Endotoxin contamination

Set the diy block heater to 37 1 °C. Allow aU reagents (except LAL) and test samples to adjust to room temperature. Draw up a plate plan as follows  

Culture media, water, and physiological buffers can be tested imdiluted. Samples containing high endotoxin must be diluted within the working range. Test several different dilutions of unknown but suspect samples. 

Dispense 10 p,l of the standards, controls, and test samples according to the plate plan. 

Dispense the substrate (20 pJ per sample) into Row H so that it can later be quickly transferred to the sample wells with a multichaimel pipetter. 

Timing is critical. Use a stopwatch and add reagents in the same order, so that all wells have identical reaction time. Keep the plate in the dry block heater to maintain constant temperature. 

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Contamination with endotoxin is an important and frustrating problem in LEH manufacturing for two reasons. Firstly, hemoglobin has a strong tendency to bind endotoxin, where one hemoglobin molecule binds to four endotoxin molecules (K 3.1 x lO M) (117). Secondly, endotoxin has amphiphilicity that enables its stable insertion into lipid bilayer. Such an interaction not only presents contamination and stability problems, but also hampers accurate quantitation of endotoxin. The best possible way to prevent endotoxin contamination is to use aseptic precautions with utmost care. All the machinery, filters, and water should be endotoxin-free. Glass and metallic components may be dry-heat sterilized at about 200° C for three... 

Workers exposed to lipopolysaccharides from office humidifiers and sewage plants (70, 71) also exhibit elevated IgG levels and, in sewage workers, this response is dose dependent. Since endotoxin contamination of cotton is well documented (14), it suggests that the increased IgG levels seen in cotton workers may be due to endotoxin exposure. The possibility that this increase is due to specific IgG is slight in view of the fact that in other diseases, high specific IgG levels do not... 

Provocative Inhalation challenge has been used in an attempt to identify byssinogenic agents (52-54, 83-86). Results of these studies have been Inconclusive, but most positive reactions appear to be due to endotoxin contamination of the dust, or to a toxic or irritant factor (52,53). 

Closure sterilization, following the cleaning cycle, is typically done by autoclaving with saturated steam. The temperatures achieved in such treatments are not sufficient to eliminate significant endotoxin contamination. 

The presence of endotoxin should be monitored by LAL method in the routine operation. Endotoxin can be removed by means of distillation, reverse osmosis, and/or ultrafiltration. Incomplete separation of mist in distillation, however, and leakage in membrane of reverse osmosis or ultrafiltration cause contamination with endotoxin. After these separation processes, of course, contamination of microbes or growth of microbes causes endotoxin contamination [2,8],... 

Nakagawa Y, Mural T, Flasegawa C, Flirata M, Tsuchiya T, Yagami T, Flaishima Y. Endotoxin contamination in wound dressings made of natural biomaterials. J. Biomed. Mater. Res. B Appl. Biomater. 2003 15 347-355. 

Endotoxin contamination has been encountered frequently in Hb solutions. Historically, the production of Hb solutions low in endotoxin content has proven challenging, in part because much of the early work was performed in academic research laboratories that were not familiar with the techniques used in the pharmaceutical industry to prevent bacterial contamination of the process stream. In addition, Hb has been reported to bind endotoxin, making it even more difficult to completely remove once present. Therefore, the production of Hb solutions must be carefully designed to minimize the introduction of endotoxin into the process stream. 

While blending times and tablet press parameters may not be fully established for early phase clinical supply manufacturing of solid oral dosages, those variables have a much lower potential to directly affect product safety than sterility, endotoxin contamination, or objectionable types and levels of particulates do for sterile, parenteral clinical supplies. Because clinical supplies can be incompletely characterized, and are usually given to patients already in weakened conditions, those processes and their related validation data necessary to guarantee patient and product safety (e.g., sterilization and aseptic fill) are expected to be in place as early as Phase I clinical supply manufacture. ... 

In vitro investigations have attempted to clarify the mechanism of immune activation but so far have provided limited data. Studies testing the hypothesis that implicated tryptophan or EBT can trigger PBMCs to release cytokines have been equivocal, although one study found that EBT activates eosinophils and induces IL-5 production from T cells. Another recent study found that certain lots of L-tryptophan could stimulate PBMCs to release granulocyte-macrophage colony-stimulating factor (GM-CSF) this response, however, was caused by endotoxin contamination and not associated with case lots of tryptophan. The mechanism of immune activation is clearly complex and may be difficult to reproduce with an in vitro assay. Similar difficulties have been encountered in the study of immune system activation in TOS. 

Wakelin SJ, Sabroe I, Gregory CD, Poxton IR, Forsythe JLR, Garden OJ et al (2006) Dirty little secrets —endotoxin contamination of recombinant proteins. Immunol Lett 106 1-7... 

If the MAb is used for the preparation of a clinical IT, an additional chromatographic purification is performed on a DEAE-Sepharose column equilibrated with PBS to remove the murine DNA and bacterial endotoxin contaminating the MAb. 

USP Chapter <797> classifies compounded sterile preparations (CSP) or pharmaceuticals into low-risk, medium-risk, and high-risk level categories based on the potential chemical, microbial, and endotoxin contamination. Radiopharmaceuticals including PET radiopharmaceuticals belong to the low-risk level group. 

Gordon T., Galdanes K. and Brosseau L. (1992) Comparison of sampling media for endotoxin contaminated aerosols. Appl. Occup. Environ. Hyg., 7, 427-436. 

PFW stored below 80 C should be tested at least twice-weekly for microbial and endotoxin contamination. 

Equipment cleaning and sanitization studies should address microbiological and endotoxin contamination for those processes intended or purported to reduce bioburden or endotoxins in the API, or other processes where such contamination may be of concern (e.g., nonsterile APIs used to manufacture parenteral products). 

Written procedures should be established and measures taken to control bioburden and endotoxin contamination of nonsterile APIs intended for use in the preparation of parenteral drug products. 

It is best to make certain all reagents are free of endotoxin contamination before attempting to isolate neutrophils. This can be done using the Limulus Amebocyte Lysate assay (as described by the manufacturer). 

Pyrogenidty (fever producing) tests are also included in the systemic toxicity category to detect material-mediated pyrogenic reactions of extracts of medical devices or materials. It is noteworthy that no single test can differentiate pyrogenic reactions that are material mediated from those due to endotoxin contamination. 

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