Endotoxin analysis

Resin evaluation of both new and used resins (titration of total binding sites, total protein capacity, flow vs. pressure, particle size distribution, total organic carbon removed by cleaning procedures, and microbial and endotoxin analysis)... 

Product component bioburden. Endotoxin analysis involves testing twenty (20) pieces. Twenty (20) vials or stoppers are filled with or immersed in WEI, mixed and sonicated. Duplicate 0.1 mL aliquots are removed and tested for endotoxin per the USP <85> Bacterial Endotoxins Test. 

Bacterial endotoxin analysis is performed on finished product where specihed in the individual monographs of the current USP. Additionally, endotoxin analysis is performed on water for injection, raw materials, and incoming components to detect contamination even in the absence of recovery of viable bacteria. Bacterial endotoxin analysis is performed per current USP, using gel clot analysis or other suitable endotoxin analysis methods (provide reference attachment number). 

Qualification of the laboratory. Qualification of the laboratory includes qualifying analysts initially by demonstrating their proficiency at preparing the necessary reagents, standards, and controls. Analyst qualification is necessary for each type of bacterial endotoxin analysis test prior to performance of any testing on actual samples. 

Inhibition enhancement validation testing is performed on each raw material and finished product as part of the validation for bacterial endotoxin analysis in accordance with the current USP and the FDA Guidelines. Limits are determined from USP monographs or the FDA Guidelines and are used in calculating the maximum valid dilution (MVD). Serial dilutions are performed of spiked and unspiked product in order to determine the dilution required to overcome inhibition or enhancement (DROIE). If the DROIE does not exceed the MVD, the test is determined to be valid. Confirmation of the DROIE is performed on three finished product or raw material lots in order to complete the product validation. Products are tested at a dilution greater than the DROIE but less than the MVD. 

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. 

Endotoxin testing. Endotoxin analysis, whenever applicable, is conducted as described in the stability protocol in (provide section number specihed in ANDA hie). 

Olenchock S.A., Lewis D.M. and Mull J.C. (1989) Effects of different extraction protocols on endotoxin analysis of airborne grain dusts. Scand. J. Work Environ., 15, 430-435. 

Bacterial products such as lipopolysaccharides (endotoxins) and cytokines (IL-2) are able to activate the contact system in vitro and in vivo (D9, H4, H7, M41). Immediately after severe trauma or after surgical intervention and particularly during sepsis, a reduction of plasma contact system proteins has been found (C10, K1, N9). Gel filtration studies of plasma demonstrated that plasma PK after activation becomes complexed with a2-M and Cl-Inh (W4). These complexes are rapidly eliminated from the circulation in vivo. In experimental studies in which pulmonary insufficiency was induced in dogs, a significant reduction of plasma kallikrein inhibitors was observed together with reduced HMK. Analysis of the relation be-... 

After the product has been filled (and sealed) in its final product container. QC personnel then remove representative samples of the product and carry out tests to ensure conformance to final product specification. The most important specifications will relate to product potency, sterility and final volume fill, as well as the absence of endotoxin or other potentially toxic substances. Detection and quantification of excipients added will also be undertaken. Product analysis is considered in Chapter 7. 

Determine endotoxin potency (EU/ng) from certificate of analysis (C of A). Calculate endotoxin units per milliliter (EU/ml), based on C of A. 

Every marketed product has a level of endotoxin tolerated based on the minimum pyrogenic dose and amount of the drug to be administered as per Food and Drug Administration (FDA) guidelines [19]. However, there are none for the more advanced chemical assays described here. Indeed there are only a few highly specialized university laboratories that currently have experience in trace chemical analysis of LPS and PG. There are no commercial testing laboratories. Simplification and automation will allow more widespread availability of these methods. 

Mielniczuk, Z., Mielniczuk, E., and Larsson, L. (1993), Gas chromatography-mass spectrometry methods for analysis of 2- and 3-hydroxylated fatty acids Application for endotoxin measurement, I. Microbiol. Meth., 17, 91-102. 

While these compounds may be useful as reference compounds for structural analysis of endotoxins and helpful for orienting investigations aiming at the establishment of the chemical structure/biological activity relationship of these substances, they are still far from representing any Lipid A fragment and even less the hydrophobic region of an endotoxin. From... 

Abstract Lipopolysaccharides are the major components on the surface of most Gram-negative bacteria, and recognized by immune cells as a pathogen-associated molecule. They can cause severe diseases like sepsis and therefore known as endotoxins. Lipopolysaccharide consists of lipid A, core oligosaccharide and O-antigen repeats. Lipid A is responsible for the major bioactivity of endotoxin. Because of their specific structure and amphipathic property, purification and analysis of lipopolysaccharides are difficult. In this chapter, we summarize the available approaches for extraction, purification and analysis of lipopolysaccharides. 

Karibian, D., Deprun, C., Carofif, M. Use of plasma desorption mass spectrometry in structural analysis of endotoxins effects on lipid A of different acid treatments. Prog Clin Biol Res 392 (1995) 103-111. 

Andra, J., Howe, J., Garidel, P., Rossle, M., Richter, W., Leiva-Leon, J., Moriyon, I., Bartels, R., Gutsmann, T., Brandenburg, K. Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins thermodynamic, biophysical and microbiological analysis. Biochem J 406 (2007) 297-307. 

Zahringer, U., Salvetzki, R., Ulmer, A.J., Rietschel, E.Th., Isolation, chemical analysis and biological investigations of natural lipid A and lipid A-antagonists. J Endotoxin Res 3 (Suppl. 1) (1996) 33. 

Ho, B., Kim, J.C., Ding, J.L. Electrophoretic analysis of endotoxin-activatied gelation reaction of Cardnoscorpius rotundicauda amoebocyte lysate. Biochem Mol Biol Inti 29 (1993) 687-694. 

Sultzer, B.M. Genetic analysis of lymphocyte activation by lipopolysaccharide endotoxin. Infect Immun 13 (1976) 1579-1584. 

Figure 1. SDS-PAGG analysis of alkaline-dissolved Bacillus thuringiensis subspecies kurstaki (BTK) and israelensis (BTI) -endotoxin at 25 yg per track (1) BTK -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.), (2) BTI 6-endotoxin from Sandoz Inc., (3) BTI (Sandoz) -endotoxin purified by DEAE-anion exchange chromatography, (4) percipitate formed after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (5) soluble fraction after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (6) BTI (Sandoz) -endotoxin purified by Sephadex G-75 gel filtration chromatography at Rf 1.35, (7) at Rf 1.58, and (8) BTI strain IFC-1 -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.). S, molecular weights as indicated X1000 for bovine serum albumin (BSA), ovalbumin (OA), trypsin, and myoglobin. Reproduced with permission from Ref. 29. Copyright 1984, Academic Press, Inc.

Ug/ml + 1 S.D. with slope of probit analysis in parenthesis. Larvae were incubated in water with BTI 6-endotoxin added. 

Figure 5. SDS-Page analysis of alkaline-dissolved Bacillus thuringiensis israelensis (BTI) 6-endotoxin from Sandoz Inc. at 25 yg per track (1) BTI 6-endotoxin as prepared in Figure 1 (Track 2), (2) soluble fraction after dialysis of BTI 6-endotoxin into pH 4.5 sodium acetate buffer, and (3) 25K component from BTI 6-endotoxin after pH 4.5 percipitation and DEAE-anion exchange chromatography. S, molecular weight markers from top to bottom bovine serum albumin (68K daltons), ovalbumin (43K), and myoglobin (16K).

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