Sterility testing sampling

Sterility test samples from the prescribed times/ locations should be divided between test samples and reserve samples, except that reserve (or retest) samples need not be taken at the same time as initial samples for the test for sterility if the above requirements for taking representative samples across the lot are met... 

Written specifications and procedures must be established for sampling of products for sterility testing. Samples for sterility testing must be selected at intervals during the operation. 

A sterility test is basically a test which assesses whether a sterilized pharmaceutical or medical product is free from contaminating microorganisms, by incubation of either the whole or a part of that product with a nuhient medium. It thus becomes a destructive test and raises the question as to its suitability for testing large, expensive or delicate products or equipment. Furthermore, by its very nature such a test is a statistical process in which part of a batch is randomly sampled and the chance of the batch being passed for use then depends on the sample passing the sterility test. 

The purpose of a sterility test is to determine the probable sterility of a specific batch. The USP lists the procedural details for sterility testing and the sample sizes required [1], The USP official tests are the direct (or culture tube inoculation) method and the membrane filtration method. 

The interpretation of sterility results is divided into two stages by the USP relative to the type of sterility failure if one occurs. If sterility failure of the test samples occurred because of improper aseptic technique or as a fault of the test itself, stage 1 may be repeated with the same sample size. Sample size is doubled in a stage 2 testing, which is performed if microbial growth is observed in stage 1 and there is no reason to believe that the test was invalid. The only absolute method to guarantee the sterility of a batch would be to test every vial or ampoule. 

AmB formulations were dispersed in phosphate-buffered saline (PBS) at different concentrations (0.1 lOOpg/mL) and incubated for five minutes at 37°C. Freshly isolated human erythrocytes were then added to a final hematocrit of 2% and incubated at the same temperature for 30 minutes. After centrifugation, the supernatant was removed and the RBC pellet was lysed with sterile water. The hemoglobin remaining in the pellet was estimated from its absorption at 560 nm recorded with a spectrophotometer. The percentage hemolysis was calculated from the difference between the hemoglobin remaining in the test samples and the control incubated with PBS alone. 

The sterility test is applicable for determining whether drug substances, preparations, or other pharmacopeial articles are sterile as defined by the compendial method. A satisfactory result only indicates that no contaminating microorganisms have been found in the sample examined rmder the conditions of the test. Therefore, the result is a function of the efficiency of the adopted sampling plan. Compendial references to sterility testing include USP 24 Chapter (71) Sterility Tests the Ph. Eur. 3rd ed.. Biological Tests 2.6.1, Sterility and the JP Xlll 45, Sterility Test. 

The number of samples tested should refleet the sampling requirements provided in the USP (71) Sterility Tests. 

Samples of drug product are sampled for sterility testing per manufacturing site SOP, Collection of Finished Product Samples, included as (provide reference attachment number). 

GUIDELINE OF NUMBER OF SAMPLING POINTS FOR AIR MONITORING OF STERILITY TESTING ROOM... 

During each working session (i.e., that uninterrupted period of time in which a sample or group of samples is tested) in which sterility testing is carried out, at least ten negative product control containers should be tested. For a direct inoculation test, these controls should be tested when possible at regular intervals during the test session. 

Review sterility test method and handling of samples. 

Sterility and pyrogenicity testing. For these tests, samples of eluates were obtained from 7 generators. Samples of the first elution and the final elution (48 hr after heavy clinical use) were obtained from two generators. All samples were... 

Unlike many dosage form specifications, the sterility specification is an absolute value. A product is either sterile or nonsterile. Historically, judgment of sterility has relied on an official compendial sterility test however, end-product sterility testing suffers from a myriad of limitations [1-4], The most obvious limitation is the nature of the sterility test. It is a destructive test thus, it depends on the statistical selection of a random sample of the whole lot. Uncertainty will always exist as to whether or not the sample unequivocally represents the whole. If it were known that one unit out of 1000 units was contaminated (i.e., contamination rate = 0.1%) and 20 units were randomly sampled out of those 1000 units, the probability of that one contaminated unit being included in those 20 samples is 0.02 [5], In other words, the chances are only 2% that the contaminated unit would be selected as part of the 20 representative samples of the whole 1000-unit lot. 

Even if the contaminated unit were one of the 20 samples selected for the sterility test, the possibility still exists that the sterility test would fail to detect... 

Production equipment that cannot be sterilized must be sanitized and disinfected by an appropriate method. This can be done by use of biocides like alcohols (70%), hydrogen peroxide, or formaldehyde-based chemicals or a combination of these. These can either be used for surface disinfections by wiping or spraying or even better by use of gas or dry fog systems for application of the disinfectants. The effect of cleaning and sanitation should be monitored. Microbiological media contact plates can be used to test critical surfaces, as inside the hot cells or glove boxes. The test samples must then be handled and monitored as radioactive contaminated units. 

Once the materials have been sterilized, interventions near either the formulation or product contact surfaces/parts should be minimized. Direct handling of these materials should only be done with sterilized tools or implements nonsterile objects, such as operator gloves, should never directly contact a sterilized surface. Sampling, filter integrity testing, process connection, and other activities should all be designed to eliminate the need for personnel exposure to sterile items. 

The test may also be conducted with natural sediment simulating the conditions in the sediment compartment. Moreover, by sterilizing the samples, the abiotic degradation under the test conditions can be determined. 

Cell and tissue implants Cell suspensions are obtained by trypsinization of confluent cell monolayers. Five microliters containing 2 X 10 cells in medium supplemented with 10% serum are introduced in the corneal micropocket. When the over expression of growth factors by stable transfection of a specific cDNA is studied, one eye is implanted with transfected cells and the other with the wild type cell line. A 0ien tissue samples are tested, samples of 2-3 mg are obtained by cutting the original fragments under sterile conditions. The angiogenic activity of tumor samples is compared with macroscopically healthy tissue. 

The detection of contamination of any type in an environmental sample from a critical environment, sterility testing or a filled container during a process simulation has become a rare event. 

Sterility is a state of absolute freedom from microbial contamination. Interestingly, the word sterile on the label of a sterile product has had a historic meaning that a sample of the product lot passed the compendial test for sterility.Today, to claim that a product is sterile involves much more than passing a sterility test. Achievement of sterility involves the combination and coordination of a wide range of activities and processes such as ... 

Concern that the small sample (usually 20 containers per lot) truly represents the entire lot. Probability statistics reveal that with such a small sample size, the extent of contamination must be significant (on the order of at least 1% of the lot) for the sample to fail the sterility test. 

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