Study on the Application of Flow Cytometry in Drug Sterility Test / 中国药师

Objective: To establish a method for the rapid detection of sterile preparations. Methods: Microbes in the sterile preparations were labeled by fluorescent dye SYTO9,and detected by flow cytometry. Meanwhile,the results from the routine examina-tion method recorded in Chinese Pharmacopoeia and those from the flow cytometry were compared. Results:The detection limit of in-jection and injection of sterile powder using flow cytometry was less than 10 cfu. It could be directly detected when the microbial con-tamination of the test samples was greater than 105cfu. When the amount of pollution was <10 cfu-104cfu, the detection time was shortened by 40%-70% when compared with that of the routine method. Conclusion:Flow cytometry used for the detection of microbi-ological contamination can shorten the time for positive result,which can be an effective supplement to the routine method.

Application of DNA-based electrochemical biosensor in rapid detection of Escherichia coli exist in licorice decoction / 中国中药杂志

A new method for detection of Escherichia coli exist in licorice decoction was developed by using DNA-based electrochemical biosensor. The thiolated capture probe was immobilized on a gold electrode at first. Then the aptamer for Escherichia coli was combined with the capture probe by hybridization. Due to the stronger interaction between the aptamer and the E. coli, the aptamer can dissociate from the capture probe in the presence of E. coli in licorice decoction. The biotinylated detection probe was hybridized with the single-strand capture probe. As a result, the electrochemical response to Escherichia coli can be measured by using differential pulse voltammetric in the presence of α-naphthyl phosphate. The plot of peak current vs. the logarithm of concentration in the range from 2.7×10² to 2.7×10⁸ CFU·mL⁻¹ displayed a linear relationship with a detection limit of 50 CFU·mL⁻¹. The relative standard deviation of 3 successive scans was 2.5%，2.1%，4.6% for 2×10²，2×10⁴，2×106：⁶ CFU·mL⁻¹ E. coli, respectively. The proposed procedure showed better specificity to E. coli in comparison to Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. In the detection of the real extractum glycyrrhizae, the results between the proposed strategy and the GB assay showed high degree of agreement, demonstrating the designed biosensor could be utilized as a powerful tool for microbial examination for traditional Chinese medicine.

Comparison of Applicability Test of Sterile Examination Method for Artemether Injection / 中国药师

Objective:To develop a more suitable sterile examination method for artemether injection in order to control its quality better. Methods:The direct inoculation and the membrane filtration was respectively used for the sterile examination of artemether in-jection. Results:In the direct inoculation method, Staphylococcus aureus, Bacillus subtilis and Clostridium perfringens grew well in the test tubes containing artemether injection in 24 h, and Escherichia coli, Aspergillus niger and Candida albicans grew well in 48 h. In the membrane filtration method, Staphylococcus aureus grew well in 24 h in the test group containing artemether injection, and the other 5 strains grew well in 48 h. Conclusion:The positive strains can grow well in the applicability test of both methods. However, due to the simpler operation and higher accuracy of membrane filtration method, it is recommended to be used for the sterile test of artemether injection.

Comparison of Applicability Test of Sterile Examination Method for Artemether Injection / 中国药师

Objective:To develop a more suitable sterile examination method for artemether injection in order to control its quality better. Methods:The direct inoculation and the membrane filtration was respectively used for the sterile examination of artemether in-jection. Results:In the direct inoculation method, Staphylococcus aureus, Bacillus subtilis and Clostridium perfringens grew well in the test tubes containing artemether injection in 24 h, and Escherichia coli, Aspergillus niger and Candida albicans grew well in 48 h. In the membrane filtration method, Staphylococcus aureus grew well in 24 h in the test group containing artemether injection, and the other 5 strains grew well in 48 h. Conclusion:The positive strains can grow well in the applicability test of both methods. However, due to the simpler operation and higher accuracy of membrane filtration method, it is recommended to be used for the sterile test of artemether injection.

