in black and white
Main menu
Home About us Share a book
Biology Business Chemistry Computers Culture Economics Fiction Games Guide History Management Mathematical Medicine Mental Fitnes Physics Psychology Scince Sport Technics

biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
Previous << 1 .. 91 92 93 94 95 96 < 97 > 98 99 100 101 102 103 .. 292 >> Next

the heat-stability exhibited by endotoxin (see next section) 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 0.5ng/kg body weight.
Endotoxin, the molecule
The structural detail of a generalized endotoxin (LPS) molecule is presented in Figure 3.37. 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
Figure 3.37. Structure of a generalized lipopolysaccharide (LPS) molecule. LPS consitutes the major structural component of the outer membrane of Gram-negative bacteria. Although LPS of different Gram-negative organisms differ in their chemical structure, each consists of a complex polysacharide component, linked to a lipid component. Refer to text for specific details
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-specific) domain is usually bacterial strain-specific.
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 isolated from different bacteria can vary somewhat. The structure of E. colis lipid A has been studied in greatest detail. Its exact structure has been elucidated, and it can be chemically synthesized.
Pyrogen detection
Pyrogens may be detected in parenteral preparations (or other substances) by a number of methods. 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 monitoring 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 1.15C. If the total increase recorded is greater than 2.65C the product has failed. However, if the response observed falls between these two limits, the result is considered inconclusive, and the test must be repeated using a further batch of animals.
This test is popular because it detects a wide spectrum of pyrogenic substances. However, it is also subject to a number of disadvantages, including:
it is expensive (there is a requirement for animals, animal facilities and animal technicians);
excitation/poor handling of the rabbits can affect the results obtained, usually prompting a false-positive result;
sub-clinical infection/poor overall animal health can also lead to false-positive results;
use of different rabbit colonies/breeds can yield variable results.
Another issue of relevance is that certain biopharmaceuticals (e.g. cytokines such as 1L-1 and TNF; Chapter 5), themselves, induce a natural pyrogenic response. This rules out use of the rabbit-based assay for detection of exogenous pyrogens in such products. Such difficulties have led to the increased use of an in vitro assay; the Limulus amoebocyte lysate (LAL) test. This is based upon endotoxin-stimulated coagulation of amoebocyte lysate obtained from horseshoe crabs. This test is now the most widely used assay for the detection of endotoxins in biopharmaceutical and other pharmaceutical preparations.
Development of the LAL assay was based upon the observation that the presence of Gram-negative bacteria in the vascular system of the American horseshoe crab, (Limulus polyphemus), resulted in the clotting of its blood. Tests on fractionated blood showed the factor responsible for coagulation resided within the crab s circulating blood cells, the amoebocytes. Further research revealed that the bacterial agent responsible for initiation of clot formation was endotoxin.
The endotoxin molecule activates a coagulation cascade quite similar in design to the mammalian blood coagulation cascade (Figure 3.38). Activation of the cascade also requires the
Active Factor
Active Factor
Factor A
Active Factor A
Coagulin & peptide C
Previous << 1 .. 91 92 93 94 95 96 < 97 > 98 99 100 101 102 103 .. 292 >> Next