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Recombinant Factor C (rFC) is the first component in the horseshoe crab clotting cascade activated by endotoxin. Recombinant Factor C (rFC) is activated by endotoxin binding, and the active enzyme then cleaves a synthetic substrate, resulting in the generation of a fluorogenic compound. In the last couple of years new assays for endotoxin detection have been developed which don't have to use the blood of horseshoe crabs by using recombinant proteins. The recombinant protein introduced a new method for efficient and reliable determination of endotoxins (LPS) based on the proprietary phage ligand technology. The LPS from the sample is specifically immobilized for detection while interfering ingredients such as proteins or beta glucans are removed by a washing step. This enables the recombinant method to precisely determine the endotoxin without matrix effects, without dilution and with a broad pH tolerance. An alternative to using recombinant Factor C is Limulus Amebocyte Lysate an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantification of bacterial endotoxins.

Background
Fred Bang reported in 1956 that gram-negative bacteria, even if killed, will cause the blood of the horseshoe crab to turn into a semi-solid mass. It was later recognized that the animal's blood cells, mobile cells called amoebocytes, contain granules with a clotting factor known as coagulogen; this is released outside the cell when bacterial endotoxin is encountered. The resulting coagulation is thought to contain bacterial infections in the animal's semi-closed circulatory system.

In 1970 the U.S. Food and Drug Administration (FDA) approved LAL for testing drugs, products and devices that come in contact with the blood. Prior to that date, much slower and more expensive tests on rabbits had been used for this purpose.

Blood is removed from the horseshoe crab's pericardium; the crabs are returned to the water. LAL manufacturers have measured mortality rates of 3% in bled crabs, however recent studies indicate that this number may be closer to 15% or even 30%. The blood cells are separated from the serum using centrifugation and are then placed in distilled water, which causes them to swell and burst ("lyse"). This releases the chemicals from the inside of the cell (the "lysate"), which is then purified and freeze-dried. To test a sample for endotoxins, it is mixed with lysate and water; endotoxins are present if coagulation occurs.

In September 2011, a recombinant factor C (rFC) endotoxin detection product was introduced - a new method for efficient and reliable determination of endotoxins (LPS) based on the proprietary phage ligand technology. An enormous advantage of a ELISA-based endotoxin detection assay is the heterogenous microtiter plate based format (96-well microplate with 8-well strips). The LPS from the sample is specifically immobilized for detection while interfering ingredients such as proteins or beta glucans are removed by a washing step. This enables the method to precisely determine the endotoxin without matrix effects, without dilution and with a broad pH tolerance. The detection reaction is based on Recombinant Factor C (rFC). In January 2013, a further assay based on Recombinant Factor C without the solid phase, being suitable for medical device and water testing was introduced.

Recombinant Factor C
In the last couple of years new assays for endotoxin detection have been developed which don't have to use the blood of horseshoe crabs by using recombinant proteins. The recombinant protein introduced a new method for efficient and reliable determination of endotoxins (LPS) based on the proprietary phage ligand technology. The LPS from the sample is specifically immobilized for detection while interfering ingredients such as proteins or beta glucans are removed by a washing step. This enables the recombinant method to precisely determine the endotoxin without matrix effects, without dilution and with a broad pH tolerance.

Overcoming inhibition and enhancement
One of the most time consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition and enhancement. Agents such as EDTA and heparin are known to affect the assay if they are present in sufficient concentrations. All assays, independent of methodology are standardized using endotoxin in water. Therefore, unless the sample is water, some components of the solution may interfere with the LAL test such that the recovery of endotoxin is affected. If the product being tested causes the endotoxin recovery to be less than expected, the product is inhibitory to the LAL test. Products which cause higher than expected values are enhancing. Overcoming the inhibition and enhancement properties of a product is required by the FDA as part of the validation of the LAL test for use in the final release testing of injectables and medical devices. Proper endotoxin recovery must be proven before LAL can be used to release product.

Masking and Demasking of Endotoxins
Low Endotoxin Recovery (LER) effects in certain common sample matrix compositions was investigated using homogeneous assays as well as the solid phase based rFC assay. We found that the velocity of masking of endotoxin can be modulated using different additive. Simple protein/buffer compositions are generating moderate masking effects, whereas surfactant/buffer conditions can support very fast masking kinetics. Mixtures simulating common formulations in bio-pharmaceuticals also show rapidly appearing masking of LPS. Consequently, endotoxin measurements in certain formulations have to be interpreted with caution.

There are still important pharmaceutical products (biologics) where a simple and direct endotoxin determination is not possible. Further examples are the determination of endotoxin in protein-containing solutions and in human body fluids such as serum, plasma or cerebrospinal fluid. In these complex fluids, endotoxins are often masked and are therefore determined too low (Low Endotoxin Recovery). In order to test such samples, time-consuming sample preparation is recommended (e.g. protease digestion), with highly variable results.

Low Endotoxin Recovery (LER) effects in certain common sample matrix compositions was investigated using homogeneous assays as well as the solid phase based rFC assay. We found that the velocity of masking of endotoxin can be modulated using different additive. Simple protein/buffer compositions are generating moderate masking effects, whereas surfactant/buffer conditions can support very fast masking kinetics. Mixtures simulating common formulations in bio-pharmaceuticals also show rapidly appearing masking of LPS. Consequently, endotoxin measurements in certain formulations have to be interpreted with caution