Sterility testing

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. 

The membrane filtration method is specified by the USP to be used whenever the nature of the product permits. The direct transfer method should 

Growth media can be made from chemical constituents in the laboratory or can be purchased either ready to use in sterile containers or as a dehydrated powder to be rehydrated and sterilized in the laboratory. The latter is considerably less expensive and has a far greater shelf life than the ready-to-use type. These media are modified if used for penicillins or cephalosporins by the addition of an appropriate amount of -lactamase to inhibit the action of the antibiotics. The amount of inhibitor must be determined experimentally. For additional information on these and other types of growth media and their formulations, see USP 24-NF 19 ( 71 ) or a manufacturer s manual such as the Difco Manual  

The number of units sampled for the test varies according to the following factors (1) the number of units per batch, (2) the fill volume of the container, and (3) the method of product sterilization. Generally, 10-20 units are randomly sampled from the lot. If the lot size is between 20 and 200 units, then n = 10. 

If the lot size is 200 units, then n = 20. However, there are many variations in sample size determination, and it is important to consult the USP, European Pharmacopeia (EP), or Japanese Pharmocopeia (JP) for detailed directions. 

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. 

One problem that may arise is when an antibiotic or preservative is the product, or part of the product under test. When this happens, the material must be inactivated or removed before sterility testing can take place. There are several methods of achieving this. Antibiotics, such as the penicillins, may be inactivated by the addition of the enzyme P-lactamase, whilst the action of sulphonamides can be blocked by the addition of / -aminobenzoic acid. Products containing preservatives or antimicrobial agents, such as benzoic acid, alcohols, or phenols, are diluted to the level at which the compound becomes ineffective. Products containing quaternary ammonium compounds can be inactivated by the addition of Tween, whilst many compounds containing heavy metals can be 

Organisms suggested as positive controls by the BP are Staphylococcus aureus (a typical aerobic micro-organism). Bacillus subtilis (aerobic spore former), Clostridium sporogenes (anaerobic spore former), and Candida albicans (yeast and fungus). 

The sampling of a number of items is discussed in the chapters on food, water and counting. It is obvious from the fact that sterility testing is destructive, that one cannot test the whole batch, and therefore sampling on a statistically significant basis must be carried out. The statistics are discussed briefly with a worked example, and also in the BP.  

A small number of simple examples are probably the best way of demonstrating the situation. 

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In-process quality control is the control exercised over starting materials and intermediates. Its importance stems from the opportunities that it provides for the examination of a product at the stages in its manufacture at which testing is most likely to provide the most meaningful information. The WHO Requirements and national authorities stipulate many in-process controls but manufacturers often perform tests in excess of those stipulated, especially sterility tests (Chapter 23) as, by so doing, they obtain assurance that production is proceeding normally and that the final product is likely to be satisfactory. Examples of in-process control abound but three of different types should suffice. 

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... 

Sterility testing Methods Antimicrobial agents 8 Further reading... 

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. 

There are three alternative methods available when conducting sterility tests. 

Fig. 23.2 Isolators used for sterility testing. The operator works within the hood which is suspended inside the cubicle the hydrogen peroxide generator which is used to sterilize the isolators is shown in the left foreground. (Courtesy of SmithKline Beecham Pharmaceuticals.)...

Specific details of the sterility testing of parenteral products, ophthalmic and other non-injectable preparations, catgut, surgical dressings and dusting powders will be found in the British and European pharmacopoeias. 

A sterility test attempts to infer the state (sterile or non-sterile) of a batch from the results of an examination of part of a batch, and is thus a stahstical operation. 

The British Pharmacopoeia makes an allowance for acddenlal contamination which may arise during the execution of a sterility test by aUowing the test to be repeated. Under these circumstances the following rules apply. 

It can be seen from the above that a sterility test can only show that a proportion of the products in a batch is sterile. Thus, the correct conclusion to be drawn from a satisfactory test result is that the batch has passed the sterility test not that the batch is sterile. 

The techniques discussed in this chapter comprise an attempt to achieve, as far as possible, the continuous monitoring of a particular sterilization process. The sterility test on its own provides no guarantee as to the sterility of a batch however, it is an additional check, and continued compliance with the test does give confidence as to the efficacy of a sterilization or aseptic process. Failure to carry out a sterility test, despite the major criticism of its inability to detect other than gross contamination, may have important legal and moral consequences. 

Absence of microorganisms Sterility test (see also Chapter 23)... 

Several enzymes have important therapeutic and other medical or pharmaceutical uses (Table 25.3). In this section, those enzymes used therapeutically will be described, with section 4 discnssing the applications of microbially derived enzymes for antibiotic inactivation in sterility testing and diagnostic assays. 

Lactamases Bacillus cereus (or other bacteria, as appropriate) Sterility testing, treatment of penicillin-induced allergic reaction 2.4.4, 4.5... 

Breeze A.S. Simpson A.M. (1982) An improved method using acetyl-coenzyme A regeneration for the enzymic inactivation of aminoglycosides prior to sterility testing. JApplBacteriol, 53, 277-284. 

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. 

Sterility testing is conducted on each lot of ophthalmic product by suitable procedures, as set forth in the appropriate pharmacopeia and validated in each manufacturer s laboratory. While the majority of ophthalmic preparations contain preservatives for multiple-dose use, sterile preparations in special containers for individual use on a single patient must be made available. This availability is especially critical for every hospital, office, or other installation where accidentally or surgically traumatized eyes are treated, as well as for patients intolerant to preservatives. 

Currently the main application of interest for parametric release is to replace the sterility test as a control method in appropriate cases (given the limited value of that test to predict sterility assurance due to statistical considerations, although it is also pointed out that a sterility test provides a final opportunity to identify a major failure, although other means should provide a more reliable way of detecting such failures). The concept is applicable to well-founded methods of sterilization where the product stability is known and development data have identified the critical process parameters. The measured parameters should be such as to ensure that correct processing of the batch provides sufficient assurance that the sterility assurance level intended has been achieved. 

Even where it is possible to omit routine or even any sterility testing of finished product, the sterility requirement should be retained in the finished product specification. 

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