Reference standard endotoxin

After weighing the dust filters, the amount of endotoxin was determined by shaking them In 10 ml of pyrogen free water and preparing serial dilutions. Llmulus lysate (Cape Cod Associates Inc.) was added to the dilutions according to the manufacturer s recommendations. The last dilution giving a stable clot was read as the Escherichia coll endotoxin equivalent concentration. Dilutions were also prepared with commercial E. coll endotoxin (. coll 026-B26, Dlfco) to assess the accuracy of the production reference standard. The values were always found to agree closely with the stated values. 

Limulus amebocyte lysate is obtained from a licensed manufacturer. Each lot of reagent is tested per USP for release. Endotoxin used in all bacterial endotoxin analysis testing is obtained from a licensed manufacturer and is standardized against the USP reference standard. 

The <85> BET first appeared in 1980 as an informational chapter in the USP XX. Significantly, the USP adopted the FDA E. coli 0113 endotoxin standard as its reference standard and assigned units of potency to it. It also included information about calibrating a control standard endotoxin (CSE) to the RSE. 

In vitro assays do not use any whole-cell or animal-based components. The fibrin clot lysis assay, as established for tissue plasminogen activators and described for alteplase in the USP, is an example of this type of potency testing [5]. By means of defined standard materials, a fibrin clot is formed and the time to complete lysis is characterized as measure of potency, compared to a reference standard with defined activity. The LAL-test is a well-established and internationally harmonized in vitro alternative to detect or quantify bacterial endotoxins, using Limulus amebocyte lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tri-dentatus) which has been prepared and characterized appropriately [5]. Two types of technique may be used for this test gel-clot techniques, which are based on gel formation and photometric techniques. 

Standardization and Testing". The final vaccine is tested for safety, potency, and residual chemicals. Safety includes testing for endotoxin and stetihty. Potency is evaluated by quantitative determination of the amount of hemagglutinin in the vaccine. Antibody to this glycoprotein is associated with protection. The single radial immunodiffusion (SKID) technique is used to standardi2e the mass of this protein in comparison to a reference preparation. 

However, the establishment of a new endotoxin standard by the World Health Organization is a recent example of successful international collaboration between the World Health Organization, the United Stated Pharmacopoeia and the European Pharmacopoeia (Poole et al. 1997). Thus this standard is available from any of these organizations to be employed as a reference in the harmonized Limulus Amoebocyte Lysate test. 

Bacterial endotoxin kinetic test or gel clot limit standard test method (provide reference number)... 

The General Tests, Processes, and Apparatus section contains 60 test methods, such as fluorometry and electrometric titration, dissolution and disintegration tests, content uniformity, and a bacterial endotoxin test. Qualitative tests, reagents and test solutions, and standard solutions are also included. The Infrared Reference Spectra of 124 chemical entities are included in an appendix and will be updated in subsequent editions. 

Naturally occurring bacterial endotoxins contain the lipid, carbohydrate, and protein makeup of the outer cell membrane of GNB (Fig. 1). However, most of the commercial endotoxin preparations have been purified by various extraction procedures and are generally free of nucleic acids, proteins, phospholipids, and other bacterial cell components. The primary chemical configuration that remains after purification is apolysaccharide structure that is covalently bound to a lipid component called Lipid A. Based on its chemical nature, which is common to various bacterial families, this substance is referred to as lipopolysaccharide (LPS). Although the terms endotoxin and LPS are often used interchangeably, most reference endotoxin standards are purified preparations that are more correctly described as LPS. 

Pyrogenicity or endotoxin content. The pyro-genicity of a specified dose of product when administered to rabbits can be assayed by a standard pharmacopoeial method but the trend is to replace this with an in vitro assay for endotoxin (Chapter 19). The capacity of the product to induce gelation of Limulus polypbemus amoebocyte lysate is determined against a reference endotoxin preparation and the result is expressed as IU of endotoxin. 

Weary M.E., Donohue G., Pearson F.C. and Story K. (1980) Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests. Appl. Environ. Microbiol., 40, 1148-1151. 

Most raw material suppliers indicate the source of the quality specifications on the label. However, it may occur, that despite there being an entry in the Ph. Eur., suppliers, perhaps due to market conditions, will sell substances that meet the requirements of a foreign pharmacopoeia or other standard. Some suppliers may allow the download of a certificate fi om their website or supply a certificate with each delivery. In other cases it may be necessary to contact the supplier to request the certificate to be sent. Such a certificate should refer to which pharmacopoeia or standard the raw material complies. It should also list the pharmacopoeia analytical data of the raw materials including relevant quantifiable impurities such as bacterial endotoxins. A certificate of analysis is signed, or refers to the authorisation by the quaUty officer of the supplier. 

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