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. 2009 Aug;75(15):5058-63.
doi: 10.1128/AEM.00122-09. Epub 2009 Jun 5.

Inactivation of Escherichia coli endotoxin by soft hydrothermal processing

Toru Miyamoto  1 , Shinya Okano, Noriyuki Kasai
Affiliations

Inactivation of Escherichia coli endotoxin by soft hydrothermal processing

Toru Miyamoto et al. Appl Environ Microbiol. 2009 Aug.

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 degrees 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 degrees C for 60 min or at 140 degrees 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 degrees C for 60 min or at 140 degrees 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.

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Figures

FIG. 1.
FIG. 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.
FIG. 2.
FIG. 2.
Schematic diagram showing the flow-type endotoxin inactivation apparatus used for the soft hydrothermal process. Components: 1, water tank; 2, plunger pump; 3, steam generator; 4, condenser; 5, backpressure regulator; 6, line heater; 7, reactor; 8, sample cages; 9, reactor heater; 10, control valve; 11, steam trap; 12, safety valve. The apparatus for the soft hydrothermal process consists of four main components: a cylindrical reactor (400-mm inner diameter, 1,260-mm length, and 158.3-liter volume) equipped with a 9-kW electric heater, a steam generator with a 24-kW electric heater, a backpressure regulator, and a reactor pressure controller. The connecting pipes are constructed of SUS304.
FIG. 3.
FIG. 3.
Temperature course of the inactivation of endotoxin with relation to the steam saturation ratio and to the flow system for 30 min. Inactivation was carried out at 120, 130, or 140°C for 30 min. The data are expressed as means ± the standard deviations (n = 3). Columns: open, LRV at a 50% steam saturation ratio; stippled, LRV at a 100% steam saturation ratio; cross-hatched, LRV at a 1,000% steam saturation ratio; solid, LRV of the flow system. The broken line represents the USP guideline boundary for depyrogenation with an LRV of >3.0. Significant individual differences were evaluated by using Tukey's honestly significant differences adjustment if the two-way ANOVA was significant (the symbols †, ‡, and § indicate P < 0.05).
FIG. 4.
FIG. 4.
Time course of the inactivation of endotoxin with relation to the steam saturation ratio and to the flow system at 130°C. Inactivation was carried out for 30, 60, or 90 min at 130°C. The data are expressed as described for Fig. 3.
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References

    1. Andersson, T., K. Hartonen, T. Hyotylainen, and M. L. Riekkola. 2002. Pressurized hot water extraction and thermal desorption of polycyclic aromatic hydrocarbons from sediment with use of a novel extraction vessel. Anal. Chim. Acta 466:93-100.
    1. Anspach, F. B. 2001. Endotoxin removal by affinity sorbents. J. Biochem. Biophys. Methods 49:665-681. - PubMed
    1. Bamba, T., R. Matsui, and K. Watabe. 1996. Effect of steam-heat treatment with/without divalent cations on the inactivation of lipopolysaccharides from several bacterial species. PDA J. Pharm. Sci. Technol. 50:129-135. - PubMed
    1. Bamba, T., R. Matsui, and K. Watabe. 1996. Enhancing effect of non-ionic surfactant on the inactivation of lipopolysaccharides by steam-heat treatment. PDA J. Pharm. Sci. Technol. 50:360-365. - PubMed
    1. Bamba, T., R. Matsui, and K. Watabe, and M. Tadanori. 1997. Enhancing effect of non-ionic surfactant on the inactivation of lipopolysaccharides by steam-heat treatment. PDA J. Pharm. Sci. Technol. 51:156-160. - PubMed

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