Inactivation of Escherichia coli Endotoxin by Soft Hydrothermal Processing

Abstract

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.

Kinetics of Hydrothermal Inactivation of Endotoxins

“…Further attempts were made to correlate available data obtained at different temperatures. The data from this study were obtained at the upper temperature limit, while the data from three previous studies were considered to extend the lower temperature limits to 200°C (25), 190°C (1), and 120°C to 140°C (28). Figure 4 shows the Arrhenius plot, which follows a linear trend with r 2 ϭ 0.9875.…”

“…Similarly, steam sterilization is about 1 order of magnitude more effective than the dry heat sterilization process. At the lower temperature limit, near 121°C, the data show that high steam saturation (28) shortens and low steam saturation (5,28) prolongs the time required to achieve depyrogenation. Furthermore, the data confirm that depyrogenation requires a higher temperature or a longer time than sterilization.…”

“…If effective dry heat depyrogenation is performed, sterilization generally is achieved as well. Depyrogenation can also be achieved by moist heat (4,5,20,28) and in condensed high-temperature water (25). The former typically involves temperatures of 121°C to 150°C under saturated conditions, and the latter has extended the temperature from 150°C to 400°C under pressures that prevent water from boiling.…”

“…Moist heat appears to have an intermediate impact on depyrogenation, as exhibited in the data of Bamba et al (5). The rate of depyrogenation begins to accelerate with the (28). As the temperature is further increased while the pressure is maintained above the saturation curve, the aqueous reaction medium begins to shift from being polar to being nonpolar (40).…”

“…Figure 8 shows the temperature (T) and time (t) semilog correlation for 3 D heat sterilization and depyrogenation. The literature data were obtained from the independent studies described above (1,5,15,21,25,28,34,35). All of the data shown in Fig.…”