Development of the methoD for microbiological purity testing of recombinant human interleukin-7-baseD proDuct

The goal of the work was justification of standardization parameters of the product based on recombinant human IL-7 by “microbiological purity” parameter and development of its measurement method with consideration of the requirements of guideline documents regarding quality of medicinal pro ducts. Suitability assessment of microbiological purity testing procedures was performed. It has been established that the studied product in standard testing methods possessed antimicrobial activity against test microorganisms Basillus subtilis ATCC 6633 (on soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic), Candida albicans АТСС 10231 (on soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic), and possessed no antimicrobial activity against test microorganisms Staphylococcus aureus ATCC 6538 (on soybean casein digest agar) and Pseudomonas aeruginosa ATCC 9027 (on soybean casein digest agar). The efficacy of lecithin and polysorbate as substances eliminating antibacterial effect in combination with 50-fold dilution of the test sample has been suggested and proven for the purpose of antimicrobial activity neutralization. The developed total aerobic microbial count measurement procedure for the test product conforms to acceptance criteria provided by SPU (2.6.12), and can be used for its microbiological purity control.

The goal of the work was justification of standardization parameters of the product based on recombinant human IL-7 by "microbiological purity" parameter and development of its measurement method with consideration of the requirements of guideline documents regarding quality of medicinal pro ducts.Suitability assessment of microbiological purity testing procedures was performed.It has been established that the studied product in standard testing methods possessed antimicrobial activity against test microorganisms Basillus subtilis ATCC 6633 (on soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic), Candida albicans АТСС 10231 (on soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic), and possessed no antimicrobial activity against test microorganisms Staphylococcus aureus ATCC 6538 (on soybean casein digest agar) and Pseudomonas aeruginosa ATCC 9027 (on soybean casein digest agar).The efficacy of lecithin and polysorbate as substances eliminating antibacterial effect in combination with 50-fold dilution of the test sample has been suggested and proven for the purpose of antimicrobial activity neutralization.The developed total aerobic microbial count measurement procedure for the test product conforms to acceptance criteria provided by SPU (2.6.12), and can be used for its microbiological purity control.

K e y w o r d s: antimicrobial activity, microbiological purity, recombinant human interleukin-7.
I nterleukin-7 (IL-7) is an immune cytokine playing a key role in T-and B-lymphocytes development and homeostasis, involved in development of dendritic cells, natural killers and cells-lymphoid tissue inducers, which are essential immunity links as well.IL-7 is capable of regulating immune system homeostasis due to the ability to maintain a balance between apoptosis and proliferation processes of thymocytes, naïve T-lymphocytes and memory cells, and thus maintaining the consisten cy of abundance and functional activity of these populations [1][2][3].
Recombinant proteins planned to be used in medicine for therapeutic or diagnostic purposes have to conform to specific requirements which differentiate them from the proteins used exclusively for research work [4,5].At the same time, specific features of the use of recombinant proteins also ap-ply specific requirements regarding their standardization.One of the elements of analytical standardization of pharmaceutical products is development of the methods for testing microbiological purity.
It should be noted that this work is part of a comprehensive work on the development of recombinant human IL-7 preparation and methods of its biological standardization.At previous study steps, we have scientifically justified composition and technology of the product based on recombinant human IL-7 in the form of nasal spray (drops); quality profile of such product [6,7], its biological (specific) standardization methods have been developed [8,9].
The goal of the work was scientific justification of standardization parameters of the product based on recombinant human IL-7 in the form of nasal spray (drops) by "microbiological purity" parameter and development of the method for this parameter measurement with consideration of the requirements of guideline documents regarding quality of medicinal products.

