Controlling Endotoxin Risk

A lot of research is being focused on efficiently removing endotoxins from biopharm solutions; more research is needed

By Vinod Jyothikumar, Ph.D., University of Virginia

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Limulus Amebocyte Lysate testing layout











In addition to the gel-clot technique, manufacturers have also developed two other techniques: turbidimetric LAL technique and the chromogenic LAL technique. These newer techniques are kinetic based, which means they can provide the concentration of endotoxin by extracting the real-time responses of the LAL assay. Turbidimetric LAL assay contains enough coagulogen to form turbidity when cleaved by the clotting enzyme, but not enough to form a clot.

The LAL turbidimetric assay, when compared to the LAL gel-clot assay, gives a more quantitative measurement of endotoxin over a range of concentrations (0.01 EU/mL to 100.0 EU/mL.). This assay is based on the turbidity increase due to protein coagulation related to endotoxin concentration in the sample. The optical densities of various test-sample dilutions are measured and correlated to endotoxin concentration helped by a standard curve obtained from samples with known amounts of endotoxin. A kinetic chromogenic substrate assay differs from gel-clot and turbidimetric reactions because the coagulogen is partially or completely replaced by a chromogenic substrate. When hydrolyzed by the pre-clotting enzyme, the chromogenic substrate releases a yellow-colored substance known as p-nitroaniline. The time required to attain the yellow substance is related to the endotoxin concentration.
When implementing Quality by Design concepts, the strategy for endotoxins testing should be based upon product and process understanding in combination with risk management to ensure consistent final product quality. The appropriate in-process testing should be used to evaluate the production process areas at risk of endotoxins formation or incursion. Many firms already have programs for monitoring incoming ingredients and components, including the processing water, for endotoxins contamination. The finished product release specification should be considered when determining in-process limits for each phase of manufacturing tested. For purposes of evaluating the relative risk of product contamination, quantitative testing may be preferable to limit testing to facilitate product quality trending and to identify and correct excursions before they exceed the specification and cause product failure. An endotoxins limit should be justified on a case-by-case basis, and will be evaluated as a part of each relevant marketing application or supplement.

Efficient removal of endotoxins from biopharm solutions is challenging and a lot of research is being focused on this area. Despite the development of chromatography and membrane adsorbers in recent years, more research is needed to ensure the removal of endotoxin is a cost-effective process. All of the methods devised for endotoxin removal possess operational limitations and result in protein loss when the endotoxin level in the therapeutic solution is high, thus, increasing operational costs. Endotoxin removal from pharmaceutical and biotechnology solutions requires strict adherence to cGMP guidelines, as well as the selection, optimization and validation of the endotoxin removal method.

Published in the January 2014 edition of Pharmaceutical Manufacturing magazine

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