Filtration sterility testing

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. 

Validation of membrane filtration and direct inoculation sterility test methods... 

Positive Control Inoculated with coli showed growth after 48 h Negative Control Filtrated sterile WFI under the same conditions of the test... 

The entire volume of the challenge filtrate is subsequently forced through a sterility test filter system and incubated in the same manner as the negative control filtrate. 

Pharmaceutical products can be sterilized by steam sterilization, dry-heat sterilization, filtration sterilization, gas sterilization, and ionizing-radiation sterilization. The USP provides monographs and standards for biological indicators required to test the validity of the sterilization process. These products must also be tested for pyrogens—fever-producing substances that arise from microbial contamination most likely thought to be endotoxins or lipopolysaccharide in the bacterial outer cell membrane. 

Ansel HC, Norred WP, Roth IL. Antimicrobial activity of dimethyl sulfoxide against Escherichia coli. Pseudomonas aeruginosa, and Bacillus megaterium. J Pharm Sci 1969 58(7) 836—839. Placencia AM, Oxborrow GS, Danielson JW. Sterility testing of fat emulsions using membrane filtration and dimethyl sulfoxide. ] Pharm Sci 1982 71(6) 704-705. 

The time lag in imposition of a legal requirement for sterility of ointments compared with solutions and suspensions was due to the absence of a reliable sterility test for the petrolatum-based ointments until isopropyl myristate was employed to dissolve these ointments and allow improved recovery of viable microorganisms by membrane filtration. Manufacturers found that, in fact, many of their ointments were sterile, but revised their manufacturing procedures to increase the assurance of sterility. 

The USP describes two general methods for conducting the test the direct transfer, or direct inoculation, method and the membrane filtration method. As the name indicates, the direct inoculation method involves the aseptic transfer of a sample of test product solution into the sterility test growth medium. To use this method, it must first be demonstrated that the product solution itself does not inhibit the growth of typical indicator microorganisms specified in the USP method. It should be self-evident why it is important to perform testing to negate the chance of product inhibition of possible microbial contaminants, as this is the purpose of the sterility test. The direct inoculation method, while not theoretically complex, requires the utmost technical precision and aseptic manipulation techniques for proper execution. As a consequence of the repetitive motions involved, it is prone to human error. 

Sterility tests on each individual batch (t n > 20 min) or quality control subbatch (q/2 < 20 min) initiated within 24 h of sterility filtration. 

Finally, it is recognized that for short-lived radiopharmaceuticals, the long incubation time of the culture media (7 days for the membrane filtration technique, 14 days for direct inoculation) means the result of the sterility test cannot be available before the product is used. In these situations, the test constitutes a control of production techniques and will give valuable information about their suitability. 

The prime purpose of sterile filtration is to produce a sterile effluent that has not been altered as a result of the process of sterilization. Within these considerations. validation must address the performance of both the filler media and the whole filtration unit including housings, seals, connections, etc., versus its practical application. As with any other sterilization process, the continued effectiveness of sterile filmuion cannot be assumed without confirmation from routine monitoring end-product sterility testing (or testing for nonsteriiity) is unsuited for this purpose. 

The method of choice for sterility testing is by membrane filtration. 

During batch preparation sterility testing can be performed on randomly selected preparations either by direct inoculation or by the filtration method. During extemporaneous preparation dummy solutions should be prepared and sterility tests performed with rapid detection methods such as the bioluminescence test (see Sect. 19.6.5) or the colorimetric detection of CO2 production in culture bottles although commonly used for blood cultures this last method was shown to be adequate for sterility testing of multicomponent admixtures [68]. 

Vacuum valve, used only in dedicated LAF units for microbiological quality control the filtration of fluids for sterility testing or bioburden control. 

Though several supplier offer commercial kits, many users prefer to prepare their own buffers for calcium phosphate transfections. This adds some time for preparation, sterile filtration and testing. However, if suitably stored, the solutions ate stable for years, so large quantities can be prepared at one time. A disadvantage of the method is that the crystal formation is extremely sensitive to differences in pH, which directly aflfects the solubility. Nevertheless, the method has been used successfully by many groups and has even shown potential for application at large scale (> 10 L). ... 

Membranes 5 cm in diameter are a convenient size for use in sterility testing. Assemble the membrane in a suitable holder (various types made on the Seitz filter principle are available) with a glass or metal cover-lid and sterilise either with steam in the autoclave or with ethylene oxide. Connect to a convenient receiver and filter the test sample through the membrane, solid samples having previously been dissolved in sterile water or saline. Filtration is rapid if a vacuum is applied. Wash the membrane through several times with water or saline and then remove it aseptically from its holder, cut it in two with the aid of sterile forceps and scissors and culture one part aerobically and the other anaerobically in the appropriate media. 

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