, Evren ALGIN YAPAR*METHOD DECISION FOR DETERMINING SPECIFIC MICROORGANISMS IN PHARMACEUTICAL PRODUCTS: AN OVERVIEW
Gökhan CENGIZ, Evren ALGIN YAPAR*
Department of Analysis and Control Laboratories, Turkish Medicines and Medical Devices Agency, 06430Çankaya, Ankara, Turkey.
INTRODUCTION
Microbiological quality control analysis in pharmaceutical products is one of the most fundamental steps in determining the quality of the pharmaceuticals. The microbiological burden of the final pharmaceutical product is important in terms of safe use and product effectiveness. In addition to the microbiological controls carried out during the manufacturing stages, ensuring the microbiological quality of the final product is the most fundamental issue in determining the quality of both the manufacturing system and the pharmaceutical product. Microbiological quality is one of the main issues of Good Manufacturing Practices and quality assurance of pharmaceutical products. Since the efficacy and safety of a product depends on its pharmaceutical quality, it is necessary to reach the quality target both in the related manufacturing stages and in the finished product. Control and proving of microbial quality depends on the application of defined tests. Microbiological quality control tests for raw materials and finished products; involves microbial enumeration tests for total aerobic microbial counts (TAMC), total yeast and mold counts (TYMC), and detection of specific microorganism. Specific microorganisms; Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella spp., Candida albicans and bile-tolerant Gram negative bacteria. Ensuring microbiological quality is very important in the use of the product due to its toxic or infectious effect. Low levels of pathogenic microorganisms can cause serious effects1. Many requirements and analysis methods have been defined in pharmacopoeias for microbiological analysis of pharmaceutical products. United States Pharmacopoeia (USP) and European Pharmacopoeia (EP) can be utilized to determine the microbiological quality of pharmacological products. According to the pharmacopoeia requirements, the products should have a certain bioburden and should not contain specific microorganisms. The microbiological quality of the products varies according to the type of the product2. In this review, microbiological quality control of pharmaceutical products is given in terms of method decision and determination of specific microorganisms, which is necessary for a comprehensive microbiological quality evaluation of pharmaceuticals.
MICROBIOLOGICAL QUALITY CONTROL METHODS IN PHARMACOPOIEAS
Internationally accepted pharmacopoeia methods are used in determining microbiological quality control. According to the EP and USP, which specifies the analysis methods and requirements of internationally accepted pharmaceutical products, the evaluation of microbiological quality in pharmaceutical products varies according to their type and route of administration. According to EP and USP, pharmaceutical products are examined in two groups as sterile and non-sterile products. Ocular and injectable products which must be sterile are subject to the microbiological quality control tests that are specified in USP 71 and EP 2.6.1 Sterility Test Section. The quality control tests of products that are not necessarily sterile are specified in USP 1111 and EP 5.1.4 Microbial Quality of Non-Sterile Pharmaceutical Preparations and Substances for Pharmaceutical Use. Quality control tests of non-sterile pharmaceutical products are subject to USP 1111 and EP 5.1.4. Bioburden is determined according to USP 61 and EP 2.6.12Microbial Enumeration Tests section; specific microorganisms in pharmaceutical products are determined according to the section of USP 62 and EP 2.6.13 Tests For Specified Microorganisms section. Microbiological quality control parameters and limits according to the administration route of pharmaceuticals are shown in Table 12,3.
Microbiological bioburden and specific micro-organisms that should not be present in the product vary according to the method of use and target. Microbiological bioburden is determined quantitatively by determining TAMC and TMYC. In addition to the microbiological bioburden, the product should not contain specific microorganisms according to the method and target of the pharmaceutical product. Specific microorganism detection is a qualitative test. While determining the microbiological quality of the final product and raw materials, the parameters specified in Table 1 should be analyzed and should be within the specified limits. While determining the microbiological quality of the product, both the microbiological and the specific microorganism quality in grams or ml of the pharmaceutical preparation are evaluated2,3.
Table 1: Microbiological quality control parameters and limits for non-sterile pharmaceutical2,3.
Specific microorganism detection in pharmaceuti-cals- Specific microorganisms are investigated in order
to determine the microbiological quality of pharmaceutical products. Recovery of media used for
specific microorganism detection is very low. For this reason, the pre-enrichment process facilitates the detection of the sought microorganism to examine the specific microorganisms. In internationally used methods, pre-enrichment is routinely applied. Pre-enrichment is the process that increasing the level of the specific microorganism. However, in addition to the microorganism sought in this process, unwanted microorganisms are also enriched. This may cause a false positive result. Bioburden, whose level is increased by the pre-enrichment process, is reduced to the sought microorganism by selective enrichment and selective subculture. Pharmacopoeia methods are generally used in the analysis of pharmaceutical products. However, the International Organization for Standardization (ISO) methods can also be used because the specific microorganisms to be analyzed are similar in cosmetic products4,5.
