used for the sterility test of biological products containing mercurides with similar characteristics.

Compared with the detection method in the prior art, the sterility test method provided by the invention adopts 0.1% peptone water solution containing sodium thiosulfate as a flushing solution. Experiments prove that the method has better flushing effect by adopting the sodium thiosulfate-containing 0.1% peptone water solution, can effectively reduce the residue of mercuride in the bacteria collection incubator, and can eliminate the bacteriostatic property of the biological product containing the mercuride; and greatly shortens the sterility test time and the amount of flushing fluid used to mitigate the risk of filter membrane damage and microbial damage on the filter membrane that can result from extensive flushing over time. Therefore, the inspection method can obtain the sterility inspection result of the biological product containing the mercuride more quickly, more effectively and more accurately.

Drawings

FIG. 1 shows recovery of Staphylococcus aureus;

FIG. 2 shows recovery of Escherichia coli;

FIG. 3 shows the recovery of Clostridium sporogenes;

FIG. 4 shows recovery of Bacillus subtilis;

FIG. 5 shows the recovery of Candida albicans;

FIG. 6 shows the Aspergillus niger recovery.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials, reagents and the like used in the following examples are all commercially available products unless otherwise specified.

Some of the reagents and media used in the present invention are shown in Table 1.

TABLE 1 partial reagents and culture media

Name (R)	Manufacturer of the product	Batch number
			Sodium thiosulfate pentahydrate	CHENGDU CHRON CHEMICALS Co.,Ltd.	2020100901
0.1% peptone water solution	BEIJING SANYAO SCIENCE & TECHNOLOGY DEVELOPMENT Co.	200917
			Thioglycollate fluid medium	BEIJING SANYAO SCIENCE & TECHNOLOGY DEVELOPMENT Co.	20210622
Trypticase soy peptone liquid medium	BEIJING SANYAO SCIENCE & TECHNOLOGY DEVELOPMENT Co.	20210409
			Pancreas casein soybean peptone agar culture dish	CHENGDU INSTITUTE OF BIOLOGICAL PRODUCTS Co.,Ltd.	CM-TSA20210208
Sa's glucose agar culture dish	CHENGDU INSTITUTE OF BIOLOGICAL PRODUCTS Co.,Ltd.	CM-SDA20210307
			Thioglycollate fluid medium	CHENGDU INSTITUTE OF BIOLOGICAL PRODUCTS Co.,Ltd.	CM-LY20210311
Trypticase soy peptone liquid medium	Chengdu biological systemCompany Limited responsibility of institute of products	CM-YD20210312

The triple bacteria-collecting incubator used in the present invention is, as not described, an EVS330 type bacteria-collecting incubator produced by Hangzhou Tailin biotechnology, Inc.

The strain used in the invention is purchased from China medical bacteria preservation management center, is prepared by passage in quality verification room of Chengdu biological products research institute, Limited liability company, and is detailed in Table 2.

TABLE 2 batch number of strains used according to the invention

Example 1

Methodology prescreening

Primary screening of the sterility test method was performed.

Method 1

Using 0.1% peptone water solution as mercuride-containing biological product sterility check flushing liquor; the test sample is 150ml, and the test is carried out for 1 time.

Taking 150ml of a test sample, wetting a filter membrane in a filter cylinder of the triple bacteria collection incubator by a small amount of 0.1% peptone water solution, and uniformly distributing all the test solution into the filter cylinder of the triple bacteria collection incubator, wherein the total filter solution of each filter cylinder is 50 ml; the peptone water was then washed 10 times with 0.1% peptone water solution (100 ml per membrane) for a total wash volume of 1000ml per membrane, and drained. Preparing 3 groups of filter cartridges (9 in total) by the same method, wherein 100ml of thioglycolate fluid culture medium is added into 6 filter cartridges, the filter cartridges are divided into 2 groups, and 1ml (less than 100cfu) of escherichia coli, staphylococcus aureus and clostridium sporogenes are respectively inoculated into each group of culture medium, wherein one group is cultured at 30-35 ℃, and the other group is cultured at 20-25 ℃; and adding trypticase soytone liquid culture medium into the other 3 filter cartridges, respectively inoculating 1ml (less than 100cfu) of bacillus subtilis, candida albicans and aspergillus niger to the culture medium, and culturing at 20-25 ℃. The results of the day-by-day observations are shown in Table 3 below.

