Sterility testing membrane filtration

Sterile pharmaceutical preparations must be tested for the presence of fungal and bacterial contamination before use (see Chapters 18 and 23). If the preparation contains an antibiotic, it must be removed or inactivated. Membrane filtration is the usual recommended method. However, this technique has certain disadvantages. Accidental contamination is a problem, as is the retention of the antibiotic on the filter and its subsequent liberation into the nutrient medium. 

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... 

Product Name) can be tested for sterility by membrane filtration with 3 X 300 rinsing fluid A after incubation of 14 days and more than one week. 

Retention is a function of the pore size distribution of the membrane, solution properties, and operating conditions. For critical applications such as sterile or virus filtration, retention should be tested with the actual solution under different operating conditions. Typically, membrane filters are tested for integrity before use to ensure the required retention is obtained during operation. Integrity tests are based on bubble point or diffusion [6]. 

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. 

The Test for Sterility may be performed in one of two ways, by direct inoculation (direct transfer) or by membrane filtration. 

Fig. 1 Generalized scheme for the Test for Sterility by the membrane filtration method.

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. 

Membrane filtration application to biopharmaceutical product development is extremely important since sterile protein-peptide products can only be prepared via sterile filtration and gamma radiation steam cannot be used under pressure. There are several excellent works in the field of sterile membrane filtration.34-36 The filter media most often tested for protein formulations with minimum adsorption and maximum compatibility are mixed esters of cellulose acetate, cellulose nitrate, polysulfone, and nylon 66. Membrane filters must be tested for compatibility with the active drug substance and selected for formulations if they have the lowest adsorption and maximum compatibility with the product. 

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. 

Additionally, 100 mL of a 0.2M phosphate buffer solution at pH7 was prepared under aseptic conditions and sterilized by membrane filtration (0.22 pm, Millipore) 5.0 mL of the solution was dispensed into pre-steriUzed serum vials and stored at 4°C. All vials were tested as sterile and apyrogenic preparations by conventional pharmaceutical procedures. 

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. 

A test for sterility is laid down in the European Pharmacopeia for all parenteral products, and two techniques for testing are described, either membrane filtration of the product with subsequent incubation of the filter in suitable culture media (which is the preferred technique), or direct inoculation of the product into the culture medium, followed by incubation at the appropriate temperature for the specified time. Suitable media are described in the European Pharmacopeia although, it is also recognized that other media may be used. In every case, however, it is necessary to demonstrate that the medium is capable of supporting the growth of microorganisms both in the presence and absence of the material to be tested. 

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 membrane filtration technique is technically more elaborate and requires that the radiopharmaceutical under test, after aseptic dilution, is passed through a membrane filter with a pore size of 0.45 m, which has been moistened with a sterile nutrient diluent. After filtration, the membrane is either transferred to a suitable culture medium or aseptically cut into two equal parts and one half placed in each of two suitable media. Incubation at the appropriate temperature is required for at least 7 days. 

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

Liquid products and water soluble solids may be tested by membrane filtration. Solids would first be dissolved in a suitable diluent, such as 1 /4 strength Ringers solution. After the liquid has been passed through the sterile filter (pore size < 0.45 pm), the filter is cut up aseptically, and pieces are placed into an appropriate culture medium for incubation. Liquid products may also be tested by adding a concentrated preparation of culture medium to a container with the product in situ. The concentration of medium added is such that the combination of medium and product gives single strength medium. The whole container is then incubated at the appropriate temperature. 

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. 

Bacterial genera used in this study were isolated from a jet fuel distribution system using selective solid media. They were transferred later to fuel/water systems containing 50 ml of JP (sterilized by a membrane filtration, as a carbon source) and 250 ml of a simplified mineral medium by a previously reported method (de Mele et al., 1979). Identification of the bacterial strains was made by phase contrast microscopy. Gram staining, and selected biochemical tests. 

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