RU2596395C1 - Method of determining microbiological purity of drugs of target therapy - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutics and microbiology. Method of determining microbiological purity of medicinal agents comprises preparation of a drug sample by diluting in tryptic soy broth or medium No. 8. Method includes seeding a sample on a liquid accumulation selective medium with subsequent incubation thereon. Then, method includes re-seeding on diagnostic culture media with subsequent biochemical identification of selected microorganisms.

EFFECT: invention reduces amount sample used and minimises loss of sample during analysis.

1 cl, 5 ex

Description

The invention relates to methods of measurement or testing using viable microorganisms, and relates to a method for determining the microbiological purity of drugs for targeted therapy.

Closest to the claimed is a method for determining the microbiological purity of individual biotechnological drugs [1], which allows you to quantify viable cells of bacteria and fungi by their ability to multiply and form colonies in a dense nutrient medium. Moreover, each colony is the result of the reproduction of one cell. To identify certain types of pathogenic microorganisms, a complex of liquid and dense nutrient media corresponding to the specific needs of the identified microorganisms is used, as well as biochemical tests to identify them.

The method includes the steps of:

- sample preparation of a sample of the drug (at least 10 g);

- adding the ground sample to 100 ml of phosphate-buffered solution and plating in 4 Petri dishes, as well as in a liquid non-selective storage medium for the determination of pathogenic bacteria;

- incubation of crops with nutrient media for the isolation of bacteria at a temperature of (32.5 ± 2.5) ° C, for the cultivation of yeast and molds at a temperature of (22.5 ± 2.5) ° C;

- reseeding on a liquid selective nutrient medium;

- reseeding on diagnostic nutrient media;

- microscopic examination;

- biochemical identification of isolated microorganisms;

- quantitative and qualitative accounting of results.

The disadvantages of the known method for determining the microbiological purity of drugs include a significant amount of the sample for analysis, the dilution of which is not fully used in the test.

Between the totality of the essential features of the proposed method and the achieved technical result, there is a causal relationship, namely: the use of trypticase-soy broth or nutrient medium No. 8 as a universal diluent (solvent) and culture medium allows to reduce the sample volume for analysis by 7 times.

The proposed technical solution differs from the known one [1] in that a reduced amount of the sample is used at the sample preparation stage, the dilution of which is fully used for analysis. Trypticase-soy broth or nutrient medium No. 8 are used as a universal diluent.

The objective of the invention is to reduce the number of samples for assessing quality in terms of "Microbiological purity", the time of sample preparation and minimizing drug losses during analysis.

The problem is solved through the use of a non-selective nutrient medium as a diluent of a reduced amount of the sample when determining certain types of pathogenic bacteria.

The nutrient media described in [1] are universal, free of inhibitors and indicators, designed for a wide range of microbiological tasks. The rich nutrient basis of these media is suitable for the cultivation of most microorganisms, including those that require growth conditions.

The proposed change allows you to 7 times reduce the number of samples for the analysis of targeted drugs on the indicator "Microbiological purity".

The essence of the proposed invention is as follows:

- a sample in an amount of 1.4 g is diluted with 14 ml of trypticase-soy broth or medium No. 8;

- 4 ml of the obtained dilution is used for the quantitative determination of aerobic bacteria and fungi, sowing in 4 sterile Petri dishes (1 ml each), 2 of which are filled with a dense nutrient medium for growing bacteria, 2 others - medium for growing yeast and mold fungi ;

- the remaining 10 ml of dilution is used to isolate E. coli.

Stage incubation and subsequent operations are performed in the same way as in the known method.

The technical result of the claimed invention is to reduce the volume of the sample, minimizing the loss of the drug in the analysis, expanding the arsenal of methods for assessing the microbiological purity of drugs targeted therapy.

The possibility of carrying out the claimed invention is shown by the following examples.

Means and methods

When proving the possibility of determining the microbiological purity of drugs for targeted therapy used:

1. Test strains of microorganisms:

- Candida albicans ATCC 10231,

- Escherichia coli ATCC 8739,

- Bacillus subtilis ATCC 6633,

- Aspergillus brasiliensis ATCC 16404.

2. Culture media:

2.1. manufactured by Biomerier, France

- trypticase soy agar;

- trypticase-soy broth;

- agar Saburo;

- McConkey Broth;

- McConkey agar.

