Biomedical Engineering Reference
In-Depth Information
Despite rigorous implementation of GMP, most biopharmaceutical preparations will be con-
taminated with low levels of Gram-negative bacteria at some stage of manufacture. These bac-
teria shed endotoxin into the product stream, which is not removed during subsequent bacterial
fi ltration steps. This is one of many reasons why GMP dictates that the level of bioburden in the
product stream should be minimized at all stages of manufacture.
The heat stability exhibited by endotoxin (see Section 7.6.1) means that autoclaving of process
equipment will not destroy endotoxin present on such equipment.
Adverse medical reactions caused by endotoxin are witnessed in humans at dosage rates as low
as 0.5 ng per kilogram body weight.
7.6.1 Endotoxin, the molecule
The structural detail of a generalized endotoxin (LPS) molecule is presented in Figure 7.7. As its
name suggests, LPS consists of a complex polysaccharide component linked to a lipid (lipid A)
moiety. The polysaccharide moiety is generally composed of 50 or more monosaccharide units
linked by glycosidic bonds. Sugar moieties often found in LPS include glucose, glucosamine,
mannose and galactose, as well as more extensive structures such as L -glycero-mannoheptose.
The polysaccharide component of LPS may be divided into several structural domains. The inner
(core) domains vary relatively little between LPS molecules isolated from different Gram-negative
bacteria. The outer (O-specifi c) domain is usually bacterial-strain specifi c.
Most of the LPS biological activity (pyrogenicity) is associated with its lipid A moiety. This
usually consists of six or more fatty acids attached directly to sugars such as glucosamine.
Again, as is the case in relation to the carbohydrate component, lipid A moieties of LPS iso-
lated from different bacteria can vary somewhat. The structure of E coli 's lipid A has been
studied in the greatest detail; its exact structure has been elucidated and it can be chemically
synthesized.
7.6.2 Pyrogen detection
Pyrogens may be detected in parenteral preparations (or other substances) by a number of meth-
ods. Two such methods are widely employed in the pharmaceutical industry.
Historically, the rabbit pyrogen test constituted the most widely used method. This entails
parenteral administration of the product to a group of healthy rabbits, with subsequent moni-
toring of rabbit temperature using rectal probes. Increased rabbit temperature above a certain
point suggests the presence of pyrogenic substances. The basic rabbit method, as outlined in
the European Pharmacopoeia, entails initial administration of the product to three rabbits. The
product is considered to have passed the test if the total (summed) increase of the temperature
of all three animals is less than 1.15
C
then the product has failed. However, if the response observed falls between these two limits
C. If the total increase recorded is greater than 2.65
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