Experimental results show that the bacteriostatic activity of the method on Candida albicans and Aspergillus niger is not eliminated, and the method cannot meet the requirement of Chinese pharmacopoeia on the applicability of the sterility test method.

Method 2

Using 0.1% peptone water solution as mercuride-containing biological product sterility check flushing liquor; the test sample is 150ml, and the test is carried out for 3 times.

Taking 150ml of a test sample, dividing the test sample into 3 groups, wetting filter membranes in filter cartridges of the triple bacteria collection incubator by a small amount of 0.1% peptone water solution, and then uniformly distributing all the test solution into the filter cartridges of the triple bacteria collection incubator, wherein the total filtrate amount of each filter cartridge is about 16.7 ml; then washed 10 times with 0.1% peptone water solution (100 ml per membrane) with a total wash volume of 1000ml per membrane and drained. Preparing 3 groups of filter cartridges (9 in total) by the same method, wherein 100ml of thioglycolate fluid culture medium is added into 6 filter cartridges, the filter cartridges are divided into 2 groups, and 1ml (less than 100cfu) of escherichia coli, staphylococcus aureus and clostridium sporogenes are respectively inoculated into each group of culture medium, wherein one group is cultured at 30-35 ℃, and the other group is cultured at 20-25 ℃; and adding trypticase soytone liquid culture medium into the other 3 filter cartridges, respectively inoculating 1ml (less than 100cfu) of bacillus subtilis, candida albicans and aspergillus niger to the culture medium, and culturing at 20-25 ℃. The results of the day-by-day observations are shown in Table 3 below.

Experimental results show that the bacteriostatic activity of the method on Candida albicans and Aspergillus niger is not eliminated, and the method cannot meet the requirement of Chinese pharmacopoeia on the applicability of the sterility test method.

Method 3

Using 0.1% peptone water solution as mercuride-containing biological product sterility check flushing liquor; the test sample is 150ml, 5 times of administration.

Taking 150ml of test sample, dividing the test sample into 5 groups, wetting filter membranes in filter cartridges of the triple bacteria collection incubator by a small amount of 0.1% peptone water solution, and then uniformly distributing all the test solution into the filter cartridges of the triple bacteria collection incubator, wherein the total filtrate volume of each filter cartridge is 10 ml; the peptone water solution was then washed 10 times with 0.1% peptone water solution (100 ml/membrane each time), with a total wash volume of 1000 ml/membrane, and filtered dry. Preparing 3 groups of filter cartridges (9 in total) by the same method, wherein 100ml of thioglycolate fluid culture medium is added into 6 filter cartridges, the filter cartridges are divided into 2 groups, and 1ml (less than 100cfu) of escherichia coli, staphylococcus aureus and clostridium sporogenes are respectively inoculated into each group of culture medium, wherein one group is cultured at 30-35 ℃, and the other group is cultured at 20-25 ℃; and adding trypticase soytone liquid culture medium into the other 3 filter cartridges, respectively inoculating 1ml (less than 100cfu) of bacillus subtilis, candida albicans and aspergillus niger to the culture medium, and culturing at 20-25 ℃. The results of the day-by-day observations are shown in Table 3 below.

Experimental results show that 6 test bacteria grow in the method, and the requirement of Chinese pharmacopoeia on the applicability of the sterility test method is met. However, a batch of test samples are put into inspection in multiple groups, so that the experimental efficiency is low and the result difference among groups is easy to occur. Furthermore, the test article is exposed to the operating environment for a long time, which may lead to false positives and thus to a deviation in the results of the sterility test.

Method 4

Using 0.1mol/L sodium thiosulfate aqueous solution as neutralizer diluent, and 0.1% peptone water solution as mercuride-containing biological product sterile inspection flushing liquor; 150ml of test sample is put into inspection for 1 time, and the neutralizer diluent and the test sample are mixed according to the ratio of 1: 1.

