RU2596399C1 - Method of determining microbiological purity of pharmaceutical substance, produced biotechnologically - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutics and microbiology. Method of determining microbiological purity of pharmaceutical substance comprises preparation of a sample of a pharmaceutical substance 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. Method includes re-seeding on diagnostic nutrient media with subsequent biochemical identification of selected microorganisms.

EFFECT: invention increases accuracy of determining microbiological purity and reduces duration of analysis of pharmaceutical substances of biotechnological origin on "Microbiological purity" and amount of used sample.

1 cl, 6 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 pharmaceutical substances produced by biotechnological methods.

Closest to the claimed is a method for determining the microbiological purity of 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 20 g);

- adding 10 g of sample to 100 ml of lactose broth, followed by incubation for 2-5 hours;

- reseeding the amount corresponding to 1 g of the drug on a liquid selective storage medium for the isolation of enterobacteria;

- adding the remaining 10 g of the crushed 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 to isolate 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 diagnostic nutrient media;

- microscopic examination;

- biochemical identification of isolated microorganisms;

- quantitative and qualitative accounting of results.

The disadvantages of this method of determining the microbiological purity of biotechnological medicines include inaccuracy, the duration of the analysis, a significant amount of test sample.

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 a medium for culturing a reduced amount of the drug allows:

- improve accuracy - by increasing the number of parallel measurements (10 Petri dishes to determine aerobic bacteria and fungi),

- reduce analysis time - by eliminating the preliminary incubation stage.

The proposed technical solution differs from the known [1] in that a reduced amount of sample is used at the stage of sample preparation. For the quantitative determination of viable bacteria and microscopic (yeast and mold) fungi, the drug is diluted with a phosphate-buffered solution, and for the isolation of certain types of pathogenic bacteria with a non-selective nutrient medium. For quantification use 10 Petri dishes.

The objective of the invention is to increase the accuracy of the quantitative determination of microorganisms, reducing the analysis time and the amount of sample to assess the quality in terms of "Microbiological purity".

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 to increase the accuracy of the result, to reduce the analysis time of pharmaceutical substances of biotechnological origin according to the indicator "Microbiological purity" and the amount of sample used.

The essence of the proposed invention is as follows:

- a sample in an amount of 1 g is diluted in 10 ml of phosphate-buffered solution;

- the obtained 10 ml is used for the quantitative determination of aerobic bacteria and microscopic fungi, sowing in 10 sterile Petri dishes (1 ml each), 5 of which are poured with a dense nutrient medium for growing bacteria, the remaining ones are medium for growing yeast and mold fungi;

- a sample in an amount of 2 g is diluted with 20 ml of trypticase-soy broth or medium No. 8.

- 10 ml of dilution is transferred to 90 ml of Mossel broth or medium No. 3 to isolate enterobacteria;

- the remaining 10 ml of dilution is used to isolate P.aeruginosa and S.aureus.

Stage incubation and subsequent operations are performed in the same way as in the known method [1].

The technical result of the claimed invention is to improve the accuracy, reduce the volume of the sample and expand the arsenal of methods for assessing the quality of pharmaceutical substances produced by biotechnological methods, according to the indicator "Microbiological purity".

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

Means and methods.

To prove the possibility of determining the microbiological purity of pharmaceutical substances used for the production of sterile preparations, we used:

1. Test strains of microorganisms:

- Candida albicans ATCC 10231,

- Escherichia coli ATCC 8739,

- Bacillus subtilis ATCC 6633,

- Aspergillus brasiliensis ATCC 16404,

- Staphylococcus aureus ATCC 6538,

- Pseudomonas aeruginosa ATCC 9027.

2. Culture media:

2.1. manufactured by Biomerier, France

- trypticase soy agar;

- trypticase-soy broth;

- agar Saburo;

- Mossel broth;

- Mossel agar;

- Byrd-Parker agar;

- cetrimide 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;

- Wednesday No. 9 of the timing;

- Wednesday No. 10 of the timing.

3. Reagents: phosphate-buffered saline (pH 7.0), sodium chloride solution 0.9%.

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 tested 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).

