Endotoxin reduction

Testing Stage Minimum Value (Min) Maximum F Value (Min) Endotoxin Reduction... 

There are no compendial cycles recommended for endotoxin reduction. The standard quoted in the USP and being enforced by the FDA is that a claim to depyrogenation should be supported by evidence that endotoxin present on the product prior to tFeaimenl has been inactivated to no mote than 1/1000 of the... 

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-... 

Contaminant-clearance validation studies are of special signibcance in biopharmaceutical manufacture. As discussed in Section 7.6.4, downstream processing must be capable of removing contaminants such as viruses, DNA and endotoxin from the product steam. Contaminant-clearance validation studies normally entail spiking the raw material (from which the product is to be purihed) with a known level of the chosen contaminant and subjecting the contaminated material to the complete downstream processing protocol. This allows determination of the level of clearance of the contaminant achieved after each purihcation step, and the contaminant reduction factor for the overall process. 

Quil-A saponin toxicity. Mice fed Quil-A-supplemented diet (a saponin that emulsifies fats and potentiates the immune responses) showed higher level of docosa-pentaenoic acid in the liver. These changes were associated with a significant reduction in the plasma PGEl and PGE2 and thrombohane-B2 levels in response to an intraperitoneal injection of a lethal dose of lipopolysaccharide endotoxin, LDjf, 20 mg/ kg. The data suggest that sesame seed oil and Quil A, when present in the diet, exerted cumulative effects that resulted in a decrease in the levels of dienoic eicosanoids with a reduction in lL-1 P and a con-commitant elevation in the levels of lL-10 that were associated with a marked increase survival in mice . ... 

FIGURE 12 (A) Endotoxin and (B) DNA reduction monitored during purification steps for... 

The purification process was scaled up 10-fold from 100 to 1000 cm2 of stacked 142 mm disks. The scale-up exhibited a good separation with a 7 log reduction in endotoxin levels. The yield was 98% with a final purity of 98.2%. This process was again scaled up with the modular configuration described previously (Fig. 3). Two 30-layer 4 m2 Q modules were used in conjunction with a 1 m2 C module. The chromatograms appeared very similar to the 1000 cm2 purification. The product purity was found to be 99.7% with < 0.002 endotoxin units per microgram of product and 0.003 pg DNA per microgram of product. The final product exceeded the clinical pharmaceutical specifications for this product. 

Belanich et al.63 reported the removal of endotoxins from protein mixtures. Endotoxins in a bacterial extract containing a protein photolyase was passed through a stack of 10 disk membranes (Q-type, Sartorius). LAL assay was used to monitor the endotoxin levels after each pass. There was over 5 log reduction in endotoxin content after three passes through the membranes. The protein content was reduced during this process, however, the enzyme s specific activity was increased 35-fold. This study also determined that the binding capacity of the membrane was greater than 2.25 million EU/cm2 of membrane area. 

Belanich, M., Cummings, B., Grob, D., Klein, J., O Konner, A., and Yarosh, D. (1996). Reduction of endotoxin in a protein mixture using strong anion-exchange membrane absorption. Pharm. Technol. 20(3), 142-150. 

Reduced binding of toxin is a primary mechanism of insect resistance to Cry proteins (Ferre and Van Rie, 2002). Resistance of the Indian meal moth to Bt was due to a 50-fold reduction in affinity of the membrane receptor (from brush borer membrane vesicles of larval midguts) for 5-endotoxin. In pinkbollworms, Bt resistance is associated with reduced binding of CrylAb to the brush borer membrane vesicle. Similarly, CrylAc resistance in diamondback moths was demonstrated to be due to dramatically reduced target binding. 

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