materials and methods
Nutrient media.Nutrient media (NM) in accordance with requirements of SPU (State Pharmacopeia of Ukraine) 2.0 (2.6.12,2.6.13) were used during the tests.The NM were prepared from dry nutrient media or individual ingredients; each nutrient medium batch was tested for sterility, growth and, when needed, inhibitory and indicator properties.The media list, characteristics, and media suitability control results are shown in Table 1.
Test microorganisms.Test microorganisms in accordance with requirements of SPU 2.0 (2.6.12,2.6.13) were used during the testing.The list of test microorganisms is shown in In order to prepare the starting suspensions of bacterial test strains, the broth cultures were diluted using buffer solution with sodium chloride and peptone рН 7.0 till formation of the suspension containing 10 3 to 10 4 CFU/ml of test microorga nism.
In order to prepare the starting suspension of test microorganism C. albicans, the culture was washed from the surface of Sabouraud dextrose agar using buffer solution with sodium chloride and peptone рН 7.0, and diluted with the same solvent till formation of the suspension containing 10 3 to 10 4 CFU/ml of test microorganism.
In order to prepare the starting suspension of test microorganism a. brasiliensis (niger), the culture was washed from the surface of Sabouraud dextrose agar using buffer solution with sodium chloride and peptone рН 7.0, which contained 0.05% of polysorbate-80, and diluted using buffer solution with sodium chloride and peptone рН 7.0 till formation of the suspension containing 10 3 to 10 4 CFU/ml of test microorganism.
Suitability testing of total aerobic microbial count measurement procedure.Total aerobic microbial count measurement procedure conforms to the requirements of SPU 2.0 (2.6.12).
Sample preparation: Transfer 10 ml of the product into a sterile volumetric vessel, make up the volume to 100 ml with sterile buffer solution containing sodium chloride and peptone рН 7.0, and mix thoroughly (sample А); transfer 10 ml of the product into a sterile volumetric vessel, make up the volume to 200 ml with sterile buffer solution containing sodium chloride and peptone рН 7.0, and mix thoroughly (sample B).For total aerobic microbial count (TAMC) measurement, inoculate 1 ml portions of the prepared sample B via double-layer technique Procedure suitability testing.The samples were prepared as specified by the method detailed above, using the sterile solvent.Five individual 10 ml portions were taken from the prepared sample B. Each portion was inoculated with monoculture of one of test microorganisms B. subtilis, S. aureus, P. aeruginosa, C. albicans, a. brasiliensis (niger), for which 0.1 ml of inoculums (containing 10 3 to 10 4 CFU/ml) were added to 10 ml of the sample.

T a b l e 2. Microorganisms used for testing the procedure suitability
In control experiment 0.1 ml of the inoculum of the same microorganism was added to 10 ml of sterile solvent.Each inoculated sample (experimental and control) in the amount of 1 ml was plated into 2 Petri dishes as specified in the procedure.Soybean casein digest agar was used for plating the samples.The inoculations in soybean casein digest agar were incubated at temperatures from 30 to 35 °C.Incubation of inoculations containing bacterial test strains was performed within 3 days, and incubation of inoculations containing fungal test strains was per-formed within 5 days.Two individual 10 ml portions were taken from the prepared sample A. Each portion was inoculated with monoculture of one of test microorganisms C. albicans, A. brasiliensis (niger), for which 0.1 ml of the inoculums (containing 10 3 to 10 4 CFU/ml) were added to 10 ml of the sample.In control experiment 0.1 ml of the inoculum of the same microorganism was added to 10 ml of sterile solvent; 1 ml of each inoculated sample (experimental and control) was plated into 2 Petri dishes as specified in the procedure.Sabouraud dextrose agar containing an antibiotic was used for plating the samples.Inoculations in Sabouraud dextrose agar were incubated at temperatures from 20 to 25 °C for 5 days.
To obtain statistically significant results, the testing was performed for three different product batches.

