INTRODUCTION
Bacterial endotoxin, composed of lipopolysaccharides (LPS) and other membrane-associated components, is derived from the outer cell membrane of Gram-negative bacteria. The LPS is an amphipathic and highly variable component. The LPS moiety contains lipid A (an acylated di-phosphorylated diglucosamine), a core keto-deoxyoctulosonate (KDO) group, and a polysaccharide tail (1). The polysaccharide tail is highly variable in composition of sugars, length, and branching, according to species and strain (2–3). The polysaccharide tail is usually used in serotyping of an isolated organism in clinical microbial identification procedures. The diglucosamine of lipid A is acylated at either the nitrogen or oxygen of each glucosamine. There may be as few as two acyl groups, or up to eight acyl groups attached to the glucosamine, and the acyl groups may be of varying length according to the originating species (4). The LPS molecules are linked together by divalent cation salt bridges (usually magnesium, Mg++) during normal growth. However, during times of low nutrient availability (such as in purified waters), where divalent cations are not freely available, the microorganism may undergo a change in its LPS linkages via the PhoQ/PhoP sensing system (5) on the cell surface and remodel the LPS linkages to use sugars instead of divalent cations to maintain membrane activity. The organism may also change the number of its lipid A acylations based on growth conditions (5).
The naturally occurring endotoxin (NOE) derived from autochthonous microbial colonization could be considered “wild type” for its particular environmental niche. Such endotoxin may vary within a single species and is dependent on the environment from which it was isolated. The USP Reference Standard Endotoxin (RSE) (USP Endotoxin RS) was derived from a batch of E. coli O113:H3 under high nutrient conditions, including divalent cations, and therefore was under no environmental stresses that would have induced any adaptations needed for survival. This material was subjected to a hot phenol (Westphal) extraction for isolation and purification purposes. In doing so, the polysaccharide “tail” of the LPS molecule is partially stripped, thus affecting the amphipathic nature of the LPS and becoming more hydrophobic in nature (6–7). As such, it does not represent the endotoxin found in the manufacturing environment with regard to its physiochemical interactions in the environment. The Control Standard Endotoxin (CSE) supplied by Limulus amebocyte lysate (LAL) manufacturers suffers from the same Westphal purification issues as the RSE. Both were meant to be calibration standards but not representative of all endotoxin characteristics.
In 2020, USP published Use of Recombinant Reagents in Bacterial Endotoxins Test 〈1085.1〉 (8) for comment. Among other recommendations, that proposed chapter stated that the potential end user must perform a comparability test using a naturally contaminated product as the matrix containing autochthonous endotoxin from the manufacturing process. In lieu of such a contaminated product, the end user may use the NOE found upstream in the water system (e.g., in the event of a failure of the water purification process, this is the endotoxin most likely to contaminate the manufacturer’s products) (9). Previous validation efforts used RSE or CSE (10–11) whose differences from actual environmental endotoxin are described above.
This article describes the methods of measuring the level of NOE in an upstream water sample and using this water to simulate a breach in the water purification system that causes contamination near the endotoxin release limit (ERL) for that product. The products chosen for analysis were derived from a proposed study by the European Directorate for the Quality of Medicines and Healthcare (EDQM), the entity responsible for publication of the European Pharmacopoeia (EP). This study was ultimately not performed by the EDQM. The products under study were Acyclovir, Gentamicin, IV Saline, and Insulin.
Source from USP:https://online.uspnf.com/uspnf/document/2_GUID-39D7842E-76C1-4CF2-B2F6-A10B2857B74A_10101_en-US
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