Taking 150ml of a test sample, uniformly mixing 0.1mol/L sodium thiosulfate aqueous solution with the test sample at a ratio of 1:1, standing for 10 minutes to fully neutralize, then wetting a filter membrane in a filter cylinder of a triple bacteria collection incubator by a small amount of 0.1% peptone aqueous solution, and then completely and uniformly distributing the test solution into the filter cylinder of the triple bacteria collection incubator, wherein the total filtrate amount of each filter cylinder is about 100 ml; then washed 10 times with 0.1% peptone water solution (100 ml per membrane) with a total wash volume of 1000ml per membrane and drained. Preparing 3 groups of filter cartridges (9 in total) by the same method, wherein 100ml of thioglycolate fluid culture medium is added into 6 filter cartridges, the filter cartridges are divided into 2 groups, 1ml (less than 100cfu) of escherichia coli, staphylococcus aureus and clostridium sporogenes are respectively inoculated into each group of culture medium, one group of the culture media is cultured at 30-35 ℃, and the other group of the culture media is cultured at 20-25 ℃; and adding trypticase soytone liquid culture medium into the other 3 filter cartridges, respectively inoculating 1ml (less than 100cfu) of bacillus subtilis, candida albicans and aspergillus niger to the culture medium, and culturing at 20-25 ℃. The results of the day-by-day observations are shown in Table 3 below.

Experimental results show that the bacteriostatic activity of the method on Candida albicans and Aspergillus niger is not eliminated, and the method cannot meet the requirement of Chinese pharmacopoeia on the applicability of the sterility test method.

Method 5

Using 0.1mol/L sodium thiosulfate aqueous solution as neutralizer diluent, and 0.1% peptone water solution as mercuride-containing biological product sterile inspection flushing liquor; 150ml of test sample is put into inspection for 1 time, and the neutralizer diluent and the test sample are mixed according to the ratio of 2: 1.

Taking 150ml of a test sample, neutralizing the test sample with a 01mol/L sodium thiosulfate aqueous solution in a ratio of 2:1, uniformly mixing and standing for 10 minutes, wetting a filter membrane in a filter cylinder of the triple bacteria collection incubator by a small amount of 0.1% peptone water solution, and uniformly distributing all the test solution to the filter cylinder of the triple bacteria collection incubator, wherein the total filtrate amount of each filter cylinder is about 150 ml; then washed 10 times with 0.1% peptone water solution (100 ml per membrane) with a total wash volume of 1000ml per membrane and drained. Preparing 3 groups of filter cartridges (9 in total) by the same method, wherein 100ml of thioglycolate fluid culture medium is added into 6 filter cartridges, the filter cartridges are divided into 2 groups, and 1ml (less than 100cfu) of escherichia coli, staphylococcus aureus and clostridium sporogenes are respectively inoculated into each group of culture medium, wherein one group is cultured at 30-35 ℃, and the other group is cultured at 20-25 ℃; and adding trypticase soytone liquid culture medium into the other 3 filter cartridges, respectively inoculating 1ml (less than 100cfu) of bacillus subtilis, candida albicans and aspergillus niger to the culture medium, and culturing at 20-25 ℃. The results of the day-by-day observations are shown in Table 3 below.

Experimental results show that the bacteriostatic activity of the method on Candida albicans and Aspergillus niger is not eliminated, and the requirement of pharmacopoeia on the applicability of the sterility test method cannot be met.

TABLE 3 test results of methods 1-5

As can be seen from the results in Table 3, in the methods 1 to 3, 0.1% peptone water solution is used as a conventional flushing liquid, the antibacterial elimination efficiency of mercury ions or mercurides remained on the filter membrane is too low, the antibacterial activity of the mercuride-containing biological products can hardly be eliminated when the dosage of the flushing liquid reaches the upper limit requirement of Chinese pharmacopoeia, and the requirement of the sterility test can be met only by putting the test samples into test in groups and reducing the antibacterial components loaded on the filter membrane, but the detection efficiency is low in this way, and the result difference among groups is easy to occur, so that the operability is not strong; the related literature suggests that mercurides in biological products are neutralized by directly using 0.1mol/L sodium thiosulfate aqueous solution neutralizer, but the method can not perform effective sterility test by the method 4 and the method 5, for the sterility test of biological products, sample amount differences exist between intermediate products and finished products at various stages, the using amount of neutralizer and neutralization time can change along with the change of test products, and the operability is not strong; in combination with the consideration of the method 1-5, the creative discovery can achieve the aim of neutralizing and flushing only mercury ions or mercurides remained on the filter membrane by adding sodium thiosulfate into 0.1% peptone water solution.

