Endotoxin elimination

The experience of application of various efferent methods of endotoxin elimination has led to the idea of creating selective anti-LPS hemosorbents with Polymyxin B as the bioUgand. The covalently immobilized Polymyxin B does not cause side effects characteristic of free Polymyxin B and keeps its therapeutic effects. G.W. Duff in 1982 demonstrated for the first time the sorption efficiency of Polymyxin B-immobilized sorbent in vitro [9]. In 1984 K. Hanasawa with co-workers developed PMX-F - a fibrous polystyrene based hemosorbent with immobilized Polymyxin B that could be used directly for blood perfusion [10]. In 1994 PMX-F sorbent was officially approved for patient treatment in Japan. Currently, there are several polymyxin immobilized (PMX-F) sorbents produced commercially for clinical use, such as Alteca - LPS Absorber (Sweden), Toray Industries hic. (Japan), etc. However these sorbents have poor hemocompatibility and are very expensive. 

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-... 

It has been concluded that hemosorption with Polymyxin hemosorbent Lyposorb is an effective method of treatment of abdominal sepsis caused by gram-negative flora. HS eliminates endotoxins and stabilizes hemodynamics. The use of Polymyxin hemosorbent Lyposorb for hemosorption in the integrated therapy of patients with abdominal sepsis significantly improves results of treatment. 

Water quality is usually defined in terms of chemical and bacteriological purity, particulate matter content, and endotoxin levels. Potable water is normally from the municipal water system, which may have been treated with chlorine to control microbiological growth. Soft water and deionized water have undergone ion exchange or similar treatment to eliminate unwanted ionic species, such as Mg2+ and/or Ca2+. Purified water, water for injection, and other types of water meeting compendial specifications are produced by ion exchange, reverse osmosis, distillation, or a combination of such treatments. 

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. 

Endotoxin should be eliminated by means of distillation, reverse osmosis, and/or ultrafiltration. Generation of endotoxin is prevented by controlling the... 

The rDNA-derived products may contain potentially harmful contaminants that are normally not present in their equivalents prepared by chemical methods, and which the purification process must be capable of eliminating, such as the endotoxins expressed in bacterial cells, cellular DNA, and viruses of animal origin. Contamination with nucleic acid from transformed mammalian cells is of particular concern due to the possible presence of potentially oncogenic DNA. 

We had sought to address two questions (a) what is the elimination half-life of DS-96 in the murine model, and does the tV2 correspond to pharmacodynamic data shown in Fig. 12.19 (b) what is the therapeutic plasma concentration of DS-96 that corresponds to full protection against endotoxemic challenge in mice. DS-96 at a dose of 200 (xg/mouse (8 mg/kg) was administered to CF1 mice via i.p. and i.v. routes. Plasma concentrations of DS-96 were determined by LC-MS/MS using a deuterated DS-96 internal standard (Nguyen et al., 2008 Shrestha et al., 2008). The elimination tV2 in mice is about 400 min (Fig. 12.22), which is consistent with the observed pharmacodynamic (in vivo efficacy) data shown in Fig. 12.19. The observed concentration-versus-time profile of DS-96 in the mouse i.p. model suggests that a plasma concentration of 0.5-1.5 txg/mL corresponds to complete protection by a dose of 200 ng/animal of LPS in the D-galactosamine-primed model of endotoxin-induced lethality. 

The DNA and endotoxin levels were monitored throughout the purification process (Fig. 12). The final optimized process was robust and able to handle variable levels of these contaminants. The process achieved a high level of purity (99%) with reasonable yields (68%). The pH shifts used in elution eliminated the need for time-consuming diafiltration steps. The entire purification was completed within approximately 75 min. It was found that this process could easily be modified to accommodate other similar recombinant proteins. This process could easily be scaled up. 

The main advantage of this method is its ability to eliminate not only impurities such as albumin, but also nucleic acids and possibly endotoxins. This methodology could also be used for the separation of IgM after a few adjustments and method optimization. Figure 25 represents an example of purification of a monoclonal IgGI. 

These impurities pose risks for the safety of proteins used as therapeutics and must be removed to a final concentration below their target limit. In addition, the product stream contacts materials such as filters and resins. Extractables, such as leachates from protein A resins, can pose an immunogenic risk to the patient and must be eliminated.75 Finally, adventitious agents such as viruses and bacterial pathogens or related contaminants such as endotoxins can lead to serious problems with the safety of the protein preparation and therefore must be minimized. Table 32.6 lists concentrations for the above impurities that are generally considered acceptable in a final protein product.76... 

Although the capture step dramatically enriches a targeted protein and removes some impurities, bulk impurities such as host cell protein, DNA, endotoxin, virus, and leaching ligand remain in the eluted pool. Additional procedures are needed to eliminate these impurities. 

Elimination of endotoxins is reduced owing to the restricted filtering function of the RES in cholestasis, (s. p. 65) Renal dysfunction may occur as a result of endotoxins or deposits of IgA immunocomplexes in the glo-merula. The kidneys are occasionally swollen and contain bilirubin deposits, (s. p. 327)... 

Finally, if E. coli is used as the production system, the therapeutic Hb must be purified rigorously. Both elimination of all non-Hb proteins and elimination of endotoxins are stringent requirements, because the latter lipoproteins are recognized pyrogens. Extensive chromatographic purification is used to accomplish this. 

This dichotomy of biological activity continues to intrigue research scientists who are attempting to modify the Lipid A component of endotoxin in such a way that its toxic effects can be reduced or eliminated while its beneficial effects are retained. ... 

A process that removes or destroys endotoxin in a solution or on a material is termed depyrogenation. Endotoxin is difficult to eliminate because it is ubiquitous... 

The elimination of endotoxin from a recombinant or fermentation product is challenging because the host organism often contributes enormous amounts of endotoxin to the bulk material. The product is usually separated from endotoxin and feedstream impurities by affinity columns. 

Endotoxin is the lipid A component of LPS (see section 2.2.1). It possesses multiple biological properties including the ability to induce fever, initiate the complement and blood cascades, activate P lymphocytes and stimulate production of tumour necrosis factor. Endotoxin is generally released from lysed or damaged cells. Care must be taken to eliminate or exclude such heat-resistant material from parenteral products and their delivery systems through a process known as depyrogenation (Chapter 20). 

When drugs are to be administered by the parenteral route, solubilization plays a more important role than in oral administration, as with the latter, a solid dosage form is equally acceptable. Therefore the endotoxin-free types, povidone K12 and povidone K 17 are recommended for parenterals. The molecular weight of both products is low enough (see Section 2.2.6) to allow rapid renal elimination without storage. In many countries in Europe, e.g. Germany and Austria, only such low-molecular povidone types with a K-value of up to 18 are approved for injection (see Section 6.1.3). 

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