无菌生产容器完整性测试方法

Microbial Ingress Test 1. Objective - To describe a procedure for verifying the integrity of container/closure seal is satisfactory by challenging with viable, motile bacteria. 2. Materials - Standard microbiological laboratory instrument - Test organism: 18-24 hours fresh culture of Escherichia Coli CMCC 44102 - Cell suspension: TSB medium - Immersion chamber - Test samples: 30 vials (sampled at beginning, middle and end of capping) of TSB filled, stopped and capped. - 0.5% peracetic acid solutions and 70% alcohol 3. Test procedure 3.1 Vials tested preparation - Fill a sufficient number of container with TSB to the normal fill volume on the production line by performing aseptic process and seal them using automatic stopper and capping equipment; - Sampled vials of TSB filled at beginning, middle and end of capping process; - Invert all vials to allow the medium to contact all internal surfaces, and then incubate them at least for totally 14 days at 30~35? and 20~25? separately; 3.2 Verification of medium growth promoting properties - At the end of incubation, check all vials and discard any vials that might have been contaminated. - After the container have incubated successfully for 14 days with no contamination, randomly select 5 capped vials and inoculate them with 0.1 ml of Escherichia Coli CMCC 44102 suspension adjusted to 10-100 CFU; - Incubate the inoculated containers at 30 to 35? for 7 days or until growth appears, whichever occurs first. - The medium in the containers is satisfactory if growth of the test organisms appears within 7 days in all the inoculated containers; 3.3Preparation of challenge microorganism suspension - Transfer a loopful of culture from a fresh slant of Escherichia Coli CMCC 44102 to a tube containing 10 ml of sterile TSB; incubate 16-18 hours at 30-35?; - Transfer the entire contents of TSB tube to bottle containing 1000 ml TSB medium and incubate 22-24 hours at 30-35?;The medium should be visibly turbid at the end of the incubation period. Use the cell suspension to challenge container/closure integrity. 3.4 Ingress test procedure - Add the freshly grown Escherichia Coli CMCC 44102 cell suspension to a suitable vessel; - Remove 1 ml of the cell suspension at the beginning of the exposure period, determine the number of viable cells/ml by standard plate count technique, and confirm that they are Escherichia Coli by performing Gram staining; Invert 30 vials and immerse them in the cell suspension, and the outer closure surface and container neck should be immersed in the cell suspension completely; - All vials must be inverted in the suspension for at least 4 hours; - At the end of exposure time, carefully remove the vials from the suspension; allow excess cell suspension to drain from the vials. Wipe them dry, and then disinfect them with a solution of 0.5% peracetic acid in 70% ethanol; Similarly disinfect two medium-filled vials that were not immersed in Escherichia Coli 44102 suspension, inoculate them with 10-100CFU of Escherichia Coli CMCC CMCC 44102 to perform growth promotion test. - Place the disinfected vials in plastic bags and incubate them at 30-35? for 7 days; - Autoclave the Escherichia Coli cell suspension and articles used in the challenge test before discarding them; 4. Reading of results - At the end time of incubation, examine the medium inside the containers for evidence of microbial growth; - Record growth (+) or no growth for each vial on recording sheet; - If growth is observed in any container, confirm that the challenge organism causes it by performing Gram staining or identification; - If all containers appear to sterile, repeat the growth promotion test on 5 vials selected at random from all vials tested. 5. Evaluation of results - Growth promotion test of the vials before challenged, after disinfected and incubated must be successful for the challenge to be valid; -The suspension of Escherichia Coli cells used to challenge sealed containers 6must contain at least 1 ×10 viable cells per ml at the beginning of the challenge; - The challenge study fails if any of the sealed vials exhibit growth of the challenge organisms and ingress was not the result of obvious physical defect in the container/closure seal.