Wednesday, June 3, 2009

Endotoxin Detection

Reckoning Recombinant FactorC

Sensitive endotoxin detection can be done with an assay based upon the recombinant factor C

The current Limulus Amoebocyte Lysate (LAL) method for endotoxin detection uses an enzymatic coagulation cascade found in the LAL that is activated by endotoxin. A new method for endotoxin detection using recombinant factor C (rFC) has been developed and evaluated. The activation of rFC is determined by the fluorescence generated by the enzymatic cleavage of a peptide-coumarin substrate. Fluorescence is measured after one-hour incubation with endotoxin standards at 37oC. The fluorescence is proportional to the endotoxin concentration and is linear in the 0.01-10 EU/ml range in log scales.

The assay detected no � (1, 3)-glucan activity and measured similar potency of endotoxin from Salmonella and Pseudomonas strains as those in LAL assays. Both the LAL assays and the assay gave similar endotoxin concentration results in various samples.

Endotoxin, also known as Lipopolysaccharide, is found in the cell membrane of Gram negative bacteria. Endotoxin can cause excessive inflammation when introduced into a host by inducing cytokine responses in macro-phages. As a result, endotoxin levels in injection materials are strictly monitored by the pharmaceutical and medical device industries.

The most widely used endotoxin detection methods are the Limulus Amebocyte Lysate (LAL) tests derived from horseshoe crab hemolymph. The LAL tests employ a serine protease catalytic coagulation cascade that can be activated by endotoxin (Figure 1). Factor C (FC), the first component in the cascade, is a protease zymogen that is activated by endotoxin binding. An alternative pathway, the Factor G (FG) pathway, can be activated by glucan binding. Downstream, these two pathways activate a proclotting enzyme into a clotting enzyme. The kinetic chromogenic LAL assay (KQCL) uses the synthetic peptide-pNA substrate that can be cleaved by the clotting enzyme, resulting in a product that exhibits a yellow color. The kinetic turbidimetric assay uses the native substrate, coagulogen that can be cleaved into coagulin. Coagulin then begins to form a gel-clot, resulting in an increase in turbidity. The densities of the yellow color (OD 405nm) and the turbidity (OD 340nm) are correlated with endotoxin concentration.

Fig. 1.

The LAL tests are very sensitive and can detect as little as femtograms of endotoxin. However, because of the alternative glucan pathway, endotoxin detections by LAL tests sometimes have false positive results in samples contaminated with glucan, such as biologicals and cellulosic materials (filters). In addition, LAL lot-to-lot variation with respect to endotoxin sensitivity has been reported. Finally, since the limulus hemolymph is the sole supply for LAL, any decrease in the population of horseshoe crabs would pressure commercial production of the LAL assay. Because Factor C only binds to endotoxin, a new endotoxin detection assay using recombinant Factor C (rFC) has been developed by Ding and Ho.

Endotoxin Sensitivity and Detection Range

Endotoxin standard curve of the assay is shown in Figure 2. The endotoxin concentration and fluorescence signal have a linear correlation in a log-log scale plot. The endotoxin detection range for a one-hour assay is 0.01-10 EU/ml. This is comparable to KQCL that has a detection range of 0.005-50 EU/ml and to kinetic turbidimetric assay that has a range of 0.01-100 EU/ml. Both KQCL and the kinetic turbidimetric assay are measured kinetically, therefore require continuous absorbance signal recording during the entire reaction time, usually in less than an hour. The endotoxin detection assay, on the other hand, is a simple endpoint assay, which only takes minutes to record data. Thus, the assay can assay more samples in less time, which can better fit into high throughput applications.

Fig. 2.

Endotoxin Specificity-No Glucan Reactivity

It has been found that the LAL based assays have non-endotoxin-specific reactivity toward glucan. This reactivity was due to the alternative Factor G pathway in the coagulation cascade (Figure 1). High concentrations of b-glucan can be used as glucan blockers to inhibit the glucan reactivity of LAL. To test whether the assay has glucan reactivity, LRM was introduced into both the KQCL assay and the assay in the absence or presence of glucan blocker. As shown in Figure 3, the KQCL assay detected a significant signal due to the glucan cross-reactivity in LRM. The assay did not detect any glucan activity. The false positive signal from the KQCL assay could be inhibited by the addition of glucan blocker. Therefore, the assay is a more specific endotoxin detection method and no glucan blocker is needed.

Fig. 3.

Endotoxin Potency in Purified Lipopolysaccharide

Endotoxin potency of different purified LPS samples was compared using two LAL methods and the recombinant C-based assay method. Figure 4 shows that similar endotoxin potency is detected in these LPS samples using KQCL, recombinant C-based and the kinetic turbidimetric assays. Compared to the KQCL assay, the recombinant C-based assay measured 25 percent lower potency (P <>E.Coli O55:B5 and 50 percent higher potency (P <0.01)>Salmonella. Along with the KQCL, it measured similar (less than 5 percent variation) endotoxin potencies in Pseudomonas. The differences in endotoxin potency measurement among the three methods are probably caused by unique variations within the assay methods. As per the guidelines in the U.S. Pharmacopoeia, the commonly acceptable variation for endotoxin measurement is a 2-fold range, or 50-200 percent. In this study, using the three endotoxin assays, the variations of endotoxin were within the acceptable 2-fold ranges.

Fig. 4.

Endotoxin Potencies in Tissue Culture Medium and Other Solutions

Several in-house samples were measured for endotoxin using both the recombinant C-based and KQCL assays. Figure 5 shows that the KQCL assay and C-based assay measure similar amount of endotoxin in various solutions. The results show that the two methods give similar, within 2-fold, endotoxin concentration in tested samples.

Fig. 5.

Endotoxin Measurement in Water and Pharmaceutical Samples

Water samples are commonly measured for endotoxin in Pharmaceutical industries to monitor water sites. Endotoxin was measured in one water sample at three different dilutions using different methods. The dilution behavior of one water sample was plotted in Figure 6. Results show that endotoxin in this water sample assayed by the three methods differed within a 2-fold range. One pharmaceutical sample that has a relative low release limit was measured by KQCL and C-based assays. Using the KQCL assay, the endotoxin level was measured close to the release limit, some lots might be failed. It is suggested that this solution has possible glucan activity; therefore, using C-based assay method can effectively solve the product failure due to endotoxin test.

Fig. 6.

Lot-to-Lot Consistency of the rFC Enzyme Solution

It has been reported that the LAL has lot-to-lot variability. This is due to that the LAL are generated from the hemolymph in horseshoe crab. Differences in lysate pool, season and environment can all contribute to the lot-to-lot variation. The recombinant C-based assay, on the other hand, is generated in well-controlled laboratory. The rFC enzyme solution is produced in cell culture as recombinant protein; the C-based assay has less lot-to-lot variability. As shown in Figure 7, the endotoxin standard curves generated using four different lots of rFC enzyme solution overlapped with each other.

Fig. 7.

It All Comes Down to the Enzymes

The recombinant C assay is a sensitive endotoxin detection assay based upon the recombinant factor C. It is a single step endpoint assay that measures the fluorescence generated by the enzymatic cleavage of a synthetic coumarin substate. The endotoxin detection limit and linear assay range are comparable to the LAL tests. It has better specificity for endotoxin detection than the LAL assays and no glucan reactivity is detected. Importantly, the assay uses recombinant enzyme produced from cell culture that will help save the diminishing horseshoe crab population.

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