Study on Sterility Test after Using Non-PVC Bivalve Soft-bag Injection in PIVAS / 中国药房

OBJECTIVE:To study sterility test after using Non-PVC bivalve soft-bag injection in PIVAS. METHODS:The test was divided into 3 groups according to the type of transfusion solution packaging and dispensing environment. Group 1 received Glucose solution using bivalve soft-bag,dispensed in PIVAS;group 2 received Glucose solution using bivalve soft-bag,dispensed in wards area;group 3 received Glucose solution using plastic bottle,dispensed in wards area. After puncturing 1,3,6,9 times (n=80),finished products placed in ward for 0,2,4,6 h(n=20),and then sterility test was conducted with membrane filtra-tion method stated in second part of Chinese Pharmacopoeia (2010 edition). Infusion contamination of 3 groups was analyzed at 9th puncture. RESULTS:The growth of bacteria was not found in group 1;the positive detection rate of group 2 and 3 were 2.5%and 3.8%(n=320). The total positive detection rates after puncturing 1,3,6,9 times were 0,0.4%,0.4%,7.5%(n=240);the positive detection rates of group 1 were all 0,those of group 2 were 0,1.25%,0,8.75%and those of group 3 were 0,0,1.25%, 13.75%(n=80). After 9 times of puncture,the positive detection rates of group 1 after placing 0,2,4,6 h were all 0,those of group 2 were 25%,5%,0,5%;those of group 3 were 5%,15%,5%,30%(n=20). CONCLUSIONS:The use of the Non-PVC bi-valve soft-bag injection in PIVAS can effectively prevent microbial contamination.

Establishment and validation of sterility test of levofloxacin lactate and sodium chloride injection / 药学实践杂志

Objective To establish a sterility test of levofloxacin lactate and sodium chloride injection .Methods The test was carried out by thin-membrane filtration method with different volume of flushing liquid or adding neutralization into culture medium according to the Appendix of Chinese Pharmacopoeia (2010 edition) .Results When 0 .1% peptone water was used as flushing liquid ,Staphylococcus aureus gave no evidence of growth until the amount of flushing liquid reached 700 ml per tube (100 ml each time) .At the same time ,all of the six positive control bacteria grew normally when there was 300 ml flushing liquid in each tube with neutralization as culture medium .Conclusion This method was simple and convenient ,suggesting that it was suitable for the sterility test of levofloxacin lactate and sodium chloride injection .

Validation for sterility test method of nitroglycerin ointment / 药学实践杂志

Objective To establish and validate a method of sterility test for nitroglycerin ointment and validate this method .Methods Ten nitroglycerin ointments of 1 g were preheated oven to 45 ℃ for sample ,and added to conical flask which containing melted span 80 ,polysorbate 80 sterile mixture and sterile glass beads ,and were shake after mixing ,the sample fully emulsified by adding to 100 ml 45 ℃ pH 7 .0 sterile sodium chloride-peptone water buffer .According to the method of mem-brane filtration ,bacteria and fungus in each membrane with 300 ml pH 7 .0 sterile sodium chloride-peptone water buffer flush , the bacteriostatic activity was eliminated .Results By the method validation ,nitroglycerin ointment sample group ,negative control group were sterile growth ,and test group in each filter of the test bacteria compared with control groups were growing well ,so the samples had no inhibitory effect or the antimicrobial effect would not take into account .Conclusion Membrane fil-tration was reliable ,which could be used for sterility test for nitroglycerin ointment .

A standardization study of pre-filtration method of the test solution in microbial test for drug products / 中国药学杂志

OBJECTIVE: To carry out standardization research on pre-filtration method for microbial test solutions of drugs and establish a new double-membrane filtration method in microbial tests. METHODS: New filters with double-membranes of different materials or pore-sizes were designed and used as pre-filters for microbial tests. The effects of test solution pre-filtration on several wild-type strains were evaluated and compared with the methods described by Chinese Pharmacopoeia. RESULTS: No statistical difference was found between the double-membrane filtration method and the pharmacopoeia method. However, filter-clogging was significantly reduced by using the double-membrane filtration method, and the double-membrane filtration method could also eliminate the antibacterial activity of the samples, thus reducing the damage or loss of microorganisms in special samples. CONCLUSION: The double-membrane filtration method meets the requirements of the Chinese Pharmacopoeia; the test results are valid and the method can be used for sterility test and microbial limit test for drug products.

