Sterility tests membrane filters

Verification of the microbial retention efficiency of the membrane filters may be undertaken using either Hquid or aerosol challenge tests. A Hquid challenge test is more stringent. Furthermore, this test can provide retention information for process conditions such as extreme moisture after sterilization or air entrained with water drops. A Hquid challenge is performed using a protocol similar to that described for Hquid filtration. 

Another standard test, which is much simpler and more convenient, is the membrane filter technique. A suitable volume of sample is filtered through a sterile, 0.45-p.m membrane filter. The filter is placed in a petri dish containing a specific growth medium (M-Endo nutrient broth, M-Endo medium) and incubated for 24 h at 35°C. If after this time the colonies show the characteristic green sheen, this is taken as positive evidence for the presence of the coliform group (see Water, sewage). 

They may also be required in industrial applications where they become part of venting systems on fermenters, centrifuges, autoclaves and freeze-dryers. Certain types of filter (membrane filters) also have an important role in sterility testing, where they can be employed to trap and concentrate contaminating organisms from solutions under... 

In the first example, procaine penicillin, an aqueous vehicle containing the soluble components (such as lecithin, sodium citrate, povidone, and polyoxyethylene sorbitan monooleate) is filtered through a 0.22 pm membrane filter, heat sterilized, and transferred into a presterilized mixing-filling tank. The sterile antibiotic powder, which has previously been produced by freeze-drying, sterile crystallization, or spray-drying, is aseptically added to the sterile solution while mixing. After all tests have been completed on the bulk formulation, it is aseptically filled. 

Figure 7.5 Apparatus for testing the microbial retention characteristics of membrane filters. The whole apparatus is sterilized, and initially the flask contains 140 mL of double-strength culture medium. The culture to be tested (100 mL) is passed through the filter with clamp 1 open and clamp 2 closed. The sides of the filter apparatus are washed with two 20 mL portions of sterile broth. Clamp 2 is then opened, the vacuum released, and clamp 1 closed. The filter apparatus is replaced by a sterile rubber stopper and the flask incubated. Absence of turbidity in the flask indicates that the filter has retained the test organism. From Brock [4]. Courtesy of Thomas D. Brock...

When required, media were supplemented with casamino acids (Difco, Detroit, MI) to a final concentration of 5 g/L. Four different media were tested hydrolysate (H), supplemented hydrolysate (SH), concentrated hydrolysate (CH), and concentrated and supplemented hydrolysate (CSH). To prevent additional medium decomposition, all media were filter sterilized using 0.22 pm Gelman membrane filters (Pall, Ann Arbor, MI). 

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]. 

The avoidance of false-negatives is addressed at length in the pharmacopeias. Each batch of medium used in the Test for Sterility must have been shown to be capable of supporting the growth of low inocula of a specified array of microorganisms. Each Test for Sterility applicable to each specific product must be validated by repeated demonstration that the viabilities of low inocula of a specified array of microorganisms are not inhibited by product traces contained in the medium (direct inoculation) or on the membrane filter. 

It is essential that the microbiological particle passage test is performed as part of the development of new sterile formulations. Because of its very specialized nature, the test is normally performed only by the filter manufacturers, who then provide limits for secondary physical tests (e.g., bubble point, pressure decay, forward flow, etc.), which can be applied to verify the pore size rating and integrity of the membrane filters. 

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. 

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