Example 2

Method optimization test

1. The preparation method comprises preparing 0.1% peptone water solution containing sodium thiosulfate with a series of concentrations, wherein the peptone water solution meets the requirements of Chinese pharmacopoeia on unconventional flushing fluid, needs to carry out toxicity test on the flushing fluid with a series of concentrations, proves that the peptone water solution has no toxic effect on microorganisms, and can be used as flushing fluid for sterility test

(1) Preparing a flushing liquid: 0.1% peptone water solution was used as a base solution, 3 batches of each sodium thiosulfate rinse solution containing 0.05, 0.10, 0.20, 0.40, 0.80mol/L were prepared for parallel testing, and autoclaved at 121 ℃. Test groups: adding 20ml of 0.05-0.80 mol/L sodium thiosulfate flushing liquid into 1ml of test bacterial liquid in each batch; control group: 20ml of 0.1% peptone water solution was taken and the same 1ml test bacteria was added. After the preparation is finished, the mixture is placed at room temperature for acting for 2 hours, and a test group and a bacteria liquid control group of each test bacterium are prepared into 2 parallel groups for calculating the average number of bacterial colonies of each group;

(2) and (3) filtering and culturing: filtering a test solution added with staphylococcus aureus, escherichia coli and bacillus subtilis by adopting a microbial limit method, respectively sticking filter membrane bacteria faces upwards to trypticase soy peptone agar culture dishes, culturing in an incubation room at 30-35 ℃ for no more than 3 days, and counting; adding candida albicans and aspergillus niger test solution, filtering, respectively sticking the test solution to a Sabouraud's dextrose agar culture dish, culturing in an incubation room at 20-25 ℃ for no more than 5 days, and counting; adding a test solution of clostridium sporogenes into the culture medium, directly inoculating the test solution into a thioglycolate fluid medium, culturing the culture medium in an incubation room at the temperature of 30-35 ℃ for no more than 3 days, and counting;

(3) and (3) calculating the recovery rate: the microorganism recovery rate is the ratio of the average number of colonies in the test group to the average number of colonies in the control group;

(4) the recovery rates are shown in FIGS. 1 to 6. The experimental results according to fig. 1-6 show that: through a microorganism recovery test, the recovery rate of the 3 batches of 0.1% peptone water solution flushing liquor containing sodium thiosulfate with each concentration is within the range of 0.5-2. Meets the requirements of Chinese pharmacopoeia, so the concentration in the range is determined to be non-toxic to microorganisms, and the washing liquid can be used as washing liquid for sterility inspection.

2. The concentration of the peptone water solution flushing liquor containing sodium thiosulfate and 0.1 percent is screened by two mercuride-containing biological products

(1) Preparing a test sample: taking a semi-finished product of the adsorption acellular diphtheria-pertussis-tetanus combined vaccine (DTaP semi-finished product, batch number: 20210804B) and a semi-finished product of the 13-valent pneumococcal polysaccharide conjugate vaccine (PCV 13 semi-finished product, batch number: T-20211101B) as research objects, screening the optimal concentration, and producing a test sample by a Chengdu biological product research institute, namely a finite responsibility company;

(2) preparation of test groups: DTaP semi-finished product group: filtering the 50 ml/membrane DTaP semi-finished product by adopting a sterility check membrane filtration method, and then flushing the filter membrane by 200ml of sodium thiosulfate flushing liquid with the concentration of 0.05, 0.10 and 0.20mol/L respectively; PCV13 semi-finished group: filtering a 50 ml/membrane PCV13 semi-finished product by the same method, and then flushing the filter membrane by 200 ml/membrane 0.1% peptone water flushing liquor containing 0.05, 0.10 and 0.20mol/L sodium thiosulfate; making 2 groups of washing concentrations, adding thioglycollate fluid culture medium into 1 group after washing, inoculating staphylococcus aureus, adding tryptone soy peptone liquid culture medium into the other 1 group, and inoculating aspergillus niger; control group: directly adding the two culture mediums into a bacteria-collecting culture device respectively, and then inoculating staphylococcus aureus and aspergillus niger respectively.

