中国药典2015版《无菌及微生物方法学验证基本原理》英文版

1、1100 Biological Tests1101 Sterility TestsTest for sterility is a method to detect whether medicines, biologies, medical devices, raw materials, excipients or other articles, which are required to be sterile according to the Pharmacopoeia, are sterile. However, a result conforming with the requiremen

2、ts only indicates that no contaminating micro-organism has been found in the sample examined under the conditions of the test.Test for sterility should be carried out in aseptic conditions. Test environment must meet the requirements of sterility testing. The whole process should be performed under

3、strictly aseptic conditions to avoid any microbial contamination. The precautions taken to avoid contamination are such that they do not affect any microorganisms that are to be revealed in the test. The cleanliness of working areas including laminar flow cabinet, working bench, background room shou

4、ld be monitored regularly according to the current national standard such as the detecting method for airborne particles, airborne microbe and settling microbe in the clean room (area) of the pharmaceutical industry. The isolator should be validated regularly according to the relevant requirements,

5、and the cleanliness of inner isolator space should also comply with aseptic requirements. The working conditions in which the tests are performed are monitored at routine inspection.Culture MediaFluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will

6、 also detect aerobic bacteria. Soya-bean casein digest medium is suitable for the culture of both fungi and aerobic bacteria.Media preparation and incubation conditionMedia for the test may be prepared as described in the below formulations, and dehydrated media or prepared media may be used provide

7、d that they have the same ingredients and comply with the requirements. Media should be sterilized using a validated process. Store at a temperature between 2C and 25C and protect from light. If the prepared media are stored in unsealed containers, they should be used within 3 weeks. If stored in su

8、itable sealed containers, the media should be used within 1 year.1. Fluid thioglycollate mediumPancreatic digest of casein15. 0 gYeast extract5. 0 gGlucose anhydrous5. 0 gL-Cystine0. 5 g Sodium thioglycollateor (Thioglycollic acid)0. 5 g(0. 3 ml)Sodium chloride2. 5 g0.1% Resazurin sodium solution (f

9、reshly prepared) 1. 0 mlAgar0. 75 gWater1000 mlMix the above ingredients except glucose and resazurin solution with water to dissolve by warming gently, adjust the pH to slightly alkaline, boil and filter, add glucose and resazurin solution, mix, adjust the pH so that after sterilization the solutio

10、n will have a pH of 7. 10. 2 at 25C. Dispense the medium in suitable containers which provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a color change (pink color) indicative of oxygen uptake at the end of the incubation period. Steriliz

11、e using a validated process. When ready for use, not more than the upper one-fifth of the medium has acquired a pink color, otherwise, the medium can be restored only once by heating the containers in a water-bath until the pink color disappears (not more than 20 minutes), cooled quickly, taken care

12、 to prevent the introduction of non-sterile air into the container.Unless otherwise specified, Fluid Thioglycollate Medium is to be incubated at 30-35C.2. Soya-bean casein digest mediumPancreatic digest of casein17.0gPapaic digest of soya-bean meal3.0gGlucose monohydrate/anhydrous2.5g/2.3gSodium chl

13、oride5.0gDipotassium hydrogen phosphate2.5gWater1000mlMix the above ingredients except glucose to dissolve by warming gently, filter, adjust the pH so that after sterilization the solution will have a pH of 7.30.2 at 25C, add glucose, dispense and sterilize.Soya-bean casein digest medium is to be in

14、cubated at 20- 25C.3. Neutralizing or inactivating mediumBefore sterilization or inoculation, add proper quantity of suitable neutralizer or inactivator or surface-active substance to each of the media described above. The quantity of neutralizer or inactivator or surface-active substance adopted in

15、 sterility test should be the same as that used in validation test.4. 0.5% glucose broth medium (used in the test of antibiotic such as Streptomycin sulfate)Peptone10. 0 gBeef powder3. 0 gGlucose5. 0 gSodium chloride5.0gWater1000mlMix the above ingredients except glucose with water to dissolve by wa

