الفهرس 
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Abstract
Pyrogens an amphiphilic chemical heterogeneous group causing evoked in body temperature after administered by mammalians not only derived from Gram-negative and Gram-positive bacteria but also from viruses and fungi. They stimulate an immune response by producing endogenous pyrogens as PG, proinflammatory cytokines interleukin-1, interleukin-6, and TNF. Endotoxin is the most potent and best known pyrogen, it is amphiphilic lipopolysaccharides (LPS) which represent the main constituent of the Gram-negative bacterial cell wall.
Gram negative bacteria considered as the major contributors to the pyrogenic response observed with contaminated pharmaceutical products. Products manufactured by bioprocessing are microbiologically controlled to ensure that the end product meets the requirements for release. All parenteral preparations as well as tissue implants or other related materials must be within acceptable endotoxin limit for human use because of their associated health hazard and serious clinical effects. So the detection and limiting endotoxin in pharmaceutical and biological products is crucial, this study conducted through comparison between rabbit Pyrogen test (RPT) and Limulus Amebocyte Lysate (LAL) test as two methods for endotoxin detection and quantification.
In the present study, The in vitro LAL test using gel-colt technique was applied for determination of endotoxin level in thirteen biological products belonging to different medication categories. While the in vivo RPT was used for detection and evaluation of pyrogenicity of four biological products from those examined by LAL test (streptokinase 1.5 MIU, erythropoietin-alpha at 2 different concentrations (2000 and 4000 IU) and 2 batches of pentavelant vaccine).
The preliminary test carried out on these products proved no interference since they exhibited no enhancement or inhibition when they were used in assay samples containing specified amounts of endotoxin using gel-clot technique. Also the sensitivity of LAL reagents used in different assays were confirmed and complied the reference acceptance criteria (0.5λ - 2λ). Most of the used products were tested at two different lysate sensitivities (0.25 and 0.125 λ). However, two tested products were evaluated at higher lysate sensitivity (0.06 λ).
Additionally in this experiment the MVD for the all tested product was calculated using either (i) the labeled lysate sensitivity and (ii) the lysate sensitivity calculated from the data obtained from the experiment carried out for confirmation of lysate sensitivity. This was conducted using five tested preparations with aim of knowing if the validity range (0.5λ - 2λ) at which the LAL reagent is used will affect the test result or not. The experiments were carried out by first determination of their endotoxin level. Then the difference (ΔE) between the endotoxin test result and acceptance endotoxin limit for human use was calculated. An artificial inoculation of endotoxin into test samples was done with amount equal to ΔE, 1.5ΔE and 2ΔE . after that the endotoxin level in the inoculating samples were re-determined.
Furthermore, the effect of pre-used animals in the sensitivity of RPT for some selected biological products was checked by using a thirty four rabbits were subjected to preliminary test. Rabbits that were passed the preliminary test were divided into six groups of different sizes, each not less than three rabbits. These groups were used more than one time for determination of pyrogenicity of certain pre-tested product that passed the RPT (i.e. these products contained no pyrogenic substances)
Finally, this study was aimed to determination of validity for the maximum weight of rabbits used in RPT that done by used eighteen rabbits passed the preliminary test and having weights ranging from 1.5 kg to 5 kg were divided into three test groups, each 6 rabbits. The three test groups were injected with human albumin preparations that were pre-tested and proved to have no pyrogenic materials. Additionally, the previously used rabbits were subjected to re-use by using the same Human albumin preparations. Furthermore, the used rabbits were subjected to third re-use using antitetanic serum preparations that were pre-tested and proved to be pyrogenic.
The result, findings revealed that the endotoxin levels of the thirteen tested biological products were within the acceptance endotoxin limit for human use. Accordingly, these tested products can be used safely for treatment and/or prophylaxis against relevant human diseases. from the results it is revealed that the use of LAL reagent with different sensitivities gave the same result for the product tested at more than one lysate sensitivity. Additionally, the total response (pyrogenicity) in RPT was calculated by summation of the differences between maximum temperature and average base temperature of the three tested rabbits. The results were interpreted using the specification criteria mentioned in the European pharmacopeia and in all cases no product produced an increase in body temperature of tested rabbits exceeded the mentioned criteria. Accordingly the tested product were not pyrogenic and can be administered safely for human.
On other hand, the MVD values were calculated used two lysate sensitivities, labeled lysate sensitivity and the actual calculated lysate sensitivity. Therefore, the resulted MVD was be different in both cases and this can affect the test result. When endotoxin was artificially added to each tested product with pre-determined endotoxin level at ΔE, 1.5ΔE and 2 ΔE (where ΔE is the difference between the endotoxin test result and acceptance endotoxin limit for human use), the results obtained were the endotoxin values obtained at the different amount of artificially added endotoxin (ΔE, 1.5ΔE and 2 ΔE) were above the acceptance endotoxin level for human use for all products determined its MVD used labeled lysate sensitivity.
The endotoxin values for all tested products determined its MVD used calculated lysate sensitivity were within the acceptance criteria for human use at ΔE artificially added endotoxin – the case which is different from the corresponding one. At artificially added endotoxin equal to 1.5ΔE and 2 ΔE the results of endotoxin level determined were out of the acceptance criteria for human use as expected.
Finally, from the results obtained from the experiments for studying the effect of pre-used animals and maximum weight of this animals in validity of RPT it can be concluded that the animals involved in RPT can be used more than once for the same test with different biological products regardless the product previously used but routine work it is recommended to validate the reuse for product under test. And the effect of both repeat use of animals as well as different animal weights in the range of weights routinely used in RPT (1.5 – 2.3 Kg) and in range exceeding the aforementioned range (2.7 – 5 Kg). The obtained results revealed that variable animal weights not less than 1.5 Kg had no effect on RPT results regardless of tested product used.
Also, results revealed that the repeated animal use even for those higher weights not affect the RPT results regardless of the tested product used.