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Depyrogenation by Dilution
Preparing sterile products requires manufacturers to control microbial quality. Sterility and endotoxin content are critical because failure to properly manage them can seriously harm, or even kill, patients. Manufacturers use various means to sterilize products, but depyrogenation—a process whereby fever-inducing materials are eliminated from materials, components, and equipment—is largely restricted to dry-heat methods or cleaning and rinsing. Dry heat is appropriate for heat-resistant glass or stainless steel items. For other materials, however, depyrogenation is accomplished by cleaning and rinsing surfaces.
Large pieces of equipment such as tanks and permanent piping customarily undergo cleaning in place (CIP). Cleaning requires various chemical agents, including strong acids and bases. Although these agents remove pyrogens, the magnitude of this removal is rarely quantified (1). The last step in cleaning these items is often a rinse with water for injection (WFI). Samples for cleaning validation, and sometimes for routine verification, may be tested for endotoxins.
Small equipment that cannot be cleaned in place such as filling needles, pumps, and utensils is commonly cleaned with milder agents. Use of these agents ensures the safety of the personnel who perform much of the cleaning process. Small items are sometimes cleaned in parts washers that achieve conditions similar to those in CIP. The final rinse of these materials is always performed with WFI. Depyrogenation of equipment surfaces is not the prime objective of these processes, however. Given the diversity of equipment materials and cleaning processes, expectations for depyrogenation are varied. Some of these processes may achieve depyrogenation effectively; others are less effective.
Elastomeric materials used in the manufacturing process or as part of primary packaging also require depyrogenation. The problem with these items is that their surfaces may adsorb and retain pyrogenic materials. Reproducible recovery of known endotoxin challenges from these surfaces is not easy; results are highly variable. During production, these items may be exposed to pyrogenic materials that differ substantially from the purified endotoxin used in laboratory evaluations.
A primary technique for controlling endotoxins is rinsing surfaces with warm WFI. This technique can sometimes be supplemented with clean steam and air to dislodge pyrogenic materials. Glass containers, for example, can be treated this way. Results, however, are often less than stellar. Private discussions with industry scientists reveal that the level of reduction is typically not more than 1–2 logs. That reduction is often accomplished using purified endotoxin at high initial concentrations (2).
Thus, a considerable amount of residual endotoxin remains on the surface after the validation exercise. Going from 106 to 103 endotoxin units (EU) per component represents a 3-log reduction. This process leaves 1000 EU on an item’s surface, but the expected limit for a depyrogenated item may be as low as 0.25 EU. To support the expectations for a validated removal by washing, manufacturers must often use surfactants and other agents to aid in endotoxin removal. When preparing elastomeric materials, it is essential to monitor their incoming endotoxin content, wash them with WFI, and dry them thoroughly. Interactions between elastomeric materials and endotoxin are reportedly strong enough to preclude effective removal by rinsing (2).
Injectable drugs must be pyrogen-free. Depyrogenation by rinsing is an important part of production processes. Our ability to demonstrate effective depyrogenation by rinsing is hampered by the materials and processes we use. Manufacturers must use methods that remove pyrogens efficiently and ensure patients’ safety.
1. PDA, “TR #7–Depyrogenation,” (PDA, Philadelphia, PA, 1985).
2. J. Agalloco – Personnel Communications – 1982 to present.