Example 1. Determination of the microbiological purity of the substance rituximab, dissolved in trypticase-soy broth, inoculated with a mixture of test strains in the amount of 50 CFU / g of each microorganism.

3 g of material were taken. The sample was dissolved in 30 ml of trypticase-soy broth. Test strains were introduced in an amount of 50 CFU / g of each microorganism.

10 ml were taken from the inoculated sample and transferred to Mossel broth.

10 ml of the prepared sample was added to 10 Petri dishes (1 ml each), 5 of which were filled with 10-15 ml of trypticase-soya agar to determine the content of aerobic microorganisms, and the remaining 5 10-15 ml of Saburo agar to isolate yeast and mold fungi.

Crops on Mossel broth, as well as the remaining 10 ml of the sample solution, were incubated at a temperature of (32.5 ± 2.5) ° С for 48 h. Petri dishes, filled with trypticase-soy agar and Saburo agar, were incubated for 120 h at temperatures (32, 5 ± 2.5) ° С and (22.5 ± 2.5) ° С, respectively.

After incubation on agarized culture media, the number of colonies of microorganisms was counted and the recovery coefficient of the isolated microorganisms was calculated.

From Mossel broth, a bacteriological loop was used to transfer to Mossel agar, incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, after which typical growth of enterobacteria colonies (Escherichia coli) was observed.

After the incubation period of the crops in 10 ml of trypticase-soy broth, the bacteriological loop carried out the transfer to Bird-Parker agar and cetrimide agar to determine Staphylococcus aureus and Pseudomonas aeruginosa, respectively, were incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, then observed the growth of colonies of these microorganisms.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, with which a sample of the pharmaceutical substance rituximab was inoculated. The recovery coefficient of aerobic microorganisms was 82%, yeast and mold fungi - 76%. Using the proposed method, enterobacteria, Staphylococcus aureus, Pseudomonas aeruginosa were isolated from a contaminated medicinal product.

Example 2. Determination of the microbiological purity of the pharmaceutical substance insulin glargine, dissolved in medium No. 8, inoculated with a mixture of test strains in the amount of 50 CFU / g of each microorganism.

3 g of material were taken. The sample was dissolved in a liquid nutrient medium No. 8 GRM. Test strains were introduced in an amount of 50 CFU / g of each microorganism. 10 ml were taken from the inoculated sample and transferred to the timing medium No. 3 to enrich enterobacteria.

10 ml of the prepared sample of the substance insulin glargine was added to 10 Petri dishes (1 ml each), 5 of which were filled with 10-15 ml of timing medium No. 1 to determine the content of aerobic microorganisms, and the remaining 5 10-15 ml of medium No. 2 Timing for the isolation of yeast and molds.

Crops on timing medium No. 3, as well as the remaining 10 ml of the sample solution, were incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes filled with timing medium No. 1 and timing No. 2 were incubated for 120 hours at temperatures (32.5 ± 2.5) ° С and (22.5 ± 2.5) ° С, respectively.

After incubation on agarized culture media, the number of colonies of microorganisms was counted and the recovery coefficient of the isolated microorganisms was calculated.

From medium No. 3, a bacteriological loop was transferred to Endo agar, incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, after which a typical growth of enterobacteria (Escherichia coli) colonies was observed.

After the incubation period of crops in 10 ml of timing medium No. 8 with a bacteriological loop, reseeding on timing mediums No. 10 and No. 9 of timing was performed to determine Staphylococcus aureus and Pseudomonas aeruginosa, respectively, were incubated for 48 hours at a temperature of (32.5 ± 2.5) ° C, after which the growth of colonies of these microorganisms was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, with which a sample of the pharmaceutical substance insulin glargine was inoculated. The recovery coefficient of aerobic microorganisms was 78%, yeast and mold fungi - 90%. Using the proposed method, enterobacteria, Staphylococcus aureus, Pseudomonas aeruginosa were isolated from a contaminated medicinal product.

Example 3. Determination of the microbiological purity of a pharmaceutical substance insulin lispro dissolved in trypticase-soy broth inoculated with a mixture of test strains in an amount of 5 CFU / g of each microorganism.

