الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 96 |  |  |
| | | from | 96 | | |
Abstract
T
he most effective method to destroy microorganisms is through ‘heat ‘, as it coagulates their proteins as well the enzymes present in them. So, sterilization (destroying and/or killing the microorganisms) process follows this principle of killing microorganisms, which can be either by applying wet (moist) heat or dry heat.
Heat sterilization methods using moist heat or dry heat sterilization technique are the effective methods used to sterilize pharmaceutical products which are heat stable (thermostable) products while thermolabile products sterilized with other methods like radiation sterilization or chemical gases sterilization.
Pyrogens are fever-inducing substances, which considered as a metabolic biproducts of microorganisms. Chemically, they are lipid in nature and associated with a polysaccharide carrier molecules. These polysaccharide carriers enhance the lipid’s solubility. Microorganisms including bacteria, yeasts and moulds are producing pyrogens in surrounding media. Endotoxins are the most potent pyrogens, have a high molecular weight and produced from the cell walls of the Gram-negative bacteria.
Therefore, all lots of solutes used to prepare parenteral products should be tested to ensure that they will not participate in undesirable amounts of endotoxin to the finished product. Nowadays we have standard practice to make valid endotoxin limits on active pharmaceutical ingredients and their additives.
In this study the effectiveness of various techniques to manufacture sterile ampules in industrial facilities at Pharmaceuticals Companies will be evaluated. The effect of the annealing temperature during the manufacturing of closed ampules will be examined on the sterilization and depyrogenation of the ampules.
The results obtained can be summarized as follows:
Glass Ampoules of that incubated for 0.5, 1, 2, 3, and 4 hrs, respectively in the UV dynamic pass box showed gel formation (+ve) when LAL test was examined representing the presence of pyrogen.
After LAL test for the 10 glass ampoules containing endotoxin gel was formed (+ve result), while the other 10 plastic ampoules containing endotoxin showed no gel formation when ethylene oxide EtO was performed.
All ampoules of the first 4 groups treated with different concentrations of NaOH as 0.1 M, 0.2 M, 0.3 M and 0.4 M at 50oC for 30 min showed gel formation (+ve) result, while the -ve results (no gel formation) of the treated ampoules only was observed for G5 and G6 for 0.5 and 1 M of NaOH, respectively, proving no pyrogen.
All ampoules of the first 5 groups treated with 0.5 M NaOH and incubated for 5, 10, 15, 20 and 25 min at 50oC showed gel formation (+ve) result, while depyrogenation occurred in the last group G6 which treated with the same concentration of NaOH and incubated for 30 min at 50oC to give (-ve) LAL test result where no gel was formed.
All ampoules of the first 5 groups treated with 0.05 M HCl and incubated for 5, 10, 15, 20 and 25 min at 100oC showed gel formation (+ve) results, while the last group G6 which treated with the same concentration of HCl but incubated longer for 30 min at 100oC gave no gel formation which means (-ve) result in the test of LAL.
After incubation period (24 hours) for the biological indicator at 55oC-60oC the color of Geobacillus stearothermophillus spores’ control vial which didn’t sterilized in autoclave changed from blue to yellow color which mean bacterial growth, while the other Geobacillus stearothermophillus spores’ vials which sterilized in autoclave have no color change (-ve) result. On the other hand, all examined endotoxin-containing ampoules showed gel formation (+ve) result meaning the pyrogen still present.
All ampoule groups No. 1,2,3,4 that have been autoclaved for periods of 1 hour to 4 hours showed gel formation (+ve) result after the test of LAL. On the other hand, only group No. 5 which have autoclaving periods of 5 hours (5 cycles) showed no gel formation (-ve) and depyrogenation occurred.
In the test of dry heat (oven) at 250oC for 30 min, after incubation period of 24 hours at 55-60oC the bacterium spores of Geobacillus stearothermophillus control vial which didn’t sterilized in dry heat oven, the color of this control vial change from blue to yellow which mean (+ve) result, while the other 5 vials of Geobacillus stearothermophillus spores’ have no color change showing negative results (-ve). In addition, all endotoxin containing ampoules showed no gel formation (-ve LAL test) representing no pyrogen.
According to effect of different annealing temperatures of closed empty ampoules in depyrogenation process and according to the holding stage time at different speeds which affect the exposure time for the ampoules to the working temperature, the presented results show endotoxin depyrogenation in ampoules examined by LAL test were varied in these tests according to oven temperature (250oC to 550oC) and the belt speeds. All belt speeds showed (-ve) result with LAL reagent with exposure temperatures started from 350oC up to 550oC, however, (+ve) result of LAL tests (pyrogen presents) were shown at 250oC of all belt speeds.
In the test of sterilization and depyrogenation using dry heat (oven) at 500oC for 105 sec., after incubation period of 24 hours at 55-60oC the bacterium spores of Geobacillus stearothermophillus control vial the color changed from blue to yellow which mean (+ve) result, while the other vials of Geobacillus stearothermophillus spores’ that exposed to dry heat (500oC for 105 sec.) have no color change showing negative results (-ve). In addition, all endotoxin containing ampoules under the same conditions show no gel formation (-ve) when LAL test was examined.
In more additional experiment of annealing temperature at 500oC for 105 sec (ordinary empty ampoule manufacturing method) on depyrogenation of different sizes (3, 5 and 10 ml) of glass empty ampoules, after LAL tests for all ampoules they showed no gel formation (-ve) results.