A Practical Approach to Depyrogenation Studies Using Bacterial Endotoxin

Peer Reviewed A Practical Approach to Depyrogenation Studies Using Bacterial Endotoxin Tim Sandle sodilatation, diarrhea, and fetal shock syndrome. Due Depyrogenation devices, such as tunnels, are used in the to the level of risk, pharmaceutical water systems and pharmaceutical industry to prepare components for asep- parenteral products are tested for pyrogens including, tic filling. To qualify such devices, various pharmacopeias or exclusively, endotoxins (3). require depyrogenation devices to be periodically chal- Despite the relative comprehensiveness of the phar- lenged with high levels of bacterial endotoxin. Although macopeial monographs for the LAL test, one key ap- the pharmacopeias state the acceptance criteria, little plication of endotoxin testing is not described in great consideration is given to the practical approach. This detail: conducting depyrogenation studies. A depyro- article discusses the theoretical concept of depyrogena- genation study is the key biological test, in addition to tion. A case study of a depyrogenation tunnel is used to thermometric tests, for the qualification of depyroge- define some of the practical aspects of a depyrogenation nation devices. Depyrogenation can be defined as the study that need to be considered. elimination of all pyrogenic substances, including bacterial endotoxin, and is generally achieved by removal or inactivation (4). Depyrogenation, like sterilization, is INTRODUCTION an absolute term that can only be theoretically demon- The bacterial endotoxin test (BET) is a relatively straight- strated because of test insensitivity. forward test and has been a pharmacopeial method Some scientists regard depyrogenation purely as since 1980, when it first appeared in the United States endotoxin destruction or inactivation, and endotoxin Pharmacopeia (USP). The test, using Limulus amebocyte removal as a distinct and unrelated process. Here the lysate (LAL) methodology, is described in detail in the former refers to inactivating or destroying any endotox- harmonized chapters in both the European Pharmaco- in present on a component, the latter to the removal of poeia (1) and the United States Pharmacopeia (2). The any endotoxin present (5). With depyrogenation inacti- test describes the detection of the most common and vation, the total destruction of the “pyroburden” is as- significant pyrogenic material found in pharmaceutical sumed; with endotoxin removal it is assumed that a sig- production: gram-negative bacterial endotoxin. LAL is nificant portion of the pyroburden has been removed. an extract from the lysed blood cells (amebocytes) of the Other scientists consider both processes to be part of horseshoe crab Limulus polyphemus, or related species. depyrogenation. The need to perform the LAL test for endotoxins This article examines the mechanism of endotoxin is well established. Endotoxins can cause, to varying inactivation by dry heat and the practical steps to be degrees depending upon potency and target site, en- taken for conducting a depyrogenation study. Depyro- dotoxemia (i.e., the presence of bacterial toxins in the genation of glassware is important in the production of blood) and septic shock (i.e., the prolonged presence of parenteral pharmaceuticals as residual pyrogens could bacteria and bacterial toxins in the body). The effects of ultimately be injected into a patient resulting in an ad- endotoxin in the human body include high fever, va- verse reaction. This is especially important as endotox- 90 Journal of GXP Compliance Tim Sandle ins are heat stable, making them resistant to most con- affected by the pH range, temperature, flow rate, ventional sterilization processes and thus necessitating and amount of electrolytes in the solution. separate tests for viable cells and endotoxin (6). • Dilution or rinsing—the amount of endotoxin is The assessment of depyrogenation involves the in- washed away or reduced using WFI. troduction of purified endotoxin of a high potency and • Distillation—functions by turning water from post-process testing to assess if a minimum of a three- a liquid to a vapor and then from vapor back to log reduction has been achieved. The concept is simi- liquid. Endotoxin is removed by the rapid boiling lar to that of steam sterilization studies, where a much that causes the water molecules to evaporate and higher level of microorganisms than would ever be the relatively larger lipopolysaccharide (LPS) mol- found in the normal environment is introduced into a ecules to remain behind. device as a worst-case challenge. In the case of steam • Adsorption (e.g., activated carbon beds, where sterilization, this is Geobacillus stearothermophilus. Depy- endotoxin is adsorbed into charcoal, or depth