Specific microorganisms are generally detected in four steps;
The pharmaceutical product is included in the analysis of specific microorganism detection, according to the groups specified in Table 1. The requirements specified in internationally applied standards meet these working steps. The media and analysis steps used during the studies are similar to the pharmacopoeia methods. Dissolve process is applied to prepare the pharmaceutical flour for the analysis steps. Generally, Buffered Sodium Chloride Peptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or Soybean–Casein Digest Broth is used in the dissolve process. If necessary, adjust to pH of 6 to 8.In the dissolve process, 10 g or ml of product is transferred in 90 ml of solvent. A surface-active agent such as 1 g per L of polysorbate 80 may be added to assist in the suspension of poorly wettable substances. For fatty products, the dissolution process is accelerated with polysorbate 80 added to the solvent. Also, after the sample is added to the solvent, if necessary, the solution is heated in a water bath and mixed (not more than 40°C).When a homogeneous solution is obtained, non-selective enrichment can be started. 10 ml sample taken from the obtained solution is used in a non-selective pre-enrichment process4-7.
Non-selective Pre-enrichment
The non-selective pre-enrichment process is applied to enrich the specific microorganism sought in the analyzed pharmaceutical product. Pre-enrichment is usually done in a medium that can produce all microorganisms. In general, Tryptic Soy Broth (TSB) medium is used for pre-enrichment. Since this medium is a general productive medium, it can produce all microorganisms. In case of an antimicrobial active substance in the content of the analyzed product, the additional chemicals can be added to the medium. 1 g or ml of product analyzed in the pre-enrichment process is incubated in TSB medium at 32.5±2.5°C for 18-24 hours. As a result of incubation, microorganisms in 1 gram of product will develop and be easier to detect. In the detection of bile-tolerant Gram-negative bacteria indicated in the Table 1, 1 gram or ml of the analyzed product is transferred to TSB medium and incubated at 22.5±2.5°C for 2-5 hours. In the detection of C. albicans indicated in Table 1, 1 gram or ml of the analyzed product is transferred to Sabouraud Dextrose Broth (SDB) medium and incubated at 32,5±2,5°C for 3-5 days. TSB media can be used as an alternative for C. albicans detection4-7.
Selective Pre-enrichment
Selective pre-enrichment process is the reduction of the microorganism obtained in the non-selective pre-enrichment process. This process is not applied in all microorganism groups as specified in standard methods. It is applied in searching for E. coli and bile-tolerant Gram-negative bacteria from specific microorganisms specified in Table 1. For E. coli, 1 ml sample taken from the TSB obtained in the non-selective pre-enrichment process is transferred to the MacConkey Broth (MCB) medium. For bile-tolerant Gram-negative bacteria, 1 ml sample taken from the TSB obtained in the non-selective pre-enrichment process is transferred to the Enterobacter Enrichment Broth (EEB) medium. MCB medium is incubated at 43±1°C for 24-48 hours, EEB medium at 32.5±2.5°C for 24-48 hours. Selective subculture for other specific microorganisms is sufficient to detect the microorganism sought. For this reason, there is no need for selective pre-enrichment4-7.
Selective Subculture
Selective subculture is transferring only the sought microorganism to a medium where it can grow. With this process, only the sought microorganisms are produced and detected. In the selective subculture process, MacConkey Agar (MCA) medium is used for E. coli detection and the MCA medium is incubated at 32.5±2.5°C for 18-72 hours; Mannitol Salt Agar (MSA) medium is used for S. aureus detection and the MSA medium is incubated at 32.5±2.5°C for 18-72 hours; Cetrimide Agar (CA) medium is used for P. aeruginosa detection and the CA medium is incubated at 32.5±2.5°C for 18-72 hours; Sabouraud Dextorose Agar (SDA) medium is used for C. albicans detection and the SDA medium is incubated at 32.5±2.5°C for 24-48 hours; Violet Red Bile Glucose Agar (VRBGA) medium is used for bile-tolerant Gram-negative bacteria detection and the VRBGA medium is incubated at 32.5±2.5°C for 18-24 hours. Colonies with typical signs of growth on the plates after incubation are taken for an identification test4-7. The above analysis steps have been evaluated in terms of international standards. The comparison is given in Table 24-11.
Identification
Identification tests are the process of verifying the positive result obtained. In this process, the colonies detected as positive on the medium are identified biologically, morphologically or biochemically. Identification tests are determination of colony morphology, determination of cellular morphology, Gram staining or other differential staining techniques, and basic biochemical reactions.
Table 2: Specific microorganism detection assessment according to standards4-11.
1Alternatively, a medium can be used Soybean Casein Lecithin Poysorbate 80 medium. 2Alternatively, a medium can be used TSB medium.