16、rming gently, adjust the pH to slightly alkaline, boil, add glucose, mix well, filter, adjust the pH so that after sterilization the solution will have a pH of 7. 20.2 at 25C, dispense and sterilize.5. Soya-bean casein digest agar mediumPancreatic digest of casein15. 0 gPapaic digest of soya-bean me

17、al5. 0 gSodium chloride5.0gAgar15.0gWater1000mlMix the above ingredients except agar to tepidly dissolve, adjust the pH so that after sterilization the solution will have a pH of 7.30.2 at 25C, add agar, heat to melting, shake well, dispense, and sterilize.6. Sabouraud dextrose broth mediumMixture o

18、f equal amounts of animal tissue pepsin hydrolyzate and Tryptone10. 0 gDextrose20. 0 gWater1000mlMix above ingredients except glucose to tepidly dissolved, adjust the pH so that pH value after sterilization at 25C is 5.60.2, add dextrose, shake well, dispense and sterilize.7. Sabouraud dextrose agar

19、 mediumMixture of equal amounts of animal tissue pepsin hydrolyzate and Tryptone10. 0 gDextrose40. 0 gAgar15.0gWater1000mlMix above ingredients except dextrose and agar to tepidly dissolve, adjust the pH so that pH value after sterilization at 25C is 5.6 0.2, add agar, heat to melting, then add dext

20、rose, shake well, dispense and sterilize.Suitability tests of mediumThe fluid thioglycollate medium, soya-bean casein digest medium and the other media used in the test for sterility, as described above, should comply with the following sterility and sensitivity tests, carried out before or in paral

21、lel with the test on the product to be examined.Sterility tests Incubate not less than 5 vessels of each batch of sterilized medium at the temperature required for each medium for 14 days. Growth of microorganisms should not occur.Test for sensitivity of mediumTest strains The viable microorganisms

22、used in the test must not be more than five generations (the dried strains obtained from Culture Collection Centre are defined as Generation 0). The suitable seed-stock technique should be used so that the microorganism characters can be maintained.Staphylococcus aureusCMCG (B) 26003Pseudomonas aeru

23、ginosaCMCC (B) 10104Bacillus subtilisCMCC (B) 63501Clostridium sporogellesCMCC (B) 64941Candida albicansCMCC (F) 98001Aspergillus nigerCMCC (F) 98003Preparation of inoculum Inoculate freshly cultured Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis into soya-bean casein digest medium

24、 or soya-bean casein digest agar medium and freshly cultured Clostridium sporogelles into fluid thioglycollate medium, incubate at 30- 35C for 18-24 hours. Inoculate freshly cultured Candida albicans into sabouraud dextrose broth medium or sabouraud dextrose agar medium, incubate at 20-25C for 24-48

25、 hours. Dilute the above cultures to produce a suspension, containing less than 100 CFU microorganisms per ml, with pH 7.0 sterile sodium chloride-peptone buffer or 0.9% sterile sodium chloride solution. Inoculate freshly cultured Aspergillus niger into sabouraud dextrose agar medium, incubate at 20

26、-25C for 5-7 days until good sporulation is obtained Wash the Aspergillus niger spore culture with 3-5 ml of pH 7.0 sterile sodium chloride-peptone buffer or 0. 9% sterile sodium chloride solution containing 0.05% (ml/ml) of polysorbate 80 and transfer it to sterile tube. Prepare spore suspension co

27、ntaining less than 100 CFU per ml with pH 7.0 sterile sodium chloride-peptone buffer or 0.9% sterile sodium chloride solution containing 0.05% (ml/ml) of polysorbate 80.Use the above suspensions within 2 hours if stored at ambient temperature, or within 24 hours if stored between 2-8C. The stable sp

28、ore suspension of Aspergillus niger may be maintained at 2- 8C for a validated period of time.Inoculation of medium Take 7 containers of fluid thioglycollate medium (containing 12 ml medium per container), inoculate 2 containers of the medium with less than 100 CFU test microorganisms of Staphylococ

