Sterility test principles

These major limitations demonstrate that reliance on end-product sterility testing alone in ascertaining the sterility of a parenteral product may lead to erroneous results. One purpose of validation in the manufacture of sterile products is to minimize this reliance on end-product testing. Three principles are involved in the validation process for sterile product. 

The following principles apply to sterility testing within a pre- and postmarketed product stability program. 

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. 

Frozen reference materials have been produced by NIST (Wise et al. 1993). These materials do not have the disadvantages of the oils or freeze-dried materials, but are more difficult to transport. Obviously they have to be kept deep-frozen during transport, which makes their use rather expensive. Since the early 1990 s a new approach in this field has been introduced. This concerned the use of wet, sterilized fish and shellfish samples. These samples, packed in glass jars or in tins, were firstly used in the QUASIMEME program as reference materials for inter-laboratory studies (de Boer 1997). Later, when it appeared that the stability was maintained for longer periods, tests for organic contaminants based on this principle were also prepared. 

However, large holes or tears can be introduced into an otherwise effective sterilizing filter and performance tests have to be designed to ensure that the complete assembly of the filter membrane and its supporting equipment are devoid of imperfections. These include bubble testing in which gas is forced under pressure through the wetted filter and the pressure required for bubbles to first appear measured. In principle this identifies the largest hole present in the complete system. 

Fig. 2.46. Basic principle of a venting filter for in situ integrity tests. Arrangement of the sterile venting filters for in-line sterilization and integrity tests (I tests). The integrity tests are carried out following the water intrusion method (WIT). In this figure, WIT is carried out only at the primary filter. The secondary filter is installed as a back-up filter (police filter). 1, Primary filter with a 0.22 pm cartridge 2, secondary filter with a 0.22 pm cartridge the secondary filter can be sterilized independent-...

Method The principle of GEC involves the intravenous administration of galactose in excess substrate in order to assess the maximum rate of hepatic metabolization. This requires a galactose concentration of 0.5 g/kg BW (serum concentration of > 45 mg/dl for at least 45 minutes), which is injected within 4—5 minutes as a 40% pyrogen-free sterile galactose solution. The bladder has to be emptied immediately prior to the injection and subsequently the urine has to be collected for exactly 5 hours to assess the respective galactose excretion (in grams). Blood samples are taken from the fingertip (or from the cubital vein into an EDTA test tube) at 5, 25 and 45 minutes after injection. We have reported in detail on how to conduct this method as well as on the analysis of GEC. (67, 69)... 

Principles Sterile products should be manufactured with special care and attention to detail, with the object of eliminating microbial and particulate contamination. Much depends on the skill, training and attitudes of the personnel involved. Even more than with other types of medicinal product, it is not sufficient that the finished product passes the specified tests, and in-process Quality Assurance assumes a singular importance. 

Another research group previously conducted tests on protein denaturation kinetics and reported the data below. Determine if the data are consistent with the principles of the kinetics of cell death phenomena. Estimate the first-order rate constant for cell death at the conditions employed in the sterilization reactor. Will the proposed process meet the standards for protein degradation ... 

The sterilization efficiency of the inlet venting Alters has to be assured by regular integrity tests. The filter integrity test should be performed automatically with the relevant data documented by the control system the principle is shown in Figure 20. The criteria for an integrity test given by the Parenteral Society [10] are summarized as ... 

In 1992, OECD published its Good Developmental Principles (GDP) (OECD, 1992) for the design of small-scale field research involving transgenic plants and micro-organisms. This document describes the use of confinement in field tests. Confinement includes measures, to avoid the dissemination or establishment of organisms from a field trial, for example, the use of physical, temporal, or biological isolation (such as the use of sterility). 

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