2.2. FBUN State Scientific Center for Applied Microbiology and Biotechnology (Obolensk, Russia):

- Wednesday No. 1 of the timing;

- Wednesday No. 2 GRM (Saburo);

- medium for the isolation of enterobacteria (Endo timing agar);

- Wednesday No. 8 of the timing;

- Wednesday No. 3 of the timing.

3. Reagents: 0.9% sodium chloride solution.

4. Consumables: sterile biological tubes; serological pipettes 1, 5, 10 ml; sterile disposable vials 125 ml; Petri dishes 90 mm sterile disposable.

5. Certified and verified equipment: incubators from Binder (Germany), laminar cabinet class II type A2 from Labconco (USA), colony counter Scan 100 from Interscience (France).

When proving the possibility of using the claimed invention, the coefficient of recovery (R) of microorganisms was calculated by the formula (1):

N is the number of colonies isolated during analysis (CFU);

N ₀ - the number of introduced cells of microorganisms (CFU).

A recovery factor of at least 50% was considered acceptable.

Example 1. Determination of the microbiological purity of the drug erlotinib tablets 100 mg.

1 g of material was taken. The sample was dissolved in 10 ml of trypticase-soy broth. Test strains were introduced in an amount of 50 CFU / g of each microorganism.

1 ml of the prepared sample was introduced into 4 Petri dishes, 2 of which were filled with 10-15 ml of trypticase-soya agar to determine the content of aerobic microorganisms, and the remaining 2 10-15 ml of Saburo agar to isolate yeast and molds.

6 ml of the sample solution was incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes, filled with trypticase-soy agar and Saburo agar, were incubated for 120 hours at temperatures (32.5 ± 2.5) ° C and (22.5 ± 2.5) ° С, respectively.

After incubation on agarized nutrient media, the number of colonies of microorganisms was counted, and the recovery coefficient of the isolated bacteria, yeast, and molds was calculated using formula (1).

After the incubation period, 1 ml of the sample solution was added to 9 ml of McConkey broth, incubated for 48 hours at a temperature of (32.5 ± 2.5) ° С, the bacteriological loop was passaged on MacConkey agar, incubated for 24 hours at temperature (32.5 ± 2.5) ° С, after which a typical growth of Escherichia coli colonies was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, which was used to inoculate a sample of the drug erlotinib, 100 mg tablets. The recovery coefficient of aerobic microorganisms was 75%, yeast and mold fungi - 101%. Using the proposed method, Escherichia coli bacteria were isolated from the contaminated drug.

Example 2. Determination of the microbiological purity of the drug trametinib tablets 1 mg.

1 g of material was taken. The sample was dissolved in 10 ml of timing medium No. 8. Test strains were introduced in an amount of 50 CFU / g of each microorganism.

1 ml of the prepared sample was introduced into 4 Petri dishes, 2 of which were filled with 10-15 ml of timing medium No. 1 to determine the content of aerobic microorganisms, and the remaining 2 were filled with 10-15 ml of timing medium No. 2 to isolate yeast and molds.

6 ml of the sample solution was incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes, filled with media No. 1 of timing and No. 2 timing, were incubated for 120 hours at temperatures (32.5 ± 2.5) ° C and (22.5 ± 2.5) ° C, respectively.

After incubation on agarized nutrient media, the number of colonies of microorganisms was counted, and the recovery coefficient of the isolated bacteria, yeast, and molds was calculated using formula (1).

After the incubation period, 1 ml of the sample solution was added to 9 ml of medium No. 3, incubated for 48 hours at a temperature of (32.5 ± 2.5) ° С, the bacteriological loop was reseed on Endo agar, incubated for 24 hours at a temperature of (32 , 5 ± 2.5) ° С, after which a typical growth of Escherichia coli colonies was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, which was used to inoculate a sample of the drug tarmetinib, 1 mg tablet. The recovery coefficient of aerobic microorganisms was 71%, yeast and mold fungi - 91%. Using the proposed method, Escherichia coli bacteria were isolated from the contaminated drug.

Example 3. Determination of the microbiological purity of the drug axitinib tablets 7 mg.

1 g of material was taken. The sample was dissolved in 10 ml of timing medium No. 8. Test strains were introduced in an amount of 5 CFU / g of each microorganism.

1 ml of the prepared sample was introduced into 4 Petri dishes, 2 of which were filled with 10-15 ml of timing medium No. 1 to determine the content of aerobic microorganisms, and the remaining 2 were filled with 10-15 ml of timing medium No. 2 to isolate yeast and molds.