rogenation follows a pattern similar to steam steriliza- filters)—this functions by attracting negatively- tion, in that the log-reduction will follow a linear plot charged endotoxin molecules to the carbon bed. (i.e., as time and temperature increase, the endotoxin This mechanism is only efficient to a small degree level decreases). After this destruction continues to oc- and is affected by a range of environmental factors. cur, it does not necessarily follow linear regression (i.e., • Hydrophobic attachment—certain materials, like it does not produce the same semi-log reduction seen polyethylene, can bind endotoxin. for bioburden reduction in sterilization devices and be- • Acid or base hydrolysis—this destroys the eight- comes more noticeable at lower temperatures) and in- carbon sugar: two-keto-three-deoxyoctonic acid stead follows a biphasic reduction (7). that links Lipid-A to the core polysaccharide and causes physiochemical changes that decrease DEPYROGENATION pyrogenicity. The separated Lipid-A loses its pyro- Before focusing on the practical aspects of depyrogena- genic activity). An example is 0.05M HCl for 30 tion studies, it is appropriate to consider the process of min. at 100 ∘C or 0.5 M NaOH at 50 ∘C for 30 depyrogenation. The following are various mechanisms min. by which depyrogenation is achieved (8-11): It is possible, with alkaline hydrolysis, that the • Ultrafiltration—the process works by excluding level of endotoxin may initially rise as part of the endotoxin by molecular weight using an ultra-fine separation process. filter that blocks molecules of 10,000 Daltons or The hydrolysis methods are frequently used for greater. This is often coupled with 0.1μm filter. depyrogenating glassware. The efficiency of this • Reverse osmosis—primarily functions as a process is often connected to the cleanliness of the size-excluding filter operating under a highly- glassware prior to treatment. pressurized condition. It will block 99.5% of endo- • Oxidation—works by peroxidation of the fatty toxin and ions or salts, but allow water molecules acid in the Lipid-A region (e.g., using hydrogen through. USP reverse osmosis (RO) can be used to peroxide). make water for injection (WFI) (whereas to meet • Ionizing radiation—a very slow and inconsistent the European Pharmacopoeia requirement it can process. only be produced by distillation); within Europe it • Ethylene oxide—functions by nucleophilic substi- is used to produce highly-purified water. tution in the glucosomne of Lipid-A. It is not the • Affinity chromatography (e.g., Diethylaminoethyl most efficient depyrogenation process, and where cellulose [DEAE] sepharose or polymyxin-B, endotoxin inactivation occurs this is normally a which binds endotoxin by using a positive charge side effect of sterilization. to attract the negatively-charged endotoxin and • Moist heat—conventional autoclaving will not then allowing its elution)—such processes are destroy endotoxin. However, the combination of Autumn 2011 Volume 15 Number 4 91 Peer Reviewed a chemical additive (e.g., hydrogen peroxide) and Various items can be depyrogenated by dry heat. The physical variations (e.g., five hours at 121∘C with a main example referred within this paper is glass vials pressure of 20 PSI and a pH of 3.8) are sometimes used for the filling of parenteral product, notwithstand- effective. Alternatively, some studies have shown ing that many of the principles might apply to other that autoclaving for prolonged periods of time or primary packaging materials providing that they can at higher temperatures can be effective at reducing withstand high temperatures. endotoxin and other pyrogenic substances (12, 13). Depyrogenation by dry heat for glass in the pharma- • Endotoxin can be inactivated by wet heat, ceutical industry is the primary endotoxin destruction although this is only effective with far lower con- method used (17). This process both sterilizes and de- centrations of endotoxin and is applied to non-heat pyrogenates and is mainly used for glass components. stable materials. Destruction of endotoxin is far Dry heat involves subjecting the components to a high more difficult to achieve, and lower log reductions level of heat (normally between 180 and 250∘C) for a when compared with dry heat are achieved (14). defined time (the higher the temperature, the shorter • Dry heat (physical destruction), such as convection the time required). The typical cycle is 250∘C for not (transfer of heat by movement of fluid or air), con- less than 30 minutes. For example, the European Phar- duction (transfer of heat from adjacent molecules), macopoeia in chapter 2.6.8 states two possible time- or irradiation (emission of heat by electromagnetic temperature combinations for depyrogenation: 60 min- radiation). utes at 200∘C

Load more