Pyrogens measurement

Much information can be gained from in-life physiological measurements such as blood pressure, electrocardiograms, body temperature, and respiratory rate. When the number of studies is reduced due to the lack of appropriate species and the potential for development of neutralizing antibodies, as much information as possible should be obtained from the few studies that are conducted. In-life measurements assume more importance in these cases. When the product is likely to contain proteins that may cause acute serum sickness type responses and pyrogens, measurements of blood pressure, body temperature, and respiratory parameters are especially important. 

Pyrogenic A fever-producing substance. The presence of these substances is determined by the Limulus Amebocyte Lysate (LAL) test and measured in EU/ml (endotoxin units per milliliter). 

The United States Pharmacopeia describes a pyrogen test using rabbits as a model (USP, 1995b). This test, which is the standard for limiting risks of a febrile reaction to an acceptable level, involves measuring the rise in body temperature in a group of three rabbits for 3 h after injection of 10 ml of test solution. 

Fixed equipment in the compounding area (nonaseptic or aseptic) is cleaned in place. This eliminates traces of prior products, particles, and pyrogens. Sterilization in place is required for the aseptic fixed equipment and is sometimes employed for the nonaseptic equipment as well as a bioburden control measure. Fixed transfer lines must be cleaned and sterilized as well, and this is accomplished independently or in conjunction with the vessels. The reuse of hoses and tubing is discouraged as cleaning and extractables cannot be confirmed beyond a single use. 

Pyrogenic contamination is detected using two tests. In the older method, rabbits are injected with product samples, and rectal temperature is measured. Compendial limits are established with respect to how much temperature increase is permitted before the product is judged to be free or contaminated with pyrogens. The newer method involves a relatively simple in vitro technique called the Limulus Amebocyte Lysate (LAL) test. It is based on the high senstivity of amebocytes of the horseshoe crab (Limulus) to the lipopolysaccharide component of endotoxins originating from Gramnegative bacteria. The LAL test is now the USP method of choice with endotoxin limits established for most SVIs. ... 

All manufactured products are vulnerable to contamination by a myriad of aerosolized contaminants, including microorganisms, pyrogenic dust, ash, pollen, smoke, hydrocarbons, and other chemicals that are omnipresent in the environment (Pig. 1). Because of the potential dangers to the patient resulting from a parenteral product containing even minute quantities of these contaminants, exceptional measures are required to exclude them fromthe finished product. 

Basically, the pyrogen test involves measuring the rectal temperature of rabbits, both prior to and after the intravenous injection of a test solution in the ear veins. If the animals exhibit febrile responses that exceed established limits, the test solution is judged to be pyrogenic. Rabbits became the animal of choice because they are relatively inexpensive, are easy to handle, and have a labile thermoregulatory mechanism. Rabbits frequently produce false-positive pyrogen... 

Fig. 1 shows the variation of 7( measured at 100°C by IGC, when treating a pyrogenic or fumed silica (Aerosil from Degussa, Germany) at 100 - 700°C. The same figure presents... 

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