3Alternatively, a medium can be used MSA medium. 4Alternatively, a medium can be used Soybean Casein Lecithin Poysorbate 80 medium.
5Alternatively, a medium can be used CA medium. 6Alternatively, a medium can be used TSB medium
Table 3: According to international standards, results considered positive in the selective subculture procedure8-12.
For the identification test, positive results specified in the standards must be observed. Identification test should be applied to detect E. coli, S. aureus, C. albicans and P. aeruginosa according to EP and USP.
However, the details of the identification tests are not specified. For other specific microorganisms, the presence of colonies during the sub-culturing process is sufficient for detection and typical colonies that should be seen on the media as a result of sub-culturing were not specified. In addition, identification tests in USP are defined by USP 1113 Microbial Characterization, Identification and Strain Typing. However, the test and positive results to be performed were not specified. For this reason, general standards can be used for identification tests. In the ISO standards, which include cosmetic products, typical colony characteristics seen with the subculture process are defined12. According to USP 1113, the first step of identification is to obtain a pure culture. This process ensures that sufficient culture is formed in the subsequent identification process. Test details are given in Table 412.
Table 4: Identification tests according to USP11,12,13.
Identification tests according to USP 1113
Gram and Spore Staining: Gram staining is a 4-step identification procedure; crystal violet (primary stain), iodine (mordant), alcohol or alcohol– acetone (decolorizer), and safranin (counterstain). Dye removal is applied at each staining step and Gram-positive organisms retain the purple-blue color, Gram-negative organisms appear red. Malachite green stain can be used in the spore staining process12.
Biochemical Screening: Basically, oxidase test, catalase test and coagulase test can be used in biochemical identification tests. Gram negative rods into non-fermenters bacteria oxidase positive; enteric bacteria are oxidase negative. Staphylococci and Streptococci are separatedfrom the calatase test. Staphylococci catalase negative, Streptococci catalase positive. Coagulase testing can be used to separate Staphylococci as coagulase negative and coagulase positive Staphylococci12.
Identification tests according to ISO standards
Identification tests for determination of below given specific microorganisms are briefly explained according to ISO standards.
Identification of Escerichia coli; Gram staining Gram-negative rods, metallic sheen under reflected light and a blue-black appearance under transmitted light colonies should be seen by subculture on Eosin-Methylene Blue Agar Medium (EMB) medium (30-35°C for 24-48 h)8.
Identification of S. aureus; Gram staining Gram-positive cocci, catalase positive test, coagulase positive test9.
Identification of P. aeruginosa; Gram staining Gram-negative rods, oxidase positive test. Inoculate the surface of the Pseudomonas agar medium for the detection of pyocyanin with suspect isolated colonies grown on cetrimide agar medium, so that individual colonies develop. Incubate at 30-35°C. Check for bacterial growth after 24 h, 48 h and 72 h. P. aeruginosa forms colonies with a red to dark brown zone due to pyorubin production10.
Identification of C. albicans; Gram staining, the microscopic observation shall reveal a violet colour, short ovoid or elongated cells, sometimes with budding cells. Germ tube production; the formation of germ tubes characterizes the presence of C. albicans11.
Method Suitability and Growth Promotion Tests
In the method suitability study, it is aimed to detect the target microorganism group. The analyzed pharmaceutical product has no negative effect on the detection of specific microorganisms. In method, suitability studies, non-selective pre-enrichment process should be performed by inoculating the target and inhibited microorganism specified in Table 5 to the sample prepared by dissolving process.
Table 5: Method suitability test target and inhibitory microorganisms4-7.
As a result of the procedures performed, the tests in which the target microorganisms were inoculated should be positive, and the tests in which the inhibited microorganisms were inoculated should be negative. Microorganism inoculation amounts in all test groups should be 10-100 colony forming units (cfu)4-7.
In the growth promotion test, all the media used in the analysis should be analyzed with the microorganisms specified in Table 5. In the growth promotion test, target microorganisms should be visible signs of growth as a result of incubation, and inhibitory microorganisms should not be a growth sign. The TSB medium used in the non-selective pre-enrichment process should be analyzed with 5 different microorganisms in the growth promotion test. Since the medium used is the general producer medium, it must be controlled with 5 different microorganisms. Control microorganisms for TSB are, S. aureus ATCC 6538, P. aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633, C. albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404. Microorganism inoculation amounts in all test groups should be 10-100 cfu/mL. Incubation temperatures and times in method suitability and growth promotion tests are shown in Table 24-7.
Alternative Identification Test Methods for Dedection of Specific Microorganisms
Alternative methods can be used besides traditional methods for the detection of specific microorganisms in pharmaceutical products. All analysis steps are similar for the detection of specific microorganisms.