(3) And (3) culture observation results: and (3) placing the bacteria collecting incubator for inoculating the DTaP semi-finished product group, the PCV13 semi-finished product group and the control group with staphylococcus aureus in an incubation chamber at the temperature of 30-35 ℃ for culturing for no more than 3 days, placing the bacteria collecting cup inoculated with the Aspergillus niger in an incubation chamber at the temperature of 20-25 ℃ for culturing for no more than 5 days, and observing and recording results.

(4) The results of concentration screening are shown in table 4:

TABLE 4 growth of test bacteria washed in 2 mercuride-containing biological products by three concentrations of washing liquid

The results in Table 4 show that the growth conditions of the test group and the control group are consistent after the peptone water solution flushing solution of 0.1% containing 0.10 and 0.20mol/L sodium thiosulfate washes, and the sterility test requirements can be met under the test conditions. The results of the two test samples were combined, and 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate was selected as the washing solution for sterile examination of the mercuride-containing biological product.

3. Selecting 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate, carrying out sterility test according to Chinese pharmacopoeia requirements, and confirming the effectiveness of the flushing liquor.

(1) Preparing a test sample: 3 batches of DTaP semi-finished product (batch numbers: 20210804B, 20210805B and 20210806B), 3 batches of PCV13 semi-finished product (batch numbers: T-20211101B, T-20211102B, T-20220203B), 150ml of test sample, and 1 test sample, wherein the test sample is produced by Limited liability company of Chengdu biological product research institute.

(2) Preparing a test sample group: taking 150ml of a test sample, wetting a filter membrane in a filter cylinder of the triple bacteria collection incubator by a small amount of 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate, and then uniformly distributing the test solution into the filter cylinder of the triple bacteria collection incubator, wherein the total filter solution of each filter cylinder is 50 ml; then washed 2 times with 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate (100 ml per membrane each time), the total washing amount is 200ml per membrane, and filtered to dryness. Preparing 3 groups of filter cartridges (9 in total) by the same method, wherein 100ml of thioglycolate fluid culture medium is added into 6 filter cartridges, the filter cartridges are divided into 2 groups, 1ml (less than 100cfu) of escherichia coli, staphylococcus aureus and clostridium sporogenes are respectively inoculated into the culture medium, one group is cultured for no more than 3 days at the temperature of 30-35 ℃, and the other group is cultured for no more than 5 days at the temperature of 20-25 ℃; and adding trypticase soytone liquid culture medium into the other 3 filter cartridges, respectively inoculating 1ml (less than 100cfu) of bacillus subtilis, candida albicans and aspergillus niger to the culture medium, and culturing at 20-25 ℃ for no more than 5 days. The results are shown in Table 5 below, observed day by day.

(3) Positive control group: directly adding thioglycollate fluid medium and trypticase soytone liquid medium into the bacteria collection culture vessel, adding test bacteria, and operating the same test group.

(4) Washing solution control group: 600ml of 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate was taken for sterility check, and test bacteria were added to operate the same test group.

(5) Sample control group: the sterility of the test article is checked, the operation is the same as that of the test group, and no test bacteria are added.

(6) Medium control group: directly adding a thioglycollate fluid culture medium and a trypticase soytone liquid culture medium into the bacteria collection culture vessel without adding test bacteria.

The results of the test groups are shown in Table 5:

TABLE 52 method applicability results table for mercuride-containing articles

From the results in Table 5, it can be seen that: the test bacteria of the two test sample test groups and the washing liquid control group grow well and are consistent with the growth of the positive control group, and the test sample control group and the culture medium control group both grow aseptically, which indicates that after the 2 mercuride-containing biological products are washed by 0.1% peptone solution washing liquid containing 0.10mol/L sodium thiosulfate, the inspection amount has no bacteriostatic action or the bacteriostatic component is negligible under the test condition, and the requirement of the fourth pharmacopoeia 1101 of China on the applicability of the sterility test method can be met.

