Packaging components used in pharmaceutical drugs and medical devices are scrupulously cleaned before use to ensure patient safety. Regulators expect drug manufacturers to demonstrate compliance with federal requirements intended to assure clean, sterile and safe drug products enter the medical marketplace.
Depyrogenation, the reduction of bacterial endotoxin, is critical in preparing packaging components for use in injectable drug products.
Pharmaceutical packaging components such as ampoules, vials and stoppers require specific depyrogenation procedures appropriate for the component’s material. The main depyrogenation processes that will be referenced are dry heat and intense washing.
The FDA defines a sterile pharmaceutical product as the probability of 1 in a million of finished products being contaminated (-6). This is referred to as the Sterility Assurance Level (SAL). There are seven basic characteristics of sterile pharmaceutical packaging components, described in Table 1. All of these characteristics must be achieved for safe pharmaceutical packaging. One particularly challenging characteristic is that materials must be free from pyrogenic endotoxin contamination.
|Safety||No Adverse toxicological concerns.|
|Sterility||Free from microbiological contamination.|
|Non-pyrogenic||Free from pyrogenic endotoxin contamination.|
|Particle-free||Free from visible particle contamination.|
|Stability||Robust chemical and phyical properties|
|Compatibility||Formulation and package.|
|Tonicity||Isotonic with biological fluids.|
Pyrogens are fever causing lipid substances which can be either internal (endogenous) or external (exogenous) to the body. The FDA has set limits on endotoxin levels for drugs distributed in the United States, which are outlined in table 2.
|Intrathecal||0.2 EU/kg body weight|
|Non-intrathecal||5.0 EU/kg body weight|
The most potent pyrogens are the endotoxins produced from the cell walls of gram-negative bacteria. This element is the most problematic for effective depyrogenation, due to their stable thermal properties, strong electrostatic properties, varying sizes, broad molecular weights, and resistance to pH change.
Depyrogenation can be defined as the reduction of pyrogenic substances, including bacterial endotoxin, and is generally achieved by removal or inactivation. There are several techniques in which depyrogenation can be achieved. The more common techniques are shown in table 3. The two most utilized techniques for pharmaceutical packaging components are dry heat exposure via a depyrogenation oven and rinsing by USP and WFI water.
|Rinsing||Endotoxins are washed away by USP and WFI water.|
|Dry Heat||Endotoxins are destroyed by exposure to high temperature.|
|Moist Heat||Traditional autoclaving will not destroy endotoxins; however, the combination of hydrogen peroxide and pressure are effective.|
|Ethylene Oxide||Endotoxins are destroyed by nucleophilic substitution in the glucosomne of Lipid-A.|
|Ultrafiltration||Filters out endotoxins by molecular weight.|
Endotoxins are strongly regulated and strictly tested for within the pharmaceutical industry. A common test to detect endotoxins from gram-negative bacteria is known as the Bacterial Endotoxins Test (BET) which has three methods.
- Method A - (gel-clot technique) is based on gel formation.
- Method B - (turbidimetric technique) is based on the development of turbidity after cleavage of an endogenous substrate.
- Method C - (chromogenic technique) is based on the development of color after cleavage of a synthetic peptide-chromogen complex.
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Three common pharmaceutical packaging components are ampoules, vials and stoppers. Each component needs a specific depyrogenation procedure due to its chemical properties.
Ampoules and vials are made of borosilicate Type I glass (USP Type I). The low coefficient of thermal expansion makes it extremely resistant to thermal shock and allows it to withstand the rigors of dry heat sterilization. A depyrogenation oven utilises hot air that is free from water vapor. Depyrogenation tunnels usually consist of three chambers. The first chamber is the “inlet chamber” that emits HEPA filtered air. This laminar flow keeps the transfer of glassware clean and protects the product from the potential “hot air back flow” coming from the hot chamber.
The second chamber is the “hot chamber” where the glassware goes through its depyrogenation cycle. The third chamber is the “cooling chamber” which cools the glassware to room temperature so it can be sent to a sterile environment with Class 100/Grade A/ISO 5 conditions. There are no exact requirements for the depyrogenation processes; however, the FDA requires that a manufacturer validate three-log reduction in endotoxin from a starting concentration of at least 1000 Endotoxin Units (EU) is performed. Table 4 displays common depyrogenation cycles that can be applied to Type I glass.
|Example||Temperature (°C)||Time (mins)|
Vial stoppers are made up of synthetic rubbers which can be butyl rubber, styrene-butadiene rubber (SBR), neoprene [poly-(2-chloro-1,3-butadiene)] and nitrile rubber. Each rubber has different chemical properties; therefore, it is critical to work with the manufacturer to fully understand these chemical properties so that there is no degradation of material during the sterilisation process. Butyl rubber, the most commonly used elastomer for pharmaceutical applications, will be the rubber addressed.
Butyl rubber is a superior oxygen and moisture barrier making it extremely durable and efficient. Butyl rubber stoppers are cleaned and depyrogenated by rinsing with USP water and if necessary a cleaning agent such as sodium hydroxide, liquid Safe-Kleen (LSK-9), or trisodium phosphate (TSP). USP water meets specific ionic, chemical and microbial requirements. A final rinse of WFI (Water for Injection) is performed. WFI meets all the requirements for USP water plus an additional bacterial endotoxin specification. After the final WFI rinse the rubber stopper is dried in a sterile environment and packaged until use.
Depyrogenation can be defined as the reduction of pyrogenic substances, including bacterial endotoxin, and is generally achieved by removal or inactivation.
Depyrogenation is a critical process that, when validated, reduces bacterial endotoxins to an acceptable level. There are many factors to consider when depyrogenating pharmaceutical packing components such as the component’s material and intended application. These are just a few of the many considerations to take into account when trying to determine the best depyrogenation technique for your applications and needs. It is important to note that the risk of endotoxins or other contaminates can still occur from other sources during the packaging stage and not the packaging component alone. Yet, with a complete understanding of all the packaging components and the preparation methods used for each, the final drug product can be preserved.
- Williams, Kevin L. Endotoxins: Pyrogens, LAL Testing and Depyrogenation. 3rd ed. New York: Informa Healthcare, 2007. Print.
- Endotoxins and Their Detection With the Limulus Amebocyte Lystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY (1982)
- “Inspection Technical Guides: Bacterial Endotoxins/Pyrogens.” Food and Drug Administration. United States Department of Health and Human Services, 20 Mar. 1985. Web. 1 Sept. 2015. http://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htm.
- “Water for Pharmaceutical Purposes.” The United States Pharmacopeia. 37th ed. Vol. 1. United States Pharmacopeial Convention, 2014. 1181-1205. Print.
- “Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers.” Food and Drug Administration. United States Department of Health and Human Services, 1 June 2012. Web. 1 Sept. 2015. http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm314718.htm.
- ISO/EN 17665 - Sterilization of Health Care Products - Moist Heat (Parts 1 and 2)