3 g of material were taken. The sample was dissolved in 30 ml of trypticase-soy broth. Test strains were introduced in an amount of 5 CFU / g of each microorganism. 10 ml were taken from the inoculated sample and transferred to Mossel broth.

10 ml of the prepared sample was added to 10 Petri dishes (1 ml each), 5 of which were filled with 10-15 ml of trypticase-soya agar to determine the content of aerobic microorganisms, and the remaining 5 10-15 ml of Saburo agar to isolate yeast and mold fungi.

Crops on Mossel broth, as well as the remaining 10 ml of the sample solution, were incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes, filled with tripticase-soy agar and Saburo agar, were incubated for 120 hours at temperatures (32 , 5 ± 2.5) ° С and (22.5 ± 2.5) ° С, respectively.

After incubation on agarized culture media, the number of colonies of microorganisms was counted and the recovery coefficient of the isolated microorganisms was calculated.

From Mossel broth, a bacteriological loop was used to transfer to Mossel agar, incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, after which typical growth of enterobacteria colonies (Escherichia coli) was observed.

After the incubation period of the crops in 10 ml of trypticase-soy broth, the bacteriological loop carried out the transfer to Bird-Parker agar and cetrimide agar to determine Staphylococcus aureus and Pseudomonas aeruginosa, respectively, were incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, then observed the growth of colonies of these microorganisms.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, with which a sample of the pharmaceutical substance insulin lispro was inoculated. The recovery coefficient of aerobic microorganisms was 59%, yeast and mold fungi - 63%. Using the proposed method, enterobacteria, Staphylococcus aureus, Pseudomonas aeruginosa were isolated from a contaminated medicinal product.

Example 4. Determination of the microbiological purity of the pharmaceutical substance rituximab, dissolved in medium No. 8, inoculated with a mixture of test strains in the amount of 5 CFU / g of each microorganism.

3 g of material were taken. The sample was dissolved in a liquid nutrient medium No. 8 GRM. Test strains were introduced in an amount of 5 CFU / g of each microorganism. 10 ml were taken from the inoculated sample and transferred to the timing medium No. 3 to enrich enterobacteria.

10 ml of the prepared sample of the substance insulin glargine was added to 10 Petri dishes (1 ml each), 5 of which were filled with 10-15 ml of timing medium No. 1 to determine the content of aerobic microorganisms, and the remaining 5 10-15 ml of medium No. 2 Timing for the isolation of yeast and molds.

Crops on timing medium No. 3, as well as the remaining 10 ml of the sample solution, were incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes filled with timing medium No. 1 and timing No. 2 were incubated for 120 hours at temperatures (32.5 ± 2.5) ° С and (22.5 ± 2.5) ° С, respectively.

After incubation on agarized culture media, the number of colonies of microorganisms was counted and the recovery coefficient of the isolated microorganisms was calculated.

From medium No. 3, a bacteriological loop was transferred to Endo agar, incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, after which a typical growth of enterobacteria (Escherichia coli) colonies was observed.

After the incubation period of crops in 10 ml of timing medium No. 8 with a bacteriological loop, reseeding on timing mediums No. 10 and No. 9 of timing was performed to determine Staphylococcus aureus and Pseudomonas aeruginosa, respectively, were incubated for 48 hours at a temperature of (32.5 ± 2.5) ° C, after which the growth of colonies of these microorganisms was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, with which a sample of the pharmaceutical substance rituximab was inoculated. The recovery coefficient of aerobic microorganisms was 93%, yeast and molds - 62%. Using the proposed method, enterobacteria, Staphylococcus aureus, Pseudomonas aeruginosa were isolated from a contaminated medicinal product.

Example 5. Determination of the microbiological purity of the pharmaceutical substance insulin lispro dissolved in medium No. 8 of the timing, inoculated with a mixture of test strains in the amount of 50 CFU / g of each microorganism.

3 g of material were taken. The sample was dissolved in a liquid nutrient medium No. 8 GRM. Test strains were introduced in an amount of 50 CFU / g of each microorganism. 10 ml were taken from the inoculated sample and transferred to the timing medium No. 3 to enrich enterobacteria.