In order to ensure that the 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate is used as the flushing liquid and can meet the validity and reliability of the sterility check of all the biological products containing mercuride, more types of biological products containing mercuride or different dosage amounts of the same type of biological products need to be selected for further verification.

Example 3

Methodology review supplemental test

(1) The 7 mercuride-containing biological products were validated for sterility testing methodology using 0.10mol/L sodium thiosulfate in 0.1% peptone water solution rinse. The specific filter capacity and rinse solution design for each product is shown in table 6:

TABLE 6 dosage and washing liquid dosage of each product

(2) The results of the validation of the sterility test methodology for the 7 mercuride-containing biologicals are shown in Table 7:

TABLE 7 methodological validation of sterility testing of mercuride-containing biologics

According to the results in table 7, the test bacteria of the 7 test article test groups and the washing liquid control group grow well and are consistent with the growth of the positive control group, and the test article control group and the culture medium control group grow aseptically, which indicates that after the 7 mercuride-containing biological products are washed by 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate, the detected amount has no bacteriostatic action or has negligible bacteriostatic components under the test condition, and can meet the requirement of the fourth rule 1101 of Chinese pharmacopoeia on the applicability of the sterility test method. Therefore, the 0.1% peptone water solution containing 0.10mol/L sodium thiosulfate can be determined as the flushing liquid, and the validity and the reliability of the sterile inspection of all the biological products containing mercuride can be met.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

Claims (10)

1. A method for sterility testing of a mercuride-containing biological product, comprising:

a0.1% peptone water solution containing sodium thiosulfate was used as a rinse solution.

2. The sterility test method according to claim 1, wherein:

the concentration of the sodium thiosulfate in the washing liquid is 0.05-0.8 mol/L.

3. The sterility test method according to claim 1 or 2, comprising:

(1) preparing a flushing liquid: weighing sodium thiosulfate pentahydrate crystals, fully dissolving with 0.1% peptone water solution, and obtaining a flushing liquid for later use;

(2) membrane filtration and washing: wetting the filter membrane by using flushing fluid, performing membrane filtration on the biological product containing mercurides, soaking the filter membrane for a period of time by using proper amount of flushing fluid, filtering, and then flushing by using the flushing fluid for multiple times, wherein the flushing process is slow and ensures that the flushing fluid covers the whole filter membrane;

(3) and (4) checking: the test was carried out according to the general 1101 sterility test method of the four pharmacopoeia of China, 2020 edition, with Staphylococcus aureus as a positive control.

4. The sterility test method of claim 3, wherein:

the concentration of the sodium thiosulfate in the washing liquid in the step (1) is 0.05-0.8mol/L, and the concentration is the ratio of the amount of the sodium thiosulfate in mol to the volume of the washing liquid in L.

5. The sterility test method according to claim 4, wherein:

the concentration of the sodium thiosulfate in the washing liquid is 0.1 mol/L.

6. The sterility test method of claim 3, wherein:

the dosage of the filter membrane flushing liquid in the step (2) is 100-500 ml/filter membrane.

7. The sterility test method of claim 3, wherein:

the proper amount of flushing liquid in the step (2) is 10-30mL per membrane flushing liquid;

the time for soaking the filter membrane by the flushing liquid is 3-5 minutes.

8. The sterility inspection method of claim 7, wherein:

the proper amount of flushing liquid is 20 mL/membrane flushing liquid;

the time for soaking the filter membrane by the flushing liquid is 5 minutes.

9. Use of the sterility test method according to any one of claims 1 to 8 in a biological product containing mercurides.

10. The use according to claim 9, wherein:

the application comprises the following steps: any biological product containing mercuride is selected, 0.1% peptone water solution containing sodium thiosulfate is used as a flushing liquid, and the dosage of the required flushing liquid is determined according to the guidelines of the applicability of the aseptic inspection method of the four-part rules 1101 of the Chinese pharmacopoeia of 2020 edition, so that the aseptic inspection requirements of the biological product are met.

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