6 ml of the sample solution was incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes, filled with media No. 1 of timing and No. 2 timing, were incubated for 120 hours at temperatures (32.5 ± 2.5) ° C and (22.5 ± 2.5) ° C, respectively.

After incubation on agarized nutrient media, the number of colonies of microorganisms was counted, and the recovery coefficient of the isolated bacteria, yeast, and molds was calculated using formula (1).

After the incubation period, 1 ml of the sample solution was added to 9 ml of medium No. 3, incubated for 48 hours at a temperature of (32.5 ± 2.5) ° С, the bacteriological loop was reseed on Endo agar, incubated for 24 hours at a temperature of (32 , 5 ± 2.5) ° С, after which a typical growth of Escherichia coli colonies was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, with which a sample of the drug axitinib, 7 mg tablets, was inoculated. The recovery coefficient of aerobic microorganisms was 93%, yeast and mold fungi - 78%. Using the proposed method, Escherichia coli bacteria were isolated from the contaminated drug.

Example 4. Determination of the microbiological purity of the drug ibrutinib capsules 4 mg.

1 g of material was taken. The sample was dissolved in 10 ml of trypticase-soy broth. Test strains were introduced in an amount of 5 CFU / g of each microorganism.

1 ml of the prepared sample was introduced into 4 Petri dishes, 2 of which were filled with 10-15 ml of trypticase-soya agar to determine the content of aerobic microorganisms, and the remaining 2 10-15 ml of Saburo agar to isolate yeast and molds.

6 ml of the sample solution was incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes, filled with trypticase-soy agar and Saburo agar, were incubated for 120 hours at temperatures (32.5 ± 2.5) ° C and (22.5 ± 2.5) ° С, respectively.

After incubation on agarized nutrient media, the number of colonies of microorganisms was counted, and the recovery coefficient of the isolated bacteria, yeast, and molds was calculated using formula (1).

After the incubation period, 1 ml of the sample solution was added to 9 ml of McConkey broth, incubated for 48 hours at a temperature of (32.5 ± 2.5) ° С, the bacteriological loop was passaged on MacConkey agar, incubated for 24 hours at temperature (32.5 ± 2.5) ° С, after which a typical growth of Escherichia coli colonies was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, which was used to inoculate a sample of the drug ibrutinib, 4 mg capsules. The recovery coefficient of aerobic microorganisms was 93%, yeast and mold fungi - 87%. Using the proposed method, Escherichia coli bacteria were isolated from the contaminated drug.

Example 5. Determination of the microbiological purity of the substance imatinib mesylate.

1 g of material was taken. The sample was dissolved in 10 ml of trypticase-soy broth. Test strains were introduced in an amount of 50 CFU / g of each microorganism.

1 ml of the prepared sample was introduced into 4 Petri dishes, 2 of which were filled with 10-15 ml of trypticase-soya agar to determine the content of aerobic microorganisms, and the remaining 2 10-15 ml of Saburo agar to isolate yeast and molds.

6 ml of the sample solution was incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes, filled with trypticase-soy agar and Saburo agar, were incubated for 120 hours at temperatures (32.5 ± 2.5) ° C and (22.5 ± 2.5) ° С, respectively.

After incubation on agarized nutrient media, the number of colonies of microorganisms was counted, and the recovery coefficient of the isolated bacteria, yeast, and molds was calculated using formula (1).

After the incubation period, 1 ml of the sample solution was added to 9 ml of McConkey broth, incubated for 48 hours at a temperature of (32.5 ± 2.5) ° С, the bacteriological loop was passaged on MacConkey agar, incubated for 24 hours at temperature (32.5 ± 2.5) ° С, after which a typical growth of Escherichia coli colonies was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, which was used to inoculate a sample of the pharmaceutical substance imatinib mesylate. The recovery coefficient of aerobic microorganisms was 91%, yeast and mold fungi - 88%. Using the proposed method, Escherichia coli bacteria were isolated from the contaminated drug.

Literature.

1. The State Pharmacopoeia of the Russian Federation XII ed. Part 1. M .: Medicine, 2007: 704.

Claims (1)

A method for determining the microbiological purity of targeted therapy drugs, comprising sample preparation of a drug sample; reseeding and incubation on a selective liquid storage medium, reseeding on diagnostic nutrient media followed by biochemical identification of the isolated microorganisms, characterized in that when sample preparation of the drug sample, trypticase-soy broth or medium No. 8 is used as a diluent.