29、cus aureus, Pseudomonas aeruginosa, and Clostridium sporogenes respectively, and the remaining uninoculated culture medium (one container) is used as blank control, then incubate for 3 days. Take 7 containers of soya-bean casein digest medium (containing 9 ml medium per container), inoculate 2 conta

30、iners of the medium with less than 100 CFU test microorganisms of Bacillus subtilis, Candida albicans and Aspergillus niger, respectively, and use the remaining uninoculated culture medium (one container) as blank control, incubate for 5 days. Observe the test containers for growth of the microorgan

31、isms every day during the incubation.Interpretation of Results No growth of microorganisms shall be observed in the blank control. If a clearly visible growth of the microorganisms occurs in an inoculated culture medium, the medium meets the requirement of the test for sensitivity of medium.Diluents

32、, Rinsing Fluids and Their Preparation MethodsPrepare diluents and rinsing fluids and sterilize using a validated process.1. 0. 1% sterile peptone solution Dissolve tepidly 1. 0g of peptone in 1000 ml of water, filter, adjust the pH to 7.10. 2, dispense and sterilize.2. pH 7.0 sterile sodium chlorid

33、e-peptone buffer Mix 3. 56 g of potassium dihydrogen phosphate, 5.77 g of anhydrous disodium hydrogen phosphate, 4.30 g of sodium chloride and 1. 00 g of peptone with 1000 ml of water to tepidly dissolve, filter, dispense and sterilize.According to the nature of the product being examined, other sui

34、table solutions which are validated may also be adopted (e. g., 0. 9% sterile sodium chloride solution).If necessary, add suitable neutralizer or surface-active substance to each of the diluents or rinsing fluids described above before or after sterilizationMethod Suitability TestIn the course of st

35、erility testing method for product to be examined, the method must be verified to ensure that the adopted method is suitable for sterility test of the product. Whenever there is a change of test procedure or drug product which might impact the test result, the testing method must be revalidated.Meth

36、od suitability test is to be conducted as described below under Test for sterility of the product being examined using exactly the same methods and the following instructions. The tests should be performed separately for each of the microorganism tested.Tested strains and preparation of inoculum Exc

37、ept for Escherichia coli CMCC (B) 44102, preparation of the strains and the inoculums of Staphylococcus aureus, Badllus subtilis, Clostridium sporogenes, Candida albicans, and Aspergillus niger is the same as described in the test for sensitivity of medium. Preparation of Escherichia coli inoculums

38、is the same as Staphylococcus aureus.Membrane filtration Filter the specified quantity of the test specimen with membrane filtration apparatus, rinse the membrane, add test microorganism with less than 100 CFU to the final portion of rinsing fluids, filter again. Add fluid thioglycollate medium or s

39、oya-bean casein digest medium to the filtration apparatus. Use another container containing the same volume of the medium, then add the same amount of test microorganisms as control. Incubate the containers at specified temperature for not more than 5 days. Repeat the procedure for each of the test

40、microorganism tested.Direct inoculation Take 6 containers containing certain required volume of fluid thioglycollate medium for direct inoculation, inoculate 2 containers of medium with less than 100 CFU test microorganisms of Staphylococcus aureus, Escherichia coli, and Clostridium sporogenes separ

41、ately. Take 6 containers of soya-bean casein digest medium complying with the required volume for direct inoculation, inoculate 2 containers of the medium with less than 100 CFU test microorganisms of Bacillus subtilis, Candida albicans and Aspergillus niger separately. Add specified quantity of the

42、 product being tested to one of the inoculated containers of each test microorganism, the other inoculated container is for control. Incubate the bacterial containers at specified temperature for not more than 5 days.Interpretation of Results Compare with the control container, if clearly visible gr

43、owth of each test microorganisms is obtained in the test containers containing the product to be tested, visually either the product possesses no antimicrobial activity under the conditions of the test, or such activity has been satisfactorily eliminated. The test for sterility of the product being

