Depyrogenation of Equipment in Sterile Pharmaceutical Manufacturing

Depyrogenation means the removal of pyrogens from pharmaceutical equipments. Pyrogens are substances that cause fever. Both exotoxins and endotoxins may be pyrogens, but the most common pyrogens are typically endogenous to their hosts, hence, they are mainly endotoxins. Endotoxins are principally lipopolysaccharide (LPS) molecules that form part of the bacterial cell walls of Gram-negative bacteria, and which are usually released following bacterial cell lysis. After they are released, the endotoxins become pyrogenic when they access the bloodstream or tissues where they are not typically found.

Depyrogenation of equipment in sterile pharmaceutical manufacturing helps to reduce the risk of contaminating pharmaceutical preparations with pathogens, and improves the safety of medicinal products.

Maximum Acceptable Endotoxin Level:

For endotoxins to cause fever, they must reach a certain critical number inside the bloodstream or tissue. Therefore, when sterilizing pharmaceutical equipments to remove pyrogens, the degree of sterility is measured in terms of the endotoxin levels. Nonetheless, the molecular weight of endotoxins usually varies a huge deal (from 10,000 to 1,000,000 Da) and so the acceptable level is measured in terms of endotoxin units (EU). A single endotoxin unit (EU) is approximately equivalent to 100 picograms (pg) of E. coli lipopolysaccharide. However, humans can experience fever when they are exposed to as low as 5 EU/kg of body weight, and the symptoms may also include increased heart rate, low blood pressure and low urine output. In fact, even very minimal quantities of endotoxins in the bloodstream can be fatal.

Depyrogenation of equipment in sterile pharmaceutical manufacturing should help to minimize the amounts of pyrogens that can access injectable drugs through equipments. The maximum permissible levels of endotoxins in drugs should be 0.25-0.5 EU/ml for sterile water (depending on intended use), 5 EU/kg body weight for non-intrathecal drugs, and 0.2 EU/kg body weight for intrathecal drugs. Before removing the pyrogens, detection methods such as rabbit test and Limulus Amebocyte Lysate (LAL) test are used to ascertain the presence of pyrogens.

Depyrogenation Methods for Pharmaceutical Equipments:

Depyrogenation of equipment in sterile pharmaceutical manufacturing is a critical part and assured by the quality control department. All the instruments that are used in the analysis of endotoxins should be depyrogenated in order to boost the accuracy of results. For instance, test tubes and other supplies (accessories) are usually depyrogenated using Dry Heat Sterilizer (DHS) at 300ºC. Vials should also be depyrogenated using depyrogenating tunnel at a dry heat level of 300ºC. Glass vials for pharmaceutical processes should be depyrogenated using a combination of the temperature of chamber and belt-speed. Pharmaceutical equipment change parts, bung and other accessories should be washed using water for injection (WFI) before being sterilized in the autoclave. Then, the rinse water for the change parts and bungs should be analyzed for endotoxins in order to confirm the removal of pyrogens. Due to the size of endotoxins, ultra-filtration is often used to perform size-based depyrogenation.

However, because of the difficulty in selecting the most appropriate membrane, the method is preferred only when the pyrogens have a weight of 300,000 Da and larger. Depyrogenation through distillation benefits from the heat stability and large molecular weight of the endotoxins. Equipments are purified using low molecular-weight solvents, which are boiled to vaporize before the condensed vapor is collected in an endotoxin-free vessel. Depyrogenation is also accomplished through acid-base hydrolysis, oxidation, sodium hydroxide, and anion and cation exchange chromatography.