The limulus amebocyte lysate reaction in studies involving E.coli endotoxin and glass vials

Abstract

Pyrogens are substances whose presence inside the body results in the generation of fever. Amongst all pyrogens, endotoxins are the most significant and of greatest concern to the pharmaceutical industry and in particular to parenteral manufacturing. Endotoxins are very stable, high molecular weight complexes and are a characteristic feature of the outer membrane of all gram-negative bacteria. Structurally they are composed of a polysaccharide covalently bound to a lipid component, called Lipid A. The numerous biological effects of endotoxins, including pyrogenicity have all been attributed to the presence of Lipid A. The ubiquity of gram-negative bacteria increases the risk of endotoxin contamination in the parenteral manufacturing environment. Although current Good Manufacturing Practices limit such an occurrence, regulatory bodies still require the manufacturer to test the individual production stages as well as the finished goods for the presence of endotoxins. The Limulus amebocyte lysate test is currently the most common method for the detection of endotoxins. The test is based on the fact that in the presence of endotoxin molecules, the amebocyte, which is the single circulating cell in the blood of the horseshoe crab, Umulus polyphemus forms a solid gel. Glassware is notoriously one of the potential sources of endotoxins. In view of this, prior to their use in parenteral manufacturing, glassware is first washed and then rendered pyrogen free via a dry heat depyrogenating cycle. In this project the kinetic turbidimetric variant of the Umulus amebocyte lysate test was used in a series of studies involving borosilicate glass vials and a standardized preparation of E.coli 055:B5 endotoxin. A method for the recovery of endotoxin adsorbed to the surface of glass vials was developed and used to quantify the pyroburden on the inner surface of unwashed vials. The method was also employed to determine whether the vial washing cycle used by a local parenteral manufacturing company brings about a significant reduction in endotoxin under different operating conditions. The results show that unwashed vials have a very low level of detectable endotoxins. The vial washing cycle has been found to bring about a reduction in endotoxin at all different operating conditions and to be influenced by speed. New, alternative methods for the detection of endotoxin have also been discussed in the light of the growing concerns on the declining populations of Umulus polyphemus.