44、examined may then be carried out using the same method and conditions of the test. If the growth of test microorganisms is not obtained, or poor, or slow in any tested container, visually comparable to that in the control container without product, then the product with the specified quantity posses

45、ses antimicrobial activity under the conditions of the Test. In such a case, modify the conditions in order to eliminate the antimicrobial activity by using a large amount of rinsing fluid, or increasing the volume of medium, or using suitable neutralizer or inactivator, or replacing the type of the

46、 membrane filter used etc. and repeat the validation test.The method suitability test may also be performed simultaneously with the test for sterility of the product being examined.Test for Sterility of the Product Being ExaminedThe test for sterility is carried out by the membrane filtration method

47、 or direct inoculation method. The membrane filtration method is used whenever the nature of the product permits. The same method and experimental conditions should be adopted in sterility test as that validated in method suitability test.If surface-active substances, neutralizers or inactivators, e

48、tc., are used during the test, their efficacy and their absence of toxicity for microorganisms must be demonstrated.Number of products to be tested It is the number of the minimum package of the product to be examined for sterility test once. Unless otherwise specified, the number of the products to

49、 be tested is specified in Table 1. The number of final product to be tested for post-marketing surveillance is specified in Table 2. The number of the products for positive control test is not included in table 1 and 2.Quantity of product to be tested It is the minimum quantity (g or ml) of product

50、s tested each minimum packaging inoculated into each medium for sterility test once. Unless otherwise specified, the minimum quantity of each container of the product for each medium are defined in Table 3. If the contents in a single container (bottle) are sufficient to inoculate two containers of

51、medium, they should be transferred to fluid thioglycollate medium and soya-bean casein digest medium respectively. When using the technique of membrane filtration, whenever permitted, the whole contents of each container should be filtered for testing.Positive control The microorganisms for positive

52、 control should be selected according to the nature of the product being examined. Staphylococcus aureus is used for the product possessing no antimicrobial activity or mainly anti-gram-positive-bacteria activity. Encherichia coli is used for the product mainly possessing anti-gram-negative-bacteria

53、 activity. Clostridium sporogenes is used for the product possessing anti-anaerobic bacteria activity. Candida albicans is used for the product possessing fungistatic activity. Preparation of the inoculums for positive control is the same as described under the method suitability test, the number of

54、 test microorganism added should be less than 100 CFU, the quantity of the product used is the same as described under the test for sterility of the product being examined for each container of medium. Incubate the positive control container for 72 hours, the growth of the test microorganisms should

55、 be well.Negative control In the course of the sterility test for examined product, a negative control should be performed with the same solvent or diluents or rinsing fluids as used for the test. There must be no growth of microorganisms.Processing of test and medium inoculationBefore opening the p

56、roduct containers to be tested, the exterior surfaces of containers must be thoroughly cleansed with a suitable disinfectant. If the containers are packaged under vacuum, sterile air should be transmitted into the container aseptically with suitable equipments (e. g., needles with sterilizing filter

57、) before the container is opened to release the contents.Unless otherwise specified, processing of test and medium inoculation should be carried out as described below.1. Membrane nitration methodSealed sterility testing system should be used in the technique of membrane filtration. The membrane use

58、d in the test for sterility has a nominal pore size which is not greater than 0. 45 m and a diameter about 50 mm. Selection of filtration membrane type should be performed according to the characters of product to be tested and solvent used. Integrity of the membrane in the apparatus should be kept

59、during the process filtration.When filtering aqueous solution, the membrane in the filter should be pre-wetted with a small quantity of rinsing fluids. When the samples tested are oily preparations, the filtration apparatus and membrane must be thoroughly dried before use. The level of the sample so

60、lution and rinsing fluid should always cover the entire surface of the membrane throughout the operation for maximal filtration efficiency. If necessary, wash the membrane by filtering through it 100 ml of rinsing fluid each time, and the total volume used should not exceed 1000 ml for one single me