Inactivation of Escherichia coli Endotoxin by Soft Hydrothermal Processing

Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250°C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130°C for 60 min or at 140°C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130°C for 60 min or at 140°C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments.Endotoxins are lipopolysaccharides (LPS) that are derived from the cell membranes of gram-negative bacteria and are continuously released into the environment. The release of LPS occurs not only upon cell death but also during growth and division. In the pharmaceutical industry, it is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities, including pyrogenicity, lethality, Schwartzman reactivity, adjuvant activity, and macrophage activation (2, 9, 12, 13, 25, 32). Endotoxins are very stable molecules that are capable of resisting extreme temperatures and pH values (3, 16, 17, 29, 30, 34, 38). An endotoxin monomer has a molar mass of 10 to 20 kDa and forms supramolecular aggregates in aqueous solutions (22, 39) due to its amphipathic structure, which makes depyrogenation more difficult than sterilization. Endotoxins are not efficiently inactivated with the regular heat sterilization procedures recommended by the Japanese Pharmacopoeia. These procedures are steam heat treatment at 121°C for 20 min or dry-heat treatment for at least 1 h at 180°C. It is well accepted that only dry-heat treatment is efficient in destroying endotoxins (3, 16, 29, 30) and that endotoxins can be inactivated when exposed to a temperature of 250°C for more than 30 min or 180°C for more than 3 h (14, 36). In the production of parenterals, it is necessary to both depyrogenate the final products and carry out sterilization to avoid bacterial contamination.Several studies have examined dry-heat treatment, which is a very efficient means to degrade endotoxins (6, 20, 21, 26, 41, 42). However, its application is restricted to steel and glass implements that can tolerate high temperatures of >250°C. For sterilization, dry heat treatment tends to be used only with thermostable materials that cannot be sterilized by steam heat treatment (autoclaving). Alternative depyrogenation processes include the application of activated carbon (35), oxidation (15), and acidic or alkaline reagents (27), but steam heat treatment would be an attractive option if it were sufficiently effective. However, the data on the inactivation of endotoxins by steam heat treatment are insufficient and contradictory. It has been reported that endotoxins were not efficiently inactivated by steam heat treatment at 121°C (19, 45). However, Ogawa et al. (31) recently reported that steam heat treatment was efficient in inactivating low concentrations of endotoxin, and that Escherichia coli LPS are unstable in aqueous solutions even at relatively low temperatures such as 70°C (see also reference 40). As mentioned above, these reports have shown that although studies have been carried out on the use of steam heat for depyrogenation, there is little agreement on its efficiency.The U.S. Pharmacopoeia (USP) recommends depyrogenation by dry-heat treatment at temperatures above 220°C for as long as is necessary to achieve a ≥3-log reduction in the activity of endotoxin, if the value is ≥1,000 endotoxin units (EU)/ml (11, 44). Due to the serious risks associated with endotoxins, the U.S. Food and Drug Administration (FDA) has set guidelines for medical devices and parenterals. The protocol to test for endotoxin contamination of medical devices recommends immersion of the device in endotoxin-free water for at least 1 h at room temperature, followed by testing of this extract/eluate for endotoxin. Current FDA limits are such that eluates from medical devices may not exceed 0.5 EU/ml, or 0.06 EU/ml if the device comes into contact with cerebrospinal fluid (43). The term EU describes the biological activity of endotoxins. For example, 100 pg of the standard endotoxin EC-5, 200 pg of EC-2, and 120 pg of endotoxin from E. coli O111:B4 all have an activity of 1 EU (17, 23).Steam heat treatment is comparatively easy to apply and control. If steam heat treatment could reliably inactivate endotoxins, it could be applied with sterilization, reducing labor, time, and expenditure. However, to our knowledge, few studies have addressed steam heat inactivation to determine the chemical and physical reactions that occur during the hydrothermal process, nor have any studies examined the relationship between the steam saturation ratio and the inactivation of endotoxins. Moreover, to date no study has been conducted on steam heat activation of endotoxins with reference to the chemical and physical parameters of the hydrothermal process.We have developed a groundbreaking method to thermoinactivate endotoxins by means of a soft hydrothermal process, in which the steam saturation ratio can be controlled. The steam saturation ratio is calculated as follows: steam saturation ratio (%) = [steam density (kg/m³)/saturated steam density (kg/m³)] × 100.The soft hydrothermal process lies in the part of the liquid phase of water with a high steam saturation ratio that is characterized by a higher ionic product (k_w) than that of ordinary water. The ionic product is a key parameter in promoting ionic reactions and can be related to hydrolysis. The ionic product of water is 1.0 × 10⁻¹⁴ (mol/liter)² at room temperature and increases with increasing temperature and pressure. A high ionic product favors the solubility of highly polar and ionic compounds, creating the possibility of accelerating the hydrolysis reaction process of organic compounds. Thus, water can play the role of both an acidic and an alkaline catalyst in the hydrothermal process (Fig. ​(Fig.1)1) (1, 37, 46). However, the soft hydrothermal process lies in the high-density water molecular area of the steam-gas biphasic field (Fig. ​(Fig.1)1) and is characterized by a lower dielectric constant (ɛ) than that of ordinary water. This process opens the possibility of promoting the affinity of water for nonpolar or low-polarity compounds, such as lipophilic organic compounds (46). We previously reported that most of the predominant aromatic hydrocarbons were removed from softwood bedding that had been treated by soft hydrothermal processing (24, 28).Open in a separate windowFIG. 1.Reaction field in the pressure-temperature relationship of water. The curve represents the saturated vapor pressure curve. The fields show where the pressure-temperature relationships are conducive to a variety of hydrothermal processing conditions, in which water has a large impact as a reaction medium. Because high-density water has a large dielectric constant and ionic product, it is an effective reaction medium for advancing ionic reactions, whereas water (in the form of steam) on the lower-pressure side of the saturated vapor pressure curve shows a good ability to form materials by covalent bonding. Small changes in the density of water can result in changes in the chemical affinity, which has the potential to advance a range of ionic and radical reactions.The purpose of the present study was to evaluate the thermoinactivation of endotoxins by the soft hydrothermal process, by controlling the steam saturation ratio, temperature, and time of treatment. There have been reports that endotoxins were thermoinactivated by steam heat treatment at 121°C in the presence of a nonionic surfactant and at over 135°C in its absence (4, 5, 10), but the minimum temperature for the inactivation of endotoxin remained unknown. This report provides the answer to this question.