Quality Assessment of Some Brands of Tetanus Toxoids Marketed in Open Markets in South-Eastern Nigeria.

Aims: This study was designed to verify the immunogenicity (potency) and the safety of tetanus toxoid vaccines marketed in three large open drug markets in South-Eastern Nigeria. Methodology: Tests for Sterility, formaldehyde concentrations, specific toxicity, endotoxin, and immunogenicity (potency) were conducted on three different brands of tetanus toxoids (Brand α - from Ariaria Drug Market, Aba-Abia State; Brand β - from Ogbete Drug Market, Enugu State; and Brand γ - from Bridge-Head Drug Market, Onitsha-Anambra State). Results: All vaccine brands studied passed the sterility testing, but did not comply with the 2011 BP specifications on free formaldehyde concentration, which stipulates that the free formaldehyde concentration should not exceed 0.02%. The three vaccine brands did not show specific, abnormal, or general toxicity, but contained different amounts of endotoxins. The result of the potency testing showed that the three brands were immunogenic and elicited specific antibodies against tetanus toxin; but brand γ was the most immunogenic since it elicited the highest titers of total IgG, IgG1, and IgG2a followed by brand α, and then brand β. Conclusion: Generally, the quality control tests carried out on these three commercial brands of tetanus toxoids marketed in Nigeria showed that they do not comply with all the pharmacopeial standards on quality and safety required for vaccines of this nature. Therefore, we conclude that some of the tetanus toxoids marketed in open drug markets in Nigeria are substandard and may be responsible for the failures of these vaccines used for immunization in the country.

Proposta de modelo de análise de risco para a implementação da Liberação Paramétrica de produtos com esterilização terminal / Proposed risk analysis model for implementation of parametric release for terminally sterilized products

A Liberação Paramétrica (L.P) é definida como a liberação de lotes de produtos submetidos à esterilização final, por meio do cumprimento de parâmetros críticos do processo de fabricação, sem a necessidade de realização do teste de esterilidade terminal A L.P é um assunto recente no Brasil e ainda não regulamentado em território nacional, de modo que, existem poucas publicações nacionais que se referem a este tema, bem como poucas informações sobre o cenário regulatório e possibilidade de aceitação da prática da L.P no país, o que torna relevante a discussão sobre o tema. Além disso, para a implementação da L.P em processos de produtos com esterilização terminal, vários aspectos relevantes devem ser considerados. No entanto, conhecer os pontos críticos do processo é essencial para a liberação de um produto no mercado de maneira paramétrica. Visando um conhecimento mais aprofundado do processo de fabricação no intuito da prática da Liberação Paramétrica, o artigo tem como objetivo (principal) propor um modelo de análise de risco para a implementação da Liberação Paramétrica de produtos com esterilização terminal. O artigo traz, ainda, uma revisão sobre a situação regulatória da L.P no Brasil, com uma breve discussão sobre a necessidade de harmonização das diretrizes para a aceitação L.P.

Parametric release is defined as the release ofterminally sterilized batches of sterile productsbased upon compliance with the defined criticalparameters of sterilization without having toperform the requirements under sterility tests. PRis a new issue in Brazil and still unregulated in thecountry. Thus, there are few national publicationsthat refer to this subject as well as limitedinformation on the regulatory scenario and theadoption of the practice of PR in the country, whichmakes it important to address the topic. In addition,for the implementation of PR processes withterminal sterilization of products several importantaspects should be considered. However, knowing thecritical points of the process is essential for releasinga product on the market in a parametric fashion.Seeking deeper understanding of the manufacturingprocess, the present article aims to propose a modelof risk analysis for the implementation of parametricrelease of products with terminal sterilization. Thearticle also reviews the regulatory status of PR inBrazil briefly discussing the importance of consistentguidelines for the adoption of PR

Antiophidian sera sterility control: topics in perspective / Controle de esterilidade de soros antiofídicos: temas em perspectiva

