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Figure 3.38. Activation of clot formation by endotoxin. The presence of endotoxin causes stepwise, sequential activation of various clotting factors present naturally within the amoebocytes of the American horseshoe crab. The net result is the generation of the polypeptide fragment coagulin, which polymerizes, thus forming a gel or clot
presence of divalent cations such as calcium or magnesium. The final steps of this pathway entail the proteolytic cleavage of the polypeptide coagulogen, forming coagulin, and a smaller peptide fragment. Coagulin molecules then interact non-covalently, forming a ‘clot’ or ‘gel’.
The LAL-based assay for endotoxin became commercially available in the 1970s. The LAL reagent is prepared by extraction of blood from the horseshoe crab, followed by isolation of its amoebocytes by centrifugation. After a washing step, the amoebocytes are lysed, and the lysate dispensed into pyrogen-free vials. The assay is normally performed by making a series of 1:2 dilutions of the test sample using (pyrogen-free) WFI (and pyrogen-free test tubes; see later). A reference standard endotoxin preparation is treated similarly. LAL reagent is added to all tubes, incubated for 1 h, and these tubes are then inverted to test for gel (i.e. clot) formation, which would indicate presence of endotoxin.
More recently a colorimetric-based LAL procedure has been devised. This entails addition to the LAL reagent of a short peptide, susceptible to hydrolysis by the LAL clotting enzyme. This synthetic peptide contains a chromogenic tag (usually paranitroaniline, pNA) which is released
free into solution by the clotting enzyme. This allows spectrophotometric analysis of the test sample, facilitating more accurate end-point determination.
The LAL system displays several advantages when compared to the rabbit test, most notably:
• sensitivity — endotoxin levels as low as a few picograms (pg) per ml of sample assayed will be detected;
• cost — the assay is far less expensive than the rabbit assay;
• speed —depending upon the format used, the LAL assay may be conducted within 1560 min.
Its major disadvantage is its selectivity — it only detects endotoxin-based pyrogens. In practice, however, endotoxin represents the pyrogen by far the most likely to be present in pharmaceutical products. The LAL method is used extensively within the industry. It is used not only to detect endotoxin in finished parenteral preparations, but also in WFI and in biological fluids such as serum or cerebrospinal fluid.
Before the LAL assay is routinely used to detect/quantify endotoxin in any product, its effective functioning in the presence of that product must be demonstrated by validation studies. Such studies are required to prove that the product (or, more likely, excipients present in the product) do not interfere with the rate/extent of clot formation (i.e. are neither inhibitors nor activators of the LAL-based enzymes). LAL enzyme inhibition could facilitate false-negative results upon sample assay. Validation studies entail, for example, observing the effect of spiking endotoxin-negative product with known quantities of endotoxin, or spiking endotoxin with varying quantities of product, before assay with the LAL reagents.
All ancillary reagents used in the LAL assay system (e.g. WFI, test tubes, pipette tips for liquid transfer, etc.) must obviously be endotoxin-free. Such items can be rendered endotoxin-free by heat. Its heat-stable nature, however, renders necessary very vigorous heating in order to destroy contaminant endotoxin. A single autoclave cycle is insufficient, with total destruction requiring three consecutive autoclave cycles. Dry heat may also be used (180°C for 3 h or 240°C for 1 h).
GMP requires that, where practicable, process equipment coming into direct contact with the biopharmaceutical product stream should be rendered endotoxin-free (depyrogenated) before use. Autoclaving, steam or dry heat can effectively be used on many process vessels, pipework, etc., which are usually manufactured from stainless steel or other heat-resistant material. Such an approach is not routinely practicable in the case of some items of process equipment, such as chromatographic systems. Fortunately, endotoxin is sensitive to strongly alkaline conditions, thus routine CIP of chromatographic systems using 1M NaOH represents an effective depyrogenation step. More gentle approaches, such as exhaustive rinsing with WFI (until an LAL test shows the eluate to be endotoxin-free) can also be surprisingly effective.
It is generally unnecessary to introduce specific measures aimed at endotoxin removal from the product during downstream processing. Endotoxin present in the earlier stages of production are often effectively removed from the product during chromatographic fractionation. The endotoxin molecule’s highly negative charge often facilitates its effective removal from the product stream by ion-exchange chromatography. Gel filtration chromatography also serves to remove endotoxin from the product. While individual lipopolysaccharide molecules exhibit an average molecular mass of less than 20 kDa, these molecules aggregate in aqueous environments, generating supramolecular structures of molecular mass 100-1 000 kDa.