However, fast alternative methods can be used for the identification test. All analysis steps should be applied prior to the identification test. These alternative methods can only be applied at the identification test step. Because pure culture is required for the identification step. Traditional methods specified in standard methods or alternative methods can be used for the identification of pure culture. Microorganisms in pure culture obtained by alternative methods can be detected by biochemical, fatty acid methyl ester analysis, MALDI-TOF mass-spectrometry, Fourier transform–Infrared (FT-IR) spectroscopy, nucleic acid extraction, PCR amplification and rRNA base sequencing and ribotyping methods. Many test kits or identification analysis systems have been developed for these methods. Traditional methods used for diagnosis may vary in the phenotypic properties of microorganism. Identification tests performed with phenotypic methods may cause false results due to the influence of microorganisms from environmental conditions. For this reason, alternative identification methods can be used. Generally, phenotypic methods require more pure microorganism cultures than other methods. In addition, the microorganism phenotype may change due to the medium used in the detection of specific microorganisms, incubation conditions, and the antimicrobial substances in the medium. For this reason, it is necessary to use alternative methods to phenotypic analysis methods13,14. All the alternative identification test methods should be confirmed by validation procedures.
Table 6: Validation of alternative qualitative tests for the presence or absence of microorganisms15.
Alternative microbiological methods are listed below15:
A. Growth-Based Methods
1. General critical aspects of methods based on early detection of growth
2. Electrochemical methods
3. Measurement of consumption or production of gas
4. Bioluminescence
5. Turbidimetry
6. Growth detection using selective and/or indicative media
B. Direct Measurement
1. Solid phase cytometry
2. Flow cytometry
3. Direct epifluorescent filtration technique (DEFT)
4. Autofluorescence
C. Cell Component Analysis
1. Phenotypic techniques
1.1. Immunological methods
1.2. Fatty acid profiles
1.3. Fourier transform infrared (FTIR) spectroscopy
1.4. Mass spectrometry
1.5. Biochemical assays based on physiological reactions
2. Genotypic techniques
2-1. Direct hybridisation
2-2. Nucleic acid amplification techniques (NAT)
2-3. Genetic fingerprinting
Validation of Alternative Microbiological Methods
Microbiological methods to be used as an alternative need to be validated in order to be applied in quality control analysis. Specific microorganism detection is a qualitative test. For this reason, the accuracy of the studies should be confirmed with certain analyzes in the validation study. This is also the case for identification tests. Alternative fast test systems are frequently used. For this reason, the validation of the systems has gained importance. The validation criteria of alternative methods for the detection of specific microorganisms are shown in Table 615.
Critical validation stages are briefly explained below.
Specificity: In an alternative qualitative test, specificity is the ability to detect only desired microorganisms, i.e., the test will not give a false positive result. This can be done using a suitable microorganism table. Detection Limit: The detection limit of an alternative qualitative method is equal to the lowest number of microorganisms that can be detected in a sample under specified analytical conditions. The microbiological limit test determines the presence or absence of microorganisms in a defined amount of the sample tested.
Robustness: The robustness of an alternative method is a measure of its capacity to remain unaffected by small but intentional changes in method parameters (e.g. incubation time or incubation temperature range).
Suitability testing: The alternative method should be applied in accordance with the specified application and with the samples to be analyzed under the responsibility of the users. It should show that the test sample does not interfere with the detection capability of the system or microbial recovery.
Equivalence testing: The equivalence testing of two qualitative methods can be done directly through the validation parameters. With this approach, testing microorganisms must be performed in sufficient replicates with low levels of inoculation (eg, less than 5 cfu).
CONCLUSION
In microbiological quality control analysis of pharmaceutical products, specific microorganism detection is very important. Detailed methods for the detection of specific microorganisms are described in the pharmacopoeias. In pharmacopoeia methods, the preparation of the product, media and incubation conditions are described in detail. In pharmaceutical products, neutralization methods of cosmetic products can be used to eliminate the antimicrobial effect. When standard methods are compared, they are mostly similar. However, the media used vary. Identification tests of microorganisms are specified in ISO standards. However, there is no definition for identification tests in pharmacopoeia methods. It is seen that general identification tests are specified only in USP. Especially in identification tests, there are deficiencies about how to determine positive results and there are deficiencies about how to determine positive results. Nowadays, alternative rapid identification tests have been developed for identification tests. However, validation is a must in order to use these systems in quality control analysis. Validation of alternative rapid test systems complicates the process. However, it shortens the analysis time. Alternative rapid test systems provide convenience in the identification phase, but these systems may also require the desired microorganism culture. For this reason, alternative rapid test systems also require traditional microbiological methods.
AUTHORS’ CONTRIBUTIONS
Both of the authors substantially and equally contributed to the conception and design of the article and interpreting the relevant literature. While Gökhan CENGIZ drafted the article, Evren ALGIN YAPAR revised it critically for important intellectual content.
CONFLICTS OF INTEREST
The authors have no conflicts to report.
REFERENCES