The aim of this work is to review the most important topics about the antiophidic sera sterility, including obtaining methods, sterilization procedures and clean room control using Vital Brazil Institute (VBI) as an example. Bibliographical research was performed through Medline, Lilacs, PubMed, ISI and the Fundação Oswaldo Cruz - RJ and VBI Libraries, from 1960 to 2009. The antiophidic sera for human use are immunobiologic products produced in Brazil by three national laboratories, including VBI. Due to the parenteral use, these products should be sterile and pyrogen-free, which demands the microbiological control during the whole fabrication process. The sterility and pyrogen tests are important steps to ensure the quality and safety of these immunobiological products. Thus, these tests are target for continue evaluation and improvement. The most interfering aspects in the consistency and analytical patterns include the proper method selection, sampling, culture conditions and validation criteria. As the national and international legal requirements are cautious with the assays validation and approval of sterile parenteral products; the intrinsic limitations for established assays still require more investigation aiming the continue improvement of the microorganism and contaminants detection methods and optimization of the analysis extent.

O objetivo deste trabalho é revisar os tópicos mais relevantes para o controle da esterilidade de soros antiofídicos, abordando-se métodos de obtenção, procedimentos de esterilização e o controle de áreas limpas utilizando como exemplo os procedimentos adotados pelo Instituto Vital Brazil (IVB). Um levantamento bibliográfico foi realizado no Medline, ISI, Biblioteca da Fundação Oswaldo Cruz-RJ e IVB, no período de 1960 a 2009. Os soros antiofídicos para uso humano são produtos imunobiológicos fabricados no Brasil por três laboratórios nacionais, dentre eles o IVB. Por serem de administração parenteral, devem ser obrigatoriamente estéreis e apirogênicos, exigindo controle microbiológico durante todo o processo de fabricação. O teste de esterilidade e apirogenia são importantes instrumentos para garantir a qualidade e segurança microbiológica desses produtos, sendo alvo de avaliações constantes para seus aprimoramentos. Os aspectos que mais interferem na sua consistência e valor analítico incluem a escolha adequada do método, amostragem, condições de cultivo e critérios de validação. À medida que os requisitos legais nacionais e internacionais se mostram rigorosos na validação de ensaios e aprovação de produtos estéreis parenterais, limitações intrínsecas ao ensaio padronizado requisitam mais investigações, objetivando o aprimoramento contínuo nos métodos de detecção de microorganisms, contaminantes e otimização do tempo total de análise.

Methodology of Sterility Test of Dexrazoxane for Injection / 中国药房

OBJECTIVE:To establish the method for sterility test of dexrazoxane for injection. METHODS: The relative procedures were conducted in accordance with the membrane-filter procedure included in sterility test specified in Chinese Pharmacopoeia (2005 edition),with Staphylococcus aureus,Pseudomonas aeruginosa,Bacillus subtilis,Clostridium sporogenes,Candida albicans and Aspergillus niger as test organisms,which were flushed with sodium chloride-peptone buffer solution (150 mL,250 mL,and 350 mL,respectively,pH 7.0) to evaluate the impact of three flushing doses on the results of sterility test. RESULTS: At a flushing dose of 150 mL,there was no growth of bacteria except Bacillus subtilis at 24～72 h;however,at a flushing dose of 250 mL or 350 mL,all bacteria strains had a good growth. CONCLUSION: The established method for sterility test of dexrazoxane for injection is accurate and reliable.

In-vitro Antibacterial Effect and Sterility Test of Danshen Injection / 中药新药与临床药理

Objective To investigate the in-vitro antibacterial effect and sterility test of Danshen injection(DI). Methods Three kinds of in-vitro antibacterial and bacteriostatic methods were used to investigate the in-vitro antibacterial effect of DI and to establish its sterility test according to the results of the antibacterial effects. By validation test, the validity of its sterility test was evaluated. Results The results of doubling dilution test showed that the minimum bacteriostatic concentration of DI is 0.098 mg/mL, and its minimum bactericidal concentration is 1. 563 mg/mL in ethanolsulfate medium and 0.195 mg/mL in nutrient meat soup medium. Rate of recovered bacterium was much more than 80% by membrane filtration method. Conclusion DI has strong in-vitro bacteriostatie effects for staphylococcus aureus and the effectual method of its sterility test is membrane filtration method.