results and Discussion
Suitability of total aerobic microbial count measurement procedure.At the first stage of the work, we have carried out suitability testing of total aerobic microbial count measurement procedure, included to the Quality Control Methods draft for recombinant human IL-7 product in the form of nasal spray (drops).
Upon inoculation into NM from the product dilution 1 : 10 in phosphate buffer solution with sodium chloride and peptone pH 7.0, the product possessed antimicrobial activity against test microorganisms B. subtilis ATCC 6633 (in soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic), C. albicans АТСС 10231 (in soybean casein digest agar and Sabouraud dextrose agar con-taining an antibiotic).It has to be mentioned that, according to the recommendations of SPU 2.0 (2.6.12),during the procedure of suitability testing using plate inoculation method, mean arithmetic value of the number of colonies obtained in the presence of test sample and in the absence of test sample (in the control experiment) for each test microorganism has to differ no more than twice.
The obtained results (Table 3) stimulated us to study the potential of elimination of the product antimicrobial effect against microorganisms in soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic, using direct inoculation method.
If growth inhibition of test microorganism is observed (CFU number decrease more than twice), justification should be presented, and the standard procedure should be modified for the purpose of obtaining the probable test results.Literature [4] describes the following approaches to elimination and/ or neutralization of antimicrobial activity: first, the increase of the volume of solvent or nutrient medium; second, addition of specific or non-specific inactivators to the solvent; third, use of the membrane filtration method.A combination of the mentioned approaches can be used.The most adequate approach, in our opinion, is the use of neutralizers; it does not require additional testing steps and involvement of additional material and technical means, which is of special importance for routine testing of product batches.In view of the product formulation (including nipagin as a preservative) and with consideration of the literature data and out own experience [4,[9][10][11][12][13], we focused on the compounds potentially exerting neutralizing effect against parabens -lecithin and polysorbate.
Thus, non-specific inactivators polysorbate-80 and lecithin, which were added to the solvent and nutrient medium, as well as dilution method were used for neutralization of antimicrobial effects.Phosphate buffer solution (PBS) with sodium chloride and peptone рН 7.0 and neutralizing fluids (NF), different by polysorbate-80 and lecithin concentrations, were used as solvents for the sample preparation.
During the study of antimicrobial effect neutralization potential, the product test samples were prepared using a sterile solution, and monoculture suspension of one of the following test microorganisms: B. subtilis ATCC 6633 or C. albicans АТСС 10231, was added to the sample so that 1 ml of the inoculated sample contained about 100 CFU of test microorganism.In the control experiment, the same quantity of microorganism monoculture suspension was added to the sterile solvent.Each inoculated sample (experimental and control) in the amount of 1 ml was plated into 2 Petri dishes with soybean casein digest agar.Incubation of inoculations was performed at temperatures from 30 to 35 ºC for no more than 3 days, and the number of colonies grown on NM was calculated.Additionally, it was suggested to use the dilution 1 : 50, which assures partial neutralization of antimicrobial effects, and receipt of reliable results of viable cells calculation.Dilution increase to 1 : 100 is not correct, as in case of using such dilution during control of the product containing maximum permissible microbial count -1000, growth of no more than 10 colonies will be observed on each dish, which essentially increases the risk to obtain either false positive or false negative test results, as well as affects reliability of the obtained results.The results of studies on neutralization of the product antimicrobial effects (Table 4 and Fig. 1-2) confirm that the suggested procedure allows us to eliminate the product antimicrobial activity effectively in antimicrobial purity testing conditions.

T a b l e 3. Results of suitability testing of total aerobic microbial count measurement procedure in the product
The product microbiological purity standardization has been established in accordance with requirements of SPU 2.0 (5.1.4)as for non-sterile medicinal products for nasal use: the product may contain total aerobic microbial count (TAMC) no more than 10 2 CFU/ml, total yeast and mold count Remark: * the data of three consecutive experiments are presented (р < 0.05) (TYMC) no more than 10 1 CFU/ml, the presence of Staphylococcus aureus in 1 ml and the presence of Pseudomonas aeruginosa in 1 ml is not permitted.

T a b l e 4. Results of studying the product antimicrobial activity neutralization potential
In order to unify the product microbiological purity testing by various parameters during TAMC and TYMC measurement, as well as during tests for detection of individual microbial species (S. aureus, P. aeruginosa), we have suggested to use the same solvent (neutralizing fluid) with the same product dilution (1 : 50).It has been established that, in suggested testing conditions the product does not exert antimicrobial effects against test microorganisms S. aureus ATCC 6538 (on soybean casein digest agar) and P. aeruginosa ATCC 9027 (on soybean casein digest agar).