10 ml of the prepared sample of the substance insulin lyspro was introduced into 10 Petri dishes (1 ml each), 5 of which were poured with 10-15 ml of timing medium No. 1 to determine the content of aerobic microorganisms, and the remaining 5 10-15 ml of medium No. 2 Timing for the isolation of yeast and molds.

Crops on timing medium No. 3, as well as the remaining 10 ml of the sample solution, were incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes filled with timing medium No. 1 and timing No. 2 were incubated for 120 hours at temperatures (32.5 ± 2.5) ° С 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 microorganisms was calculated using formula (1).

From medium No. 3, a bacteriological loop was transferred to Endo agar, incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, after which a typical growth of enterobacteria (Escherichia coli) colonies was observed.

After the incubation period of crops in 10 ml of timing medium No. 8 with a bacteriological loop, reseeding on timing mediums No. 10 and No. 9 of timing was performed to determine Staphylococcus aureus and Pseudomonas aeruginosa, respectively, were incubated for 48 hours at a temperature of (32.5 ± 2.5) ° C, after which the growth of colonies of these microorganisms was observed.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, with which a sample of the pharmaceutical substance insulin lispro was inoculated. The recovery coefficient of aerobic microorganisms was 70%, yeast and mold fungi - 73%. Using the proposed method, enterobacteria, Staphylococcus aureus, Pseudomonas aeruginosa were isolated from a contaminated medicinal product.

Example 6. Determination of the microbiological purity of a pharmaceutical substance insulin glargine, dissolved in trypticase-soy broth, inoculated with a mixture of test strains in an amount of 5 CFU / g of each microorganism.

3 g of material were taken. The sample was dissolved in 30 ml of trypticase-soy broth. Test strains were introduced in an amount of 5 CFU / g of each microorganism. 10 ml were taken from the inoculated sample and transferred to Mossel broth.

10 ml of the prepared sample was added to 10 Petri dishes (1 ml each), 5 of which were filled with 10-15 ml of trypticase-soya agar to determine the content of aerobic microorganisms, and the remaining 5 10-15 ml of Saburo agar to isolate yeast and mold fungi.

Crops on Mossel broth, as well as the remaining 10 ml of the sample solution, were incubated at a temperature of (32.5 ± 2.5) ° C for 48 hours. Petri dishes, filled with tripticase-soy agar and Saburo agar, were incubated for 120 hours at temperatures (32 , 5 ± 2.5) ° С 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 microorganisms was calculated using formula (1).

From Mossel broth, a bacteriological loop was used to transfer to Mossel agar, incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, after which typical growth of enterobacteria colonies (Escherichia coli) was observed.

After the incubation period of the crops in 10 ml of trypticase-soy broth, the bacteriological loop carried out the transfer to Bird-Parker agar and cetrimide agar to determine Staphylococcus aureus and Pseudomonas aeruginosa, respectively, were incubated for 48 h at a temperature of (32.5 ± 2.5) ° С, then observed the growth of colonies of these microorganisms.

Result: during the test, a quantitative determination of bacteria, yeast and molds was carried out, with which a sample of the pharmaceutical substance insulin glargine was inoculated. The recovery coefficient of aerobic microorganisms was 90%, yeast and mold fungi - 64%. Using the proposed method, enterobacteria, Staphylococcus aureus, Pseudomonas aeruginosa were isolated from a contaminated medicinal product.

A comparative analysis of the proposed solution with the existing method shows that the invention allows to objectively, accurately and quickly conduct a qualitative and quantitative determination of microorganisms, provided for by the requirements of regulatory documents.

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 a pharmaceutical substance produced in a biotechnological way, comprising sample preparation of a sample of a pharmaceutical substance; reseeding and incubation on a liquid accumulating selective medium, reseeding on diagnostic nutrient media followed by biochemical identification of the isolated microorganisms, characterized in that when sample-preparing a sample of a pharmaceutical substance, trypticase-soy broth or medium No. 8 is used as a diluent.