DEPYROGENATION
than both the moist heat and caustic treatments. Whilst dry heat treatment was the most effective method, the mean log endotoxin reduction after both moist and caustic treatment was 1.7 and thus there was no significant difference between these two depyrogenation methods.
Discussion
This study examined the depyrogenation effectiveness of three different treatments on glassware. The results indicated that dry heat treatment consistently depyrogenated all of the spiked bottles with a greater than 3-log reduction in endotoxin. This was expected as dry heat is a generally accepted method of depyrogenation. This is notwithstanding that the mechanism of endotoxin destruction by dry heat not having been definitively studied, although it is probably due to indiscriminate incineration of molecules due to the very high temperatures. Ludwig and Avis [15] suggest a free radical mediated oxidation reaction, catalyzed by trace metals from the manufacturing process.
With the moist heat method it was demonstrated in this study that a conventional sterilizing autoclave cycle was considerably less effective in reducing endotoxin concentrations than dry heat.
The 3-log
depyrogenation target was not achieved for any of the moist heat treated bottles, and the post-treatment endotoxin levels in all of the test bottles were above 4 EU/ml.
Endotoxin is renowned for its thermostability,
and moist heat treatment by conventional autoclaving is ineffective for depyrogenation.
As a way forward, successful depyrogenation by
autoclaving in the presence of hydrogen peroxide has been reported, destruction in this case is thought due to the oxidation of the fatty acids in the lipid A portion of the lipopolysaccharide [16]. Furthermore, autoclaving for longer periods has also been shown to depyrogenate successfully. These are avenues that could be explored in future studies; however neither approach is practicable for the typical laboratory.
Treatment of spiked bottles with sodium hydroxide demonstrated a negative effect on endotoxin; however the 3-log depyrogenation target could not be consistently achieved. Whilst the endotoxin in a number of bottles was reduced to a level below the acceptance limit, this was inconsistent. It should be noted that the caustic wash was performed by hand; hence the inconsistencies in depyrogenation may be linked to the variability of mixing. The mechanism of destruction during caustic treatment is due to the hydrolysis of ester and amide linkages found in the lipid A portion of endotoxin.
The alkaline hydrolysis of ester
linkages resulting in an alcohol and acid salt is called saponification and can be enhanced by heating [17]. Therefore a greater reduction in endotoxin may have been achieved by including a heating stage in the caustic treatment.
Conclusion
Dry heat was determined to be the most effective method of depyrogenation performing significantly better than both the moist heat and caustic treatments. This study therefore supported the general findings in most literature. Dry heat consistently achieved a greater than 3-log endotoxin reduction. The moist heat and caustic treatment protocols in this study were not capable of consistent depyrogenation. Neither did the two alternative methods achieve the same quantifiable level of endotoxin destruction compared to dry heat.
The vials used in this study were rinsed with Water for Injection before treatment. Purchased glass bottles often contain impurities and need rinsing. Recycled glassware may have chemical and biological deposits
17
www.americanpharmaceuticalreview.com 5. 6. 7. 8. 9.
that could distort or mask endotoxin residues present. These impurities and residues may affect the depyrogenation of the glass and should be removed prior to treatment; there is thus scope for the development of procedures to prepare glassware for depyrogenation. Undertaking measures to reduce endotoxin is an important part of microbiological control, in relation to pharmaceutical processing and for laboratories required to prepare materials or to test components. This relates to the risk of endotoxin. The pathological effects of endotoxin, when injected, are a rapid increase in core body temperature followed by extremely rapid and severe shock, often followed by death before the cause is even diagnosed [18].
References 1.
2.
Akers, M. J., Ketron, K. M. and Thompson, B. R. (1982): ‘F Value Requirements for the Destruction of Endotoxin in the Validation of Dry Heat Sterilization / Depyrogenation Cycles’, Journal of Parenteral Science and Technology, Vol. 36, No1, January-February 1982, pp23-27
Baines, A. (2000): ‘Endotoxin Testing’ in Baird, R. M., Hodges, N. A. and Denyer, S. P. Handbook of microbiological Quality Control, Taylor & Francis, pp144-167
3. Williams, K.L. (2001). Endotoxins: Pyrogens, LAL testing and Depyrogenation 2nd Edition. Drugs and the Pharmaceutical Sciences Volume 111. Marcel Dekker Inc., New York, USA. Chapters 1, 2, 7 & 8.
4.
Hecker, W., Witthauser, D. & Staerk, A. (1994). Validation of dry heat inactivation of bacteria endotoxins. PDA Journal of Pharmaceutical Science and Technology 48 (4): 197-204.
Rietschel, E.T. & Brade, H. (1992). Bacterial Endotoxins. 1992: 26-33
Scientific American August
Cooper, J.F. (2001). The bacterial endotoxins test: past, present and future. European Journal of Parenteral Sciences 6 (3): 89-93
Baird, R. (1988): ‘Validation of Dry Heat Tunnels and Ovens’, Pharmaceutical Engineering, Vol. 8, No.2, pp31-33
Guy, D. (2003): ‘Endotoxins and Depyrogenation’ in Hodges, N. and Hanlon, G., Industrial Pharmaceutical Microbiology: Standards and Controls, Euromed, pp12.1 – 12.15
Sandle, T. (2004). Three aspects of LAL testing: Glucans, depyrogenation and water system qualification. PharMIG News 16: 3-12
10. Nakata, T. (1993): ‘Destruction of typical endotoxins by dry heat as determined using LAL assay and pyrogen assay’, J Parenter Sci Technol, Sep-Oct;47(5):258-64
11. 12. 13.
Sandle, T. (2011): “A Practical Approach to Depyrogenation Studies using Bacterial Endotoxin”, Journal of GXP Compliance, Autumn 2011
Plant, I. (1993): ‘Destruction of typical endotoxins by dry heat as determined using LAL assay and pyrogen assay’, J Parenter Sci Technol, Sep-Oct;47(5):258-64
Cooper, M.S. (1996). Bacterial Endotoxins. The Microbiological Update 14 (1): 1-4
14. Novitsky, T.J. (1996). Limulus amebocyte lysate (LAL) assays in Automated Microbial Identification and Quantitation Technologies for the 2000s (ed. W.P. Olson). CRC – Taylor & Francis, Illinois, USA. pp. 277-298
15.
Ludwig, J.D. and Avis, K.E. (1990). Dry heat inactivation of endotoxin on the surface of glass. Journal of Parenteral Science and Technology 44 (1): 4- 12
16. Weary, M. & Pearson, F. (1988). A manufacturers guide to depyrogenation. Biopharm 1 (4): 22-29
17. 18.
Caroff, M. & Kariban, D. (2003). Structure of bacterial lipopolysaccharides. Carbohydrate Research 338: 2431-2447
Tours, N. and Sandle, T. Comparison of dry-heat depyrogenation using three different types of Gram-negative bacterial endotoxin, European Journal of Parenteral and Pharmaceutical Sciences, Volume 13, No.1, 2008, pp17-20+
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132 |
Page 133 |
Page 134 |
Page 135 |
Page 136 |
Page 137 |
Page 138 |
Page 139 |
Page 140 |
Page 141 |
Page 142 |
Page 143 |
Page 144 |
Page 145 |
Page 146 |
Page 147 |
Page 148 |
Page 149 |
Page 150 |
Page 151 |
Page 152 |
Page 153 |
